PhD and Master's Theses
PhD, Master's, and other theses by former students of the Laboratory for Fluorescence Dynamics (LFD) and students advised by LFD members, from 1987 to present. Access to PDF documents is password protected due to copyright restrictions. Reprints can be requested from the authors.
2021
- The phenotypic investigation of mitochondria in cancer.PhD in Biomedical Engineering, University of California, Irvine, 2021.
Advisor: Digman, MichelleMetastasis remains the leading cause of cancer mortality but its mitochondrial dysregulation - from its morphology and motility to its metabolic influence - in modulating cancer’s progression is poorly understood. This dissertation describes the development and use of complex microphysiological culture systems to place cancer in its proper environmental context for mitochondrial phenotypic discoveries. FLIM of NADH is used to probe the metabolic differences between invasive and non- or less-invasive cancer cells and elucidate potential invasion-specific therapeutic targets. This dissertation also introduces Mitometer, an algorithm for fast, unbiased, and automated segmentation and tracking of mitochondria in live-cell two-dimensional and three-dimensional time-lapse images. Mitometer finds that mitochondria of triple-negative breast cancer cells are faster, more directional, and more elongated than those in their receptor-positive counterparts. Furthermore, Mitometer shows that mitochondrial motility and morphology in breast cancer, but not in normal breast epithelia, correlate with metabolic activity. Mitometer is then applied to investigate cellular contractility in the influence of mitochondrial phenotype. The goal of this dissertation as a whole is to accentuate the importance of creating new assays and techniques with the impact of context and translatability in mind. - Characterization of local conformational structure and mechanical properties of chromatin using fluorescence lifetime imaging microscopy and phasor analysis.Master in Biomedical Engineering, University of California, Irvine, 2021.
Advisor: Digman, MichelleChromatin is a complex of macromolecules that plays a very important role in packaging the long strand of DNA inside the eukaryotic nucleus. Chromatin enables and regulates many DNA functions and interactions with other molecules including DNA transcription, replication, and repair. During the cell division, chromatin ensures proper division and transfer of the genetic material to daughter cells. To fulfill these roles, chromatin must constantly undergo reconfiguration and reorganization of its structure. Therefore, local conformational structure and mechanical properties of chromatin play a crucial role in creating the highly dynamic structure of chromatin. Furthermore, recent studies link mechanobiology of the nucleus, specifically nuclear stiffness and deformability, to cancer metastasis. Due to the fact that chromatin structure is highly compact and dynamic, visualizing and studying its structure, remodeling and mechanical properties is very difficult. In this research, we showed that Fluorescence Lifetime Imaging Microscopy (FLIM) and phasor analysis can be used as a powerful technique to study local conformational structure and mechanical properties of chromatin during mitotic cell division and in metastatic tumor cells. More specifically, the findings from the lifetime values revealed information about the size, stiffness, deformability, and accessibility of the minor grooves in chromatin. The findings from this study confirmed significant structural differences between metaphase and interphase chromatin, but they also revealed significant variations within chromatin at any stage. We postulated that these variations are largely sequence dependent. Two types of regions with distinct conformational and mechanical properties were identified in chromatin. One of them had a significantly longer lifetime which was indicative of more rigid and larger binding area. These regions were attributed to GC-rich minor grooves. The other regions with significantly shorter lifetime, indicating softer and smaller binding sites, were associated with AT-rich minor grooves. Moreover, the lifetime of metaphase and interphase chromatin differed more significantly in GC-associated grooves. Therefore, we concluded that there were far more AT-rich minor grooves accessible for binding in both metaphase and interphase chromatin to the extent that the effect of chromatin condensation during metaphase did not cause very significant differences in the lifetime values. Finally, the fluorescence lifetime analysis revealed that chromatin in highly aggressive metastatic tumors like MB231 and MFC7 was significantly softer and more deformable than non-tumorigenic cells like MCF10A. There was a correlation between softness and deformability of chromatin and the metastatic aggressiveness of the tumors.
2020
- Fluorescence lifetime imaging microscopy and LAURDAN spectral imaging for dynamically investigating osteoclast differentiation.PhD in Biomedical Engineering, University of California, Irvine, 2020.
Advisor: Michelle A DigmanOsteoclasts, the multinucleated bone-resorbing cells, are involved in the destructive breakdown of bones in many diseases such as osteoporosis and rheumatoid arthritis. Designing an efficient and specific therapeutic strategy to these diseases would depend on understanding osteoclasts differentiation. Although gene expression quantification and biochemical techniques have been used extensively to study osteoclast differentiation, they lack the capability to dynamically examine live osteoclasts at the single-cell level. In this thesis, we explored the practicality of the two minimally invasive microscopy techniques, NAD(P)H Fluorescence Lifetime Imaging and LAURDAN spectral imaging, in observing cellular metabolic profiles and membrane dynamics respectively during osteoclast differentiation. In addition to establishing the practicality of these two imaging platforms, our report offered a deeper understanding regarding the roles of metabolism and membrane dynamics in osteoclasts differentiation and pathogenesis of osteoclasts-associated diseases. - Fluorescence based adaptive optics and multidimensional fluorescence microscopy.PhD in Biomedical Engineering, University of California, Irvine, 2020.
Advisor: Enrico GrattonStudying the structure and interactions of molecules and cells in their native environments has always been a challenge in the life sciences. When an organism of interest is physically out of reach or invisible to the naked eye, scientists have historically turned to reductionist experiments that first isolate the organism from a complex environment and then study the bulk signal from a large population of cells in tissue or in bacterial colonies. However, the structure, function, and interactions of a population of cells can vary at a subpopulation and even single-cell level. Non-invasive tools that can study a population in relevant conditions, such as in tissue, capable of resolving single cells in a large region of interest are necessary to improve treatment of disease and understanding of physiological phenomena.
Microscopy – in particular multiphoton fluorescence microscopy – holds enormous potential to discover new science by elucidating biological properties with subcellular resolution. When paired with adaptive optics, multiphoton microscopy can image deep into highly scattering tissue, resolving structures at previously inaccessible depths. The biochemical composition of a sample can be determined with fluorescence lifetime microscopy and hyperspectral microscopy, giving information about cell state and metabolism. Image correlation spectroscopy and single molecule tracking methods can yield information about the dynamic sample properties such as transport mechanisms and diffusion of genetically encoded fluorescent proteins.
The work presented in this thesis is separated into three chapters. The first chapter describes the development of a novel deep tissue multiphoton microscope, the AO DIVER, which uses a network of guide stars to perform millimeter-scale 3D adaptive optics imaging and enable millimeter-scale imaging. In the second chapter, the deformable mirror used in the AO DIVER is used to create a high speed 3D scanning instrument, capable of scanning axially and laterally at rates of up to 40 kHz. Finally, in the third chapter, a program for simultaneous unmixing of hyperspectral and lifetime fluorescence images is described and implemented in Pseudomonas aeruginosa biofilms, elucidating a depth dependent physiological change in bacterial biofilms.
2019
- The spectral phasor approach as a potential diagnostic tool for breast cancer.Master in Biomedical Engineering, University of California, Irvine, 2019.
Advisor: Enrico GrattonBreast cancer is a significant concern within the United States, causing over 200,000 new cases and 40,000 deaths each year. A new potential solution in the form of the spectral phasor approach to data analysis, paired with the noninvasive imaging approach of DOSI, presents itself as a safe, robust, and cost-effective, answering some of the concerns presented by current imaging techniques. By performing a Fourier transform to translate a stack of absorption coefficients at varying wavelengths to a single value or cluster of points on a spectral phasor plot, we are able to visualize the data in a clearly displayed format. In this paper, we explore the spectral phasor approach to analyze data gathered from both a single point on a patient, and multiple points in a grid on a patient as means to a diagnosis. Despite the potential that exists in the solution that this approach presents for quick and simple diagnosis, significantly more data is required to determine the reliability and reproducibility of this method. - Assessment of embryo health and circulating tumor cell metabolism using the phasor-FLIM approach.PhD in Biomedical Engineering, University of California, Irvine, 2019.
Advisor: Michelle A DigmanCellular functional and structural changes associated with metabolism are essential for understanding healthy tissue development and the progression of numerous diseases. Quantitatively monitoring of metabolic processes would spur medical research towards developing precise diagnostic tools, treatment methods, and preventive strategies for reducing the impact of the diseases. Unfortunately, established methods for this purpose are either destructive or require the use of exogenous agents. Recent work has highlighted the potential of endogenous two-photon excited fluorescence as a method to monitor subtle metabolic changes. In this thesis, we apply two-photon fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores for label-free metabolic imaging in pre-implantation embryos and other biological samples.
We exploited the intrinsically fluorescent coenzyme reduced nicotinamide adenine dinucleotide (NADH), an endogenous probe extensively used for metabolic imaging. We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. Using phasor-FLIM, we identified unique metabolic states that distinguish embryonic stem cells from differentiating progeny.
We also apply the phasor-FLIM and hyperspectral microscopy to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological and non-invasive caliber for embryo quality. We identify the unique spectroscopic trajectories at different stages of mouse pre-implantation development which can be further used to distinguish pre-implantation embryo quality using an artificial intelligence algorithm at the early compaction stage with 86% accuracy. Furthermore, we showed the heterogeneity and changes in the normal pre-implantation embryos and aneuploidy embryos treated with the spindle assembly checkpoint inhibitor during embryo division can be rapidly distinguished at blastocyst stage via spectra phasor.
Finally, we designed rapid and label-free single leukemia cell identification platform that combines high-throughput size-based separation of hemocytes, and leukemia cell identification through phasor approach and phasor-FLIM to quantify changes between free/bound NADH as an indirect measurement of metabolic alteration in living cells.
These examples illustrate the potential of fluorescence lifetime imaging microscopy for unveiling complex physiological processes. Detailed image analysis and combined microscopy modalities will continue to reveal and quantify fundamental biology that will support the advance of biomedicine.
2018
- Fluorescence and phosphorescence lifetime imaging microscopy for spatial mapping of tumor behavior.PhD in Biomedical Engineering, University of California, Irvine, 2018.
Advisor: Michelle A DigmanFluorescence intensity measurements in cellular microscopy have become a valuable tool for resolving cellular structures and quantifying protein dynamics. Furthermore, the fluorescence lifetime can reveal additional information about the local molecular environment because the lifetime of a fluorophore is sensitive to changes in excited state reactions such as dynamic quenching. The spatial resolution afforded by these photoluminescence-based methods also enables the interrogation of non-homogeneous phenomenon in complex biological systems. Here, we applied lifetime imaging methods to spatially map variations in tumor behavior.
Therapeutic resistance is associated with tumor heterogeneity necessitating the development of techniques that can non-invasively identify distinct functional subpopulations. Thus, we studied glioblastoma heterogeneity using the lifetime of endogenously fluorescent NADH. With this label-free method, we identified a significant difference in the lifetime signature between tumor mass cells and stem-like tumor initiating cells. Furthermore, we were able to distinguish between the two subpopulations in a mouse xenograft model as well as monitor the transition in populations driven by culture conditions.
In addition to cellular heterogeneity, in vivo nutrient and oxygen gradients can also drive phenotypic variations illustrating the need for accurate recapitulation of varying local environments. Thus, we developed a method to non-invasively monitor cellular respiration in real time. Using tumor spheroids as the 3D model, we characterized the oxygen gradient using phosphorescence lifetime imaging microscopy and found lower oxygen concentration at the center of the spheroid relative to the periphery.
Finally, we analyzed changes in the fluorescence lifetime of a FRET biosensor to better understand the spatial regulation of Rac1 within a spheroid model. While the important regulatory roles of Rac1 in migration and in response to hypoxia have been studied separately in 2D, spheroid models contain both migrating cells along the surface and a hypoxic core. We found higher levels of Rac1 activation at the core relative to the surface of the spheroid, revealing how Rac1 may be spatially regulated within tumors in vivo.
Overall, we have used changes in the molecular environment detected by lifetime imaging techniques to spatially map functional subpopulations of cells, oxygen concentration, and protein activation to gain insight on tumor behavior. - Analyses of the cellular signaling responses to DNA damage and DNA repair factor recruitment using fluorescence lifetimes and fluctuations.PhD in Biomedical Engineering, University of California, Irvine, 2018.
Advisor: Michelle A DigmanGenome integrity is continually challenged by threats including DNA replication errors, toxic metabolic byproducts, and exposure to exogenous genotoxins. Responding to and repairing damaged DNA requires coordinating a number of critical cellular events including activating DNA repair, facilitating chromatin rearrangement, and delaying cell cycle progression. Although the factors important for DNA repair have been identified, how these activities are coordinated in the nucleus and the long-term cellular-wide consequences of DNA repair are not well characterized.
Nicotinamide adenine dinucleotide (NAD) is a coenzyme involved in both cellular metabolism and DNA repair. Thus, analyses of NAD species could provide new insight into DNA damage signaling as well as damage-induced metabolic responses to various types of DNA lesions. Here, we use the phasor approach to fluorescence lifetime imaging microscopy in combination with genetically encoded fluorescent biosensors to measure metabolic changes in single cells with high spatiotemporal resolution. We found that the absence of the reversionless 3-like translesional synthesis DNA repair protein promotes p53-mediated upregulation of oxidative phosphorylation (oxphos) in cisplatin-treated H1299 lung carcinoma cells and increases cell sensitivity to this chemotherapeutic treatment. Furthermore, it has been well-documented that depletion of NAD+ leads to an overall metabolic collapse, but it is not clear whether or not regulating metabolism can overcome cell death pathways as a survival mechanism. We observe a PARP-dependent decrease in NAD species and an increased metabolic reliance on oxphos. In all, our analyses revealed a previously unrecognized long-term effect of DNA repair signaling on energy metabolism in DNA damaged cells.
Although the patterns of DNA repair protein redistribution following DNA damage have been systematically documented, understanding the complex response to damage requires the characterization of the molecular dynamics of these proteins with high spatiotemporal resolution. By using spatial pair cross-correlation function analysis in two-dimensions, we were able to visualize the barriers to molecular motion of DNA repair proteins in response to laser microirradiation-induced DNA damage.
In summary, my project utilized cutting-edge fluorescence dynamics techniques to reveal a connection between the DNA damage response and cellular metabolism and to develop a new method to characterize molecular diffusion in response to DNA damage. - Acute alcohol exposure shifts metabolism of breast cancer cells.Master in Biomedical Engineering, University of California, Irvine, 2018.
Advisor: Michelle A DigmanAlcohol consumption has been recognized as a risk factor for breast cancer. It is positively correlated with the progression of breast cancer. Ethanol is studied to shift the metabolism of breast cancer cells. According to the Warburg Effect, cancer cells predominantly produce their energy through a high rate of glycolysis. I hypothesized that with acute ethanol exposure the breast cancer cells will shift their metabolism from oxidative phosphorylation to glycolysis. The metabolic shift of cancer cells changes the ratio of free and protein bound NADH, which is an essential coenzyme in cellular metabolic pathway. As such, investigating the fluorescent lifetime of free and protein bound NADH of breast cancer cells through Fluorescence Lifetime Imaging Microscopy (FLIM), I report the preliminary results on the metabolic shifts of two breast cancer cell lines: MDA-MB-231 and MCF-7 due to acute alcohol exposure. - Nanoscale changes for macroscale results: modulation of cancer metabolism and adhesion by the substrate.PhD in Chemical and Biochemical Engineering, University of California, Irvine, 2018.
Advisor: Michelle A DigmanCell-to-substrate interactions are known to regulate important cellular processes such as migration, proliferation, and intercellular signaling. Cancer cells remodel the surrounding collagen matrix and promote collagen fibrillogenesis for invasion. This increases substrate density and activates the mechanosensing pathway for events such as adhesion and migration. Recent studies indicate that metabolism is able to affect cancer invasion and that mitochondria are recruited towards the invading edges of the cell. There seems to be a link between the mechanosensing and metabolism pathway in promoting invasion; however, it is unclear how these are regulated and affects mitochondria trafficking towards the protruding edges. In this thesis, focal adhesion (FA) dynamics within NIH3T3 cells were studied using the Number and Molecular Brightness analysis and were found to adapt to the underlying nanotopography by modulating their adhesion size and turnover dynamics leading to a change in cell speed. This laid the foundational studies within cancer cells. In MDA-MB231 breast cancer cells, raster imaging correlation spectroscopy (RICS) was used to study focal adhesion dynamics in response to substrate topography and collagen density. The increase of substrate density and addition of nanolines showed a decrease in FA protein dynamics and increased tension, respectively. In addition, Fluorescent lifetime imaging microscopy (FLIM) was used to measure metabolic signatures within MDA-MB231 cells. A shift from oxidative phosphorylation (OXPHOS) to glycolysis (GLY) with increasing collagen density was observed. Since the increase of substrate density is known to upregulate focal adhesion formation, actin polymerization, migration, and invasion, a large amount of ATP is consumed. Rac1 activation was stimulated to promote membrane formation and actin polymerization to observe mitochondria recruitment. Mitochondria transport speed increased when Rac1 was activated compared to when it was inactivated, specifically in breast tumor cells. This shows that mitochondria are transported to regions of high energy consumption and sustain processes needed for invasion. In addition, metabolism of the invading protrusions was found to shift towards a GLY metabolic signature. Results indicate that collagen density, metabolism and mitochondria trafficking all play an important role in regulating cancer cell invasion. When developing therapies, these parameters should be considered in order to effectively prevent metastasis. - Altered energy metabolism and nucleating aggregates found in normal cells as a consequence of the cell-to-cell transfer of the pathogenic polyglutamine aggregate from HD diseased cells.Master in Biomedical Engineering, University of California, Irvine, 2018.
Advisor: Michelle A DigmanHuntington’s disease (HD) is a late-onset autosomal neurodegenerative disease caused by the abnormal expansion of polyglutamine (polyQ) in the Huntington gene with the mitochondrial dysfunction as an early pathological mechanism. Individuals carrying 7-35 glutamine repeats are considered normal while above 41 repetitions will always lead to HD. Compelling evidence shows that the cell-cell transfer of the mutant Huntingtin (mHTT) protein aggregates may play an essential role in the pathogenesis of HD. Most recently in our lab, we showed that energy metabolism is altered in polyQ expressing cells. Yet many questions remain: 1) Does the transfer of the polyQ aggregates occur between cells? 2) If so, do the Huntingtin proteins of normal length increase protein aggregation in normal length Huntingtin expressing cells? 3) Is there any influence in energy metabolism as a consequence of the transfer of the pathogenic polyQ aggregate from infected cells? In this research, mHTT aggregates transfer intermediated NADH fluorescence lifetime change was measured using fluorescence lifetime imaging microscopy (FLIM) coupled with phasor analysis. Results obtained here suggest a metabolic shift from oxidative phosphorylation (OXPHOS) to more glycolytic state caused by the internalization of mHTT aggregates in HEK293 cells, which may lead to oxidative stress and cell death. Nuclear FLIM analysis shows a lifetime shift towards a lower fraction of bound NADH, which indicates a possible transcriptional dysregulation for infected cells. In addition, we performed Number and Brightness (N&B) analysis to map the oligomerization in live cells induced by mHTT aggregates. As seen in the results, there is a significant accumulation of endogenous HTT proteins after the internalization of extracellular mHTT aggregates. Altogether, the FLIM and N&B analysis used here provide a better understanding of the metabolic dysfunction and protein aggregation mediated by mHTT aggregate, which can be useful for further research in the field of neurodegenerative disease. - Multiphoton imaging and phasor approach to identify new biomarkers in Huntington Disease.PhD in Biomedical Engineering, University of California, Irvine, 2018.
Advisor: Michelle A DigmanNeurodegenerative diseases occur when brain cells (neurons) start to deteriorate. Changes in these cells will lead to dysfunction and eventual cell death. This will lead to mild symptoms like problems with coordination, psychiatric disorders, or memory loss; and as more neurons die the symptoms also progressively worsen. World Health Organization (WHO) indicates up to 1 billion people worldwide are affected by various types of neurodegenerative diseases. In this study, I focused on Huntington disease (HD), a model to study neurodegeneration that is caused by a glitch in a single gene called huntingtin gene (HTT). Huntington disease is an autosomal dominant inherited neurodegenerative disease characterized by movement, cognitive and emotional disorders. We all carry HTT; however, the normal length of DNA trinucleotide, CAG, that codes for glutamine are between 10–35. The expanded repeats of above 40 or more will lead to HD. Using advanced functional imaging technique called Two-Photon Fluorescence Lifetime Imaging Microscopy (2P-FLIM), and spectral and temporal phasor approach, spectro-temporal phasor map in living mammalian cells and animal tissue was obtained. Using this sophisticated imaging technique, I have developed new methods and identified novel biomarkers that can help detect Huntington disease early on. This can also help for evaluating the efficacy of treatment. The novel method established in this work is noninvasive and can be performed at the single cell level. Phasor transformation used here simplifies the FLIM and spectral measurements by providing a graphical global view of the process at each pixel and avoids some of the complexity of the multi-exponential analysis. In this way, using a fit free approach that can be applied to both time and frequency domain measurements, Fluorescence Lifetime and spectral emission can be analyzed. It is hoped that this work shed a light on understanding the mechanism of Huntington disease and for new drug discovery and early diagnosis of the disease. The approach introduced in this work can also be applied as a method for understating similar neurodegenerative diseases. This work is supported in part by NIH grant P41 GM103540, NSF BEST IGERT and UC PDY grant.
2017
- Patient-derived induced pluripotent stem cells for Alzheimer's disease model and 3D culture of neurons on a multi-electrode array.Master in Biomedical Engineering, University of California, Irvine, 2017.
Advisor: Michelle A DigmanInduced pluripotent stem cells (iPSC) have opened a new direction on study of Alzheimer’s Disease due to their pluripotency. Differentiation of iPSC to neurons has been cultured in 2D successfully with solid proof including morphological, electrophysiological and immunological, but the yield is low and unpredictable. However, 3D culture of neurons might be better approach human physiology. As a result, differentiation of iPSCs to neurons in a 3D environment has become a new research topic. We obtained 3 different iPSC cell lines from fibroblasts of, non-demented, mild cognitive impaired and demented human subjects. We differentiated these iPSCs first to neural stem cells (NSC) by neural induction, then into neurons on multi-electrode arrays to measure network electrical activity. We also cultured rat hippocampal neurons in both 2D and 3D environment of Matrigel on the MEAs to compare their average spike rate and average inter spike interval. The 3D cultures of rat neurons indicate a need for further optimization of the 3D matrix. The low yields of neuroprogenitors and neurons from human iPSC suggest a need for better control of the initial generation of the pluripotency of the iPSC.
2016
- Label-free fluorescence lifetime imaging microscopy (FLIM) to study metabolism and oxidative stress in biological systems.PhD in Biomedical Engineering, University of California, Irvine, 2016.
Advisor: Enrico GrattonStudy of cellular metabolism and its influence on physiological functions and pathology along with investigation of oxidative stress in pathogenesis are essential for fundamental biology as well as biomedical research. Optical imaging offers the opportunity to assess these indices non-invasively. In this work we apply two-photon fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores for label-free metabolic and oxidative stress imaging in a wide range of biological samples. Analysis of FLIM data was performed by applying the ‘fit-free’ phasor approach where each pixel of the image is transformed to its corresponding phasor on the phasor plot. Biological systems are a rich resource of autofluorescent biomolecules. Their fluorescence lifetimes are sensitive to alteration of normal physiology, making them attractive endogenous probes. We discovered one such endogenous fluorophore with characteristic long fluorescent lifetime. We hypothesized these long lifetime species (LLS) to be fluorescent products of lipid oxidation by reactive oxygen species (ROS), rendering them biomarkers of oxidative stress. To correlate the long lifetime species (LLS) with lipid droplets, we performed simultaneous FLIM and two coherent nonlinear microscopy techniques: third harmonic generation (THG) imaging microscopy and coherent anti-Stokes Raman scattering (CARS) microscopy that are sensitive to lipids. We went one step further to characterize the chemical nature of this discovered species by classical Raman spectral analysis. We show application of this technique in cancer, induced pluripotent stem cell derived cardiomyocytes, as well as in freshly excised mice adipose tissue. The identified endogenous biomarker unfolds opportunities of performing non-invasive measurements of oxidative stress in vivo.
We also exploited the autofluorescent coenzyme reduced nicotinamide adenine dinucleotide (NADH), an endogenous probe extensively used for metabolic imaging. We performed NADH-FLIM to study the metabolic status of a vascularized three-dimensional tumor microenvironment in a microfluidic based platform. We could identify metabolically dissimilar regions, as well as identify metabolic response to anticancer drug.
Finally, we explored NADH-FLIM of a different class of organisms – bacteria. We show for the first time, FLIM-phasor fingerprint of clinically important bacteria. We discovered interesting bacterial phasor trajectories at different growth phases as well as response to antibiotics, all at single cell resolution. - Characterizing lateral diffusion of EGFR using advanced fluorescence spatial correlation functions.Master in Biomedical Engineering, University of California, Irvine, 2016.
Advisor: Michelle A DigmanEpidermal growth factor receptor (EGFR) has various functionalities in the cell cycle and also plays an important role in cellular transport. EGFR is found to be localized on the cell membrane before its activation. Although, there are numerous studies that focus on the transport and signaling mechanism of EGFR mediated cellular transport, only few studies have been done in the area characterizing its lateral diffusion. In our work, we focus on characterizing EGFR lateral diffusion prior to and after activation of the receptor by epidermal growth factor using Total Internal Fluorescence Reflection Microscopy (TIRF). TIRF microscopy uses camera based data collection and novel pixel based correlation methods enable us to analyze and study a diffusing population. One can record whole cell, and analyze and decipher the predominant diffusion mechanisms presented in the cell using the techniques like pair-correlation function, image mean square displacement, number and brightness, etc. This thesis presents the very first trial to correlate EGFR movement within a cell using these techniques. - Exploring lipids with nonlinear optical microscopy in multiple biological systems.PhD in Biomedical Engineering, University of California, Irvine, 2016.
Advisor: Enrico Gratton and Eric O PotmaLipids are crucial biomolecules for the well being of humans. Altered lipid metabolism may give rise to a variety of diseases that affect organs from the cardiovascular to the central nervous system. A deeper understanding of lipid metabolic processes would spur medical research towards developing precise diagnostic tools, treatment methods, and preventive strategies for reducing the impact of lipid diseases. Lipid visualization remains a complex task because of the perturbative effect exerted by traditional biochemical assays and most fluorescence markers. Coherent Raman scattering (CRS) microscopy enables interrogation of biological samples with minimum disturbance, and is particularly well suited for label-free visualization of lipids, providing chemical specificity without compromising on spatial resolution. Hyperspectral imaging yields large datasets that benefit from tailored multivariate analysis. In this thesis, CRS microscopy was combined with Raman spectroscopy and other label-free nonlinear optical techniques to analyze lipid metabolism in multiple biological systems. We used nonlinear Raman techniques to characterize Meibum secretions in the progression of dry eye disease, where the lipid and protein contributions change in ratio and phase segregation. We employed similar tools to examine lipid droplets in mice livers aboard a spaceflight mission, which lose their retinol content contributing to the onset of nonalcoholic fatty-liver disease. We also focused on atherosclerosis, a disease that revolves around lipid-rich plaques in arterial walls. We examined the lipid content of macrophages, whose variable phenotype gives rise to contrasting healing and inflammatory activities. We also proposed new label-free markers, based on lifetime imaging, for macrophage phenotype, and to detect products of lipid oxidation. Cholesterol was also detected in hepatitis C virus infected cells, and in specific strains of age-related macular degeneration diseased cells by spontaneous Raman spectroscopy. We used synthesized highly-deuterated cholesterol to track its compartmentalization in adrenal cells, revealing heterogeneous lipid droplet content. These examples illustrate the potential of label-free nonlinear optical microscopy for unveiling complex physiological processes by direct visualization of lipids. Detailed image analysis and combined microscopy modalities will continue to reveal and quantify fundamental biology that will support the advance of biomedicine. - High frequency digital frequency domain fluorescence lifetime imaging system.Master in Biomedical Engineering, University of California, Irvine, 2016.
Advisor: Enrico GrattonA system and method is provided for improved fluorescence lifetime measurement. A high frequency digital heterodyning technique is described in which photon counting mode detectors are sampled at a rate slightly slower than a digitally pulsed excitation signal. A digital mixer produces a difference frequency which contains the same information than the high frequency signal. This lower frequency is still suitable for fluorescence lifetime measurements in microscopy. The digital algorithm provides phases and modulations of detected photons binning them into phase steps and time windows. The digital heterodynig algorithm was implemented in a very low cost FPGA (Field Programmable Gate Array).
2015
- Spatial-temporal dynamics and metabolic alterations of p53 upon cellular stress.Master in Biomedical Engineering, University of California, Irvine, 2015.
Advisor: Michelle A Digmanp53 is a tumor suppressor protein that plays a very important role in determining the fate of damaged cells. Depending on the extent of damage, p53 being a transcription factor, induces target genes that are involved in cell repair mechanisms and apoptosis. In doing so, it prevents proliferation of abnormal cells that could lead to tumorigenesis. Primarily existing in its monomeric or dimeric form, when activated, it binds to DNA as a tetramer. The localization of these tetramers in cells has never been mapped. Since p53 is mutated in 50% of human cancers, its ability to tetramerize efficiently and hence bind to the DNA is disrupted leading to tumor progression. Here we use the Number and Brightness (N&B) analysis, a powerful method to measure protein oligomerization pixel by pixel from raster scanned images thereby providing spatial maps of p53 aggregates. The research described here shows, for the first time, the oligomerization maps of the p53 protein and its mutant counterparts to establish the crucial role of p53 tetramers in tumor suppression. In addition, p53 also regulates the metabolism of the cell by modulating important metabolic pathways upon cellular stress. To determine whether this switch is indicative of the balance between apoptosis and DNA repair, the phasor approach to lifetime imaging microscopy (FLIM) was employed to detect the free and bound lifetime of reduced nicotinamide adenine dinucleotide (NADH). The shifts in lifetime are informative of the level of stress and give an indication whether the cell is undergoing cell cycle arrest or apoptosis. The ratios of free and bound NADH obtained from this data may be used as a marker of transcriptional activity. The N&B and FLIM results together provide an insight to p53 activation and this information can be further exploited to improve the field of cancer research. - Estrogen receptor alpha dynamics and function in mammalian cells.Master in Biomedical Engineering, University of California, Irvine, 2015.
Advisor: Michelle A DigmanThe role of estrogen receptors (ER) is highly dependent on their sub-cellular localization and concentration. Here, we propose an approach to detect molecular transport, diffusion and localization of the estrogen receptor ? by measuring the time cross-correlation between pairs of locations and the average number of molecules by means of fluorescence fluctuations in mammalian cells. From this data we find that there is concentration dependence for the localization of the estrogen receptor and that 17-β-Estradiol (E2) reduces the apparent diffusion of the receptor. In addition, we use fluorescence lifetime imaging, a label-free, non-invasive imaging method to demonstrate changes in the glucose metabolic pathway in ER-positive breast cancer cells. We observe a higher free to bound NADH ratio in high glucose conditions, reflecting and increased glycolysis/oxidative phosphorylation ratio. Furthermore, E2 is able to potentiate metabolic adaptation and cell viability depending on the glucose availability. Taking advantage of a wide array of available biophysical analysis techniques may provide additional useful information for estrogen receptors and in breast cancer research.
2013
- 3D cell-ECM dynamics revealed by innovative fluorescence microscopy methods.PhD in Developmental & Cell Biology, University of California, Irvine, 2013.
Advisor: Enrico GrattonThe projects presented in this thesis targeted questions related to different aspects of the 3D cell-ECM interactions that are associated with cell migration mechanism, including focal adhesion dynamics, actin dynamics, and ECM remodeling. Focal adhesions play an important role in connecting ECM to actin cytoskeleton. In 2D, the coordination of focal adhesions binding/unbinding to the ECM and actin dynamics facilitates cell migration. Moving from 2D to the more physiologically relevant 3D environment, however, focal adhesions and actin cytoskeleton distribution change greatly. It is not clear whether the cell migration mechanism that involves focal adhesion and actin based on the observation of 2D cell monolayers applies to the cells migrating in the 3D ECM.
To expand our understanding of cell-ECM dynamics in 3D, we developed novel fluorescence microscopy methods that are capable of capturing the protein dynamics in a live cell in 3D. These methods are based on nSPIRO and various FCS approaches. With the new methods that provide higher spatial-temporal resolution compared to other state-of-the-art techniques, we showed, for the first time, molecular level details of focal adhesion and actin dynamics in cells cultured in the 3D environment. Compared to the 2D cell monolayer, although the distribution of focal adhesion and actin may be different, their dynamics at molecular level share several similarities. - Lifetime and spectral phasors: exploiting Laurdan's fluorescence to characterize cell membranes.PhD in Biomedical Engineering, University of California, Irvine, 2013.
Advisor: Enrico GrattonCell membranes constitute the barrier between the cell interior and the external world. The composition and physical properties of cell membranes influence many cellular functions One biophysical parameter found to be especially critical is membrane fluidity; changes occurring in membrane fluidity play a key role in regulation of membrane properties under physiological conditions and in pathogenesis of disease (such as motility and metastatic potential of cancer cells). Laurdan is a fluorescent probe commonly used to image model and biological membranes. Laurdan is very sensitive to membrane packing, and therefore to membrane fluidity.
Here, we develop two novel imaging techniques to exploit Laurdan's fluorescence properties. Both techniques are based on the use of the phasor approach to analyze fluorescence images. By Fourier transformation of Laurdan lifetime decay or emission spectrum into a phasor we obtain two phasor coordinates for each pixel of an image. This approach provides a highly resolved, fast and fit-free graphical and quantitative analysis of imaging data.
First, we apply the phasor approach to fluorescence lifetime images of Laurdan in live cells. This method gives us the ability to resolve in vivo membranes with different properties such as water content and cholesterol content. We demonstrate this analysis in NIH3T3 cells using Laurdan as a biosensor to monitor changes in membrane fluidity during cell migration. Then, we apply the phasor approach to Laurdan spectral images. We use this approach to perform a phasor analysis of membrane heterogeneity in NIH3T3 and HEK293 live cells. Taking advantage of the greater sensitivity to small changes of the phasor method, we are able to detect highly packed micro-domains in the cell membrane and to monitor changes in membrane packing due to acute and chronic cholesterol manipulation in live cells. - Living in a digital world: features and applications of FPGA in photon detection.PhD in Biomedical Engineering, University of California, Irvine, 2013.
Advisor: Enrico GrattonOptical spectroscopy and imaging outcomes rely upon many factors; one of the most critical is the photon acquisition and processing method employed. For some types of measurements it may be crucial to acquire every single photon quickly with temporal resolution, but in other cases it is important to acquire as many photons as possible, regardless of the time information about each of them. Fluorescence Lifetime Imaging Microscopy belongs to the first case, where the information of the time of arrival of every single photon in every single pixel is fundamental in obtaining the desired information. Spectral tissue imaging belongs to the second case, where high photon density is needed in order to calculate the optical parameters necessary to build the spectral image. In both cases, the current instrumentation suffers from limitations in terms of acquisition time, duty cycle, cost, and radio-frequency interference and emission. We developed the Digital Frequency-Domain approach for photon acquisition and processing purpose using new digital technology. This approach is based on the use of photon detectors in photon counting mode, and the digital heterodyning method to acquire data which is analyzed in the frequency domain to provide the information of the time of arrival of the photons . In conjunction with the use of pulsed laser sources, this method allows the determination of the time of arrival of the photons using the harmonic content of the frequency domain analysis. The parallel digital FD design is a powerful approach that others the possibility to implement a variety of different applications in fluorescence spectroscopy and microscopy. It can be applied to fluorometry, Fluorescence Lifetime Imaging (FLIM), and Fluorescence Correlation Spectroscopy (FCS), as well as multi frequency and multi wavelength tissue imaging in compact portable medical devices. It dramatically reduces the acquisition time from the several minutes scale to the seconds scale, performs signal processing in a digital fashion avoiding RF emission and it is extremely inexpensive. This development is the result of a systematic study carried on a previous design known as the FLIMBox developed as part of a thesis of another graduate student. The extensive work done in maximizing the performance of the original FLIMBox led us to develop a new hardware solution with exciting and promising results and potential that were not possible in the previous hardware realization, where the signal harmonic content was limited by the FPGA technology. The new design permits acquisition of a much larger harmonic content of the sample response when it is excited with a pulsed light source in one single measurement using the digital mixing principle that was developed in the original design. Furthermore, we used the parallel digital FD principle to perform tissue imaging through Diffuse Optical Spectroscopy (DOS) measurements. We integrated the FLIMBox in a new system that uses a supercontinuum white laser with high brightness as a single light source and photomultipliers with large detection area, both allowing a high penetration depth with extremely low power at the sample. The parallel acquisition, achieved by using the FlimBox, decreases the time required for standard serial systems that scan through all modulation frequencies. Furthermore, the all-digital acquisition avoids analog noise, removes the analog mixer of the conventional frequency domain approach, and it does not generate radio-frequencies, normally present in current analog systems. We are able to obtain a very sensitive acquisition due to the high signal to noise ratio (S/N). The successful results obtained by utilizing digital technology in photon acquisition and processing, prompted us to extend the use of FPGA to other applications, such as phosphorescence detection. Using the FPGA concept we proposed possible solutions to outstanding problems with the current technology. In this thesis I discuss new possible scenarios where new FPGA chips are applied to spectral tissue imaging. - Deep Imaging Via Enhanced-Photon Recovery (DIVER).PhD in Biomedical Engineering, University of California, Irvine, 2013.
Advisor: Enrico GrattonImaging at high resolution in depth in turbid media such as tissues represents one of the most daunting tasks in optical biological imaging. The development of multi-photon microscopy and other technological improvements of the last two decades extended the achievable imaging depth in turbid media to 1 millimeter. Based on multi-photon excitation, my project, the DIVER (Deep Imaging Via Enhanced-photon Recovery) is a novel approach that allows imaging in turbid media up to 4 millimeters deep. The system is also suitable for live animal imaging, and it has been successfully employed to visualize the murine colon and small intestine, as well as the vasculature in subcutaneous xenograft tumors. The DIVER has also the capability to resolve images in depth using the fluorescence lifetime as a mechanism of contrast. The transmission geometry of the DIVER allows the efficient acquisition of second harmonic generation (SHG) with fractions of the power required in traditional two-photon microscopes.
2012
- Characterization of breast cancer using an endogenous tumor spectral marker: high-spectral-resolution diffuse optical spectroscopy and its clinical applications.PhD in Biomedical Engineering, University of California, Irvine, 2012.
Advisor: Enrico GrattonOptical imaging has enjoyed a large following in cancer in general and breast cancer in particular (i.e., diffuse optical imaging, DOI and diffuse optical tomography, DOT). Optical imaging biomarkers emerge from modeling specific near-infrared (NIR) absorption signatures that are sensitive indicators of important molecular concentration and disposition. We have developed Diffuse Optical Spectroscopic Imaging (DOSI) by increasing spectral information content for the purpose of increasing access to molecular targets and states. Malignancy-specific optical imaging biomarkers may be important because the above-mentioned changes in tumor hemoglobin, water and lipids are a necessary but not a sufficient condition to classify therapeutic response. We note that for all therapeutic imaging assessments (i.e., mammography, ultrasound, MRI, PET) that the same case is true for their respective contrast mechanisms. By a novel spectral analysis method, we have discovered the presence of absorption signatures that are unique to malignant lesions. A reproducible absorption spectrum (Specific Tumor Component, STC) with several distinct spectral features emerges when compared with the normal absorption spectra (the flat line near zero) measured from the normal tissue of these subjects plus an additional 21 patients without any evidence of malignancy. These data demonstrate the existence of a spectral signature that acts as an optical biomarker for malignancy. We are not aware of any other such biomarker that combines high specificity with ease of application in the imaging field.
This DOSI-measured malignancy-specific biomarker STC provides an ideal non-invasive surrogate biomarker for breast lesion detection and differentiation. Although STC offers both spectroscopic and quantitative information for breast malignancy, this method relies on complicated data analysis and lacks of standardization. Thus, it is still far from a clinical reality. In order to carry out a quantitative assessment of its potential in becoming a standardized clinical detection modality for tumor detection/prediction/prognosis, the longitudinal temporal stability of signatures must be evaluated and the detection limit must be set. The overall clinical goal is to evaluate the possibilities for STC detection method to become a future clinical practice. Building the linkage between pre-existing detection modalities (pathological biomarkers, DCE-MRI) and novel spectral signature detection is essential. The medical interpretation of the findings from conventional tools will shed light on the understanding and further employment of STC biomarker. Similarly, STC detection with a high diagnosis sensitivity and specificity could be very well an adjunct method for traditional modalities. - Inclined selective plane illumination microscopy: an efficient microscopy method.PhD in Biomedical Engineering, University of California, Irvine, 2012.
Advisor: Enrico GrattonThere is a current need in biomedical sciences to perform live imaging with higher spatial and temporal resolution. To achieve this goal in a comprehensive manner, three-dimensional acquisitions are necessary. Ideal features of a modern microscope system should include high imaging speed, high contrast ratio, low photo-bleaching and photo-toxicity, good resolution in a 3D context and mosaic acquisition for large samples. Considering the importance of collecting data in live sample increases the technical challenges required to solve these issues. This work presents a practical ver- sion of a microscopy method, Selective Plane Illumination Microscopy re-introduced by Huisken et al in 2004. This method is gaining importance in the biomedical field, but has been limited by difficulties associated with unconventional microscope construction using two objectives and sample preparation. Based on the selective plane illumination principle but with a design similar to the Total Internal Reflection Fluorescence microscope,Dunsby demonstrated the Oblique Plane Microscope (OPM) using a single objective which uses conventional sample preparation protocols. However, the instrument of Dunsby was not intended to be part of a commercial microscope. In this work we describe a system with the advantages of OPM and that can be used as an adaptor to commonly used microscopes, such as IX-71 Olympus, simplifying the construction of the OPM and increasing performance of a conventional microscope. We named our design inclined Selective Plane Illumination Microscope (iSPIM). - Breast cancer spatial heterogeneity in near-infrared spectra and the prediction of neoadjuvant chemotherapy response.PhD in Biomedical Engineering, University of California, Irvine, 2012.
Advisor: Enrico GrattonBreast cancer accounts for more than 20% of all female cancers. Many of these patients receive neoadjuvant chemotherapy (NAC) to reduce the size of the tumor before surgery and to anticipate the efficacy of treatments for after the procedure. Breast cancer is a heterogeneous disease that comes in several clinical and histological forms. The prediction of the efficacy of chemotherapy would potentially select good candidates who would respond while excluding poor candidates who would not benefit from treatment. In this work we investigate the possibility of noninvasively predicting chemotherapy response prior to treatment based on optical biomarkers obtained from tumor spatial heterogeneities of spectral features measured using Diffuse Optical Spectroscopy. We describe an algorithm to calculate an index that characterizes spatial differences in broadband near-infrared absorption spectra of tumor-containing breast tissue. Patient-specific tumor spatial heterogeneities are visualized through a Heterogeneity Spectrum (HS). HS is a biomarker that can be attributed to different molecular distributions within the tumor. To classify lesion heterogeneities, we built a Heterogeneity Index (HI) from the HS by weighing specific absorption bands. It has been shown that NAC response is potentially related to tumor heterogeneity. Therefore, we correlate the HI obtained prior to treatment with the final response to NAC. In this thesis we also present a novel digital parallel frequency domain system for tissue imaging. The systems employs a supercontinuum laser with high brightness, and a photomultiplier with a large detection area, both allowing a deep penetration with extremely low power on the sample. The digital parallel acquisition is performed through the use of the Flimbox and it decreases the time required for standard serial systems that need to scan through all modulation frequencies. The all-digital acquisition removes analog noise, avoids the analog mixer and it does not create radiofrequencies. The detection scheme allows a very sensitive acquisition due to the low S/N. We are obtaining absorption spectra with a high number of FDPM wavelength points, thus we expect to increase the accuracy of spectra. The more detailed spectra should improve the characterization of breast tumor spatial heterogeneities, thus the prediction of chemotherapy outcome.
2011
- Explorations of the properties of two-dimensional photonic crystals: fluorescence enhancement effect and near-infrared resonant modes.Bachelor in Physics and Astronomy, University of California, Irvine, 2011.
Advisor: Enrico Gratton and Laura C EstradaA fluorescence enhancement effect has been reported in previous publications when photonic crystals (PhC’s) are illuminated with excitation light of the wavelength corresponding to their particular resonant modes. This effect, along with a shift in resonant mode wavelength with the addition of aqueous media on top of the crystal, was observed in this series of experiments. In order to prove that the fluorescence enhancement observed is due to a property of the PhC and not a property of the dye’s interactions with the PhC surface, number and brightness (N&B) experiments were performed. These experiments indicate that the fluorescence enhancement observed is due to a property of the crystal. Resonant modes were also explored in the near-infrared region of the electromagnetic spectrum.
2010
- Phasor approach to fluorescence lifetime microscopy to measure stem cell differentiation.Laurea in Bioengineering, University of Padua, Italy, 2010.
Advisor: Alfredo RuggeriIn this thesis work, the phasor approach to fluorescence lifetime images (FLIM) has been used to measure stem cells differentiation. This optical method is non invasive and it allows dynamic investigation in vivo. The objective of the thesis has been, not only measuring the stage of differentiation, but also identifying factors and chemical changes involved in stem cells division. - Quantifying the pre-corneal tear film thickness and its components.PhD in Biomedical Engineering, University of California, Irvine, 2010.
Advisor: Enrico GrattonTear film stability and its interaction with the corneal surface play an important role in maintaining ocular surface integrity and quality of vision. Dry Eye Syndrome (DES) refers to abnormalities of tear film secretion and/or stability diagnosed by conventional methods such as the Schirmer test and tear break-up time (TBUT). Several different physical methods have been developed to measure non-invasively the structure and function of the tear film including high-speed videokeratography and dynamic wavefront aberrometry. Interferometry and optical coherence tomography are amongst new proposed methods to measure tear film thickness that have remained in research phase due to their complex, bulky and expensive instrumentation.
Here we present Fluctuation Analysis of Spatial Image Correlation (FASIC), a non-invasive method for evaluating the complex dynamics of the tear film surface by spatial correlation analysis. With this technology, a series of images are obtained using a laser illumination and a cMOS camera. The spatial auto-correlation is calculated for every frame. We have developed a mathematical model to obtain the thickness of the tear film from the sinusoidal background which appears in the spatial auto-correlation image. The model includes the macroscopic dynamics of small lipid droplets in the tear film. Consistent data with live animal model and human clinical study has been obtained. - Protein aggregation in neurodegenerative disease.PhD in Biomedical Engineering, University of California, Irvine, 2010.
Advisor: Enrico GrattonProtein misfolding and aggregation are the causes of several neurodegenerative diseases which affect an increasing number of people (i.e., Alzheimer's, Parkinson's, Huntington's and prion disease). In most cases, the mechanism of protein aggregation in solution has been studied in detail, while in cells the mechanism remains unknown, especially because of the difficulty to observe intermediates of aggregation (oligomers) directly in live cells. The techniques used until now to determine the aggregation stages are antibodies labeling and electron microscopy, requiring lysing or fixation of cells.
In this work, a new method to observe aggregation in live cells is introduced: Number and Brightness (N&B) fluctuation spectroscopy analysis. For the first time, this technique has been used to observe, map and quantify the aggregation of proteins in live cells. In particular, the aggregation of the exon1 of Huntingtin (Httex1p) model system of Huntington's disease (HD), has been studied. Huntingtin is the protein responsible for HD, a late-onset neurodegenerative disease. Using N&B fluctuation spectroscopy analysis performed simultaneously on the entire cell, it is possible to observe, for periods of hours, the kinetics of aggregate formation. The N&B analysis method is a very powerful in determining the aggregate size and localization of the aggregates, and in establishing a model for Httex1p aggregation in live cell.
To our knowledge, this is the first time oligomers have been observed, quantified and localized in live cells.
Other fluorescence methods (i.e. FRAP, FCS, FLIM) have been applied to study the structure of the inclusion bodies, the diffusion and the interaction of Httex1p in the cell. We were able to observe the recruitment of Httex1p by the inclusion body from all the compartments of the cell. FLIM highlighted the presence of a "phasor-fingerprint" of the inclusion bodies both in tissues and cells, suggesting that multiple scattering in the inclusion body results in a delay in the emission.
The information obtained from the different fluorescence-based techniques gives a better understanding of Httex1p dynamic in live cells. In this work, we exploit the combination of different fluorescence methods to obtain a comprehensive description of the aggregation process in live cells.
2009
- Analysis of flow using spatial-temporal correlation.PhD in Biomedical Engineering, University of California, Irvine, 2009.
Advisor: Enrico GrattonMeasuring and mapping the flow of small particles is important in fields ranging from medicine to microfluidics. This dissertation presents two new techniques for measuring flow using spatial-temporal correlation of optical signals. Both are based on the idea that as small particles move past a light source, the pattern of reflected light fluctuates. Correlation analysis can be used to extract the velocity of the particles from the pattern of fluctuations. The first technique presented here, Near-Infrared Spatial-Temporal Image Correlation Spectroscopy (NIR-STICS) is a camera based technique aimed towards measuring blood flow. NIR-STICS uses a series of image correlations to extract the rate of flow. It can measure average velocities and can also map flow over an area by dividing the image into sub regions. NIR-STICS is useful for detecting turbulent flow. The second technique, Scanning Laser Image Correlation (SLIC) improves upon the speed and resolution of NIR-STICS and can be applied to flow over a range of sizes. SLIC is faster than NIR-STICS because in SLIC a laser scans only the regions of an image where flow is present, not the entire image. SLIC uses pair-correlation analysis to extract the rate for flow and can identify spatial and temporal variation in flow. This dissertation includes computer simulations of SLIC and NIR-STICS as well as measurements in microfluidic channels and in a zebra fish animal model.
2008
- PALM. Photo-activated localization microscopy.Laurea in Physics and Biological Technologies, University of Trento, Italy, 2008.
Advisor: Enrico Gratton and Renzo AntoliniINTRODUCTION. Photo-Activated Localization Microscopy (P.A.L.M.) invented by E. Betzig (Science 2006), optically resolves selected subsets of photo-activatable fluorescent probes within cells at mean separations of less than 25 nanometers. It involves serial photo-activation and subsequent photobleaching of numerous sparse subsets of photo-activated fluorescent protein molecules. Individual molecules are localized at near molecular resolution by determining their centers of fluorescent emission via a statistical fit of their point-spread function.
The position information from all subsets is then assembled into a super-resolution image, in which individual fluorescent molecules are isolated at high molecular densities... - Development of a fluorescence lifetime based method to detect and analyze single molecule reactions in solution.PhD in Physics, University of Illinois at Urbana-Champaign, 2008.
Advisor: Enrico GrattonTransitions between protein conformations have been found to be essential to the biological function of many proteins. These conformational transitions can be observed by the effects of Förster Resonance Energy Transfer (FRET) on fluorescence emission. We present a new fluorescence lifetime analysis method called phasor trajectory analysis (PTA) which can be used to observe these conformational transitions under single molecule conditions and on the millisecond timescale. To maximize the precision obtained from the small number of photons available for this analysis, we developed a mathematical model for digital frequency domain lifetime acquisition, which was then used to derive the hardware parameters affecting precision. Using this information, we developed a new lifetime acquisition system which makes optimal use of the photons emitted by the sample, and provides two fully independent lifetime channels.
This new hardware was used to observe the conformational dynamics of a calmodulin sample labeled with a FRET pair, and encapsulated within 100nm lipid vesicles. A toolbox of analysis techniques was developed for PTA, and was used to quantitatively describe the transitions and fluctuations of the conformation of calmodulin. Analysis was done in a model-free manner, and also by applying known parameters about the system to extract more specific information. Using the information obtained, a conformational model was developed to describe the dynamic behavior of calmodulin's conformation in terms of its binding with calcium. In addition, measurements conducted in the presence of a peptide derived from Ca 2+ /calmodulin-dependent protein kinase II were used to examine the properties of calmodulin's conformational dynamics while interacting with its binding targets.
2007
- FLIMBox: a novel fluorescence lifetime approach for the confocal microscope.PhD in Biomedical Engineering, University of California, Irvine, 2007.
Advisor: Enrico GrattonWe designed a novel fluorescence lifetime acquisition module, named the FLIMBox, for use in laser scanning confocal microscopes (LSMs), and based on Field Programmable Gate Array (FPGA) technology. The basic challenge of measuring fluorescence lifetimes is to accurately determine the distribution of times between fluorophore excitation and the emission of photons on the nanosecond timescale. The implementation was based on a digital heterodyning frequency domain method which allows the use of modulation frequencies on the order of 100MHz to measure fluorescence lifetime decays. The FLIMBox improves the duty-cycle, stability, and ease of use over analog frequency-domain techniques, and at a significantly lower cost in comparison to time-domain techniques. A theory general to all photon counting, fluorescence lifetime instruments was developed to examine the system parameters affecting the uncertainty in measuring lifetime with a frequency domain analysis. According to this theory, the FLIMBox was then optimized to achieve efficiency comparable to TCSPC systems. Two-photon and commercial single-photon LSMs were converted into FLIM systems using the FLIMBox. The acquired FLIM information which can be used to identify spatial heterogeneity of lifetime and FRET was analyzed with the phasor method, as shown in the two demonstrations. A FRET based probe was used to show the correlations between results reported by the intensity FRET method and the lifetime information available in the two parallel donor and acceptor channels provided by the FLIMBox. An analysis method was also presented by which complicated effects such as photoconversion can be compensated for by using the lifetime information available in both channels to extract the non-photo-converted portion of the lifetime signal. - Intrinsic spectroscopic tumor markers revealed by double-differential analysis of near-infrared absorption spectra.PhD in Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 2007.
Advisor: Enrico GrattonNear infrared (NIR) optical methods provide contrast based on quantitative functional changes in tissue. NIR techniques have shown to be sensitive to changes in breast physiology from disease by quantifying total hemoglobin (oxyhemoglobin and deoxyhemoglobin), oxygen saturation, fat content, and water content. Tumors display changes in these parameters; however, it is debatable whether they are specific "signatures" of cancer. Thus differential diagnosis (benign vs. malignant) solely based on changes in normal tissue components has been difficult. The question we address is: are there unique spectral differences between the normal and tumor-containing breast tissues besides spectral differences resulting from tissue composition? To this end we have developed a new approach to spectral analysis of broadband near-infrared (NIR, 650-1000nm) spectra. This is a self-referencing double-differential method which requires high spectral resolution. Application of this method to absorption spectra from tumor-containing tissue reveals specific tumor components (STC). The STC are characterized by specific NIR absorption bands. These absorption bands are tumor specific in at least two important ways: (1) spatial specificity--STC absorption bands are localized to the tumor-containing region. We quantify the spectral changes by characterizing the spectral features in an index called the Specific Tumor Component (STC) Index. The STC index identifies the regions of the breast with tumors. (2) lesion-type specificity--STC spectral shape are different for cancer and fibroadenoma lesions. To discriminate benign and malignant lesions we developed a spectral separation method which exploits the entire wavelength region of the STC spectra. We found that by normalizing the amplitude and weighting wavelength regions we could achieve the best discrimination. The spectral separation method separates lesion types. Currently the biochemical origin of the STC spectrum is unknown, however based on spectral regions of absorption near 760, 930, and 980nm, we identify these biomarkers with changes in state or addition of lipid and/or water. - Near infrared spectroscopy for breast cancer detection and planning of a new non-contact instrument.Laurea in Physics and Biomedical Technologies, University of Trento, Italy, 2007.
Advisor: Renzo Antolini and Enrico Gratton - Frequency domain near-infra-red spectroscopy in brain perfusion measurements: development of a non-contact scanning system.Laurea in Physics and Biological Technologies, University of Trento, Italy, 2007.
Advisor: Renzo Antolini and Enrico Gratton
2006
- Optical characterization of ultrasmall, hydrogen-terminated and carboxyl-functionalized silicon nanoparticles in aqueous environments.PhD in Physics, University of Illinois at Urbana-Champaign, 2006.
Advisor: Enrico GrattonThe primary theme of this dissertation is to characterize the optical and chemical properties of ultrasmall (~1 nm) silicon nanoparticles (Si-np) in aqueous environments, focusing on their potential for use as luminescent markers in biophysical and biological applications. Two systems are presented in detail: hydrogen-terminated Si-np prepared through electrochemical dispersion of a crystalline Si wafer and carboxyl-functionalized Si-np prepared via thermal hydrosilylation of surface Si-H bonds with an ω-ester 1-alkene. Chemical and physical characterizations are done using nuclear magnetic resonance, size exclusion chromatography, and infrared spectroscopy. Optical characterization is done via absorption and steady-state photoluminescence (PL) and using capillary electrophoresis coupled with laser-induced fluorescence detection.
The behavior of the hydrogen-terminated Si-np is studied over time as-prepared in isopropanol and during treatments with water, NaOH, HCl, and H2O2. The PL spectra show three distinct, near-Gaussian states with a FWHM ~0.45 eV and their respective emissions in the UV-B (~305 nm), UV-A (~340 nm), and ‘hard-blue’ (~400 nm) regions of the spectrum. The ‘hard-blue’ emission is shown to have a simple pH dependence with a pKa ~3, demonstrating the possibility of using Si-np as environmental probes. These results offer some promise for tailoring the PL properties of ultrasmall Si-np through control of their surface chemistry.
In the second part, three central elements establish that the carboxyl-functionalized Si-np have excellent potential for use as a luminescent marker in aqueous systems. First, they are shown to be ultrasmall, with a diameter of ~1 nm, comparable to that of common organic fluorophores. Second, they are shown to have narrow PL in the near-UV with a nearly-symmetric lineshape and a FWHM as small as 30 nm. Third, it is shown that standard chemical means can be used to functionalize the Si-np with carboxyl groups, giving them stability in polar (e.g. aqueous) environments and providing a route for attachment to various biomolecules through their reaction with primary amines. The capability for direct coupling with organic species through a strong and stable Si-C linkage is a defining characteristic of Si-np and offers tremendous flexibility with possible surface passivations and functionalities for this class of luminophores. - Frequency-domain near-infrared spectroscopy in the human brain.PhD in Biochemistry, University of Illinois at Urbana-Champaign, 2006.
Advisor: William W MantulinThe purpose of my dissertation is to describe the development of frequency-domain near-infrared spectroscopy (FD-NIRS) into a non-invasive and safe functional brain imaging tool for clinical and research applications. FD-NIRS is a great tool for studying cerebral physiology because we can study hemodynamic processes in the brain in real-time, where data is collected multiple times per second over time periods of hours continuously. The development of FD-NIRS has been driven by the need to non-invasively explore the anatomy and physiology of the brain from the surface of the head. Since tissues are highly transparent in the near-infrared spectral region, near-infrared light can travel through tissues. The light is treated as probability distribution of photons that are traveling through highly-scattering tissues by diffusion. FD-NIRS instrumentation measures the scattering and absorption coefficients of tissues at 758 nm and 830 nm in the near-infrared spectral window. By obtaining the absorption coefficients of the brain, we can measure brain tissue oxygen saturation and cerebrovascular hemodynamics in real-time.
Monitoring brain tissue oxygen supply, demand, delivery and consumption can be achieved by quantifying the absolute values of oxy- and deoxy-hemoglobin and how these quantities change over time during rest, functional activation, pathology, and disease. My dissertation describes the development, signal analysis, validity, and clinical feasibility of FD-NIRS and how useful the information may be for general health knowledge and preventive medicine. - "Cat"-ology: spectrally resolved neurophotonics in the mammalian brain and phantom studies.PhD in Physics, University of Illinois at Urbana-Champaign, 2006.
Advisor: Enrico GrattonPhysicists provide significant contributions to the field of Biology and Medical Sciences by applying basic physics principles to the field. Specifically, in this work, we probed the light-matter interactions in the NIR region to understand physiological processes in the mammalian brain. We sought to improve on existing principles and propose a new technique by which we can decipher these processes spectrally. This technique touted to be independent of the light transport regime allowed us to examine the hemodynamics and neuronal activity. The aim was then to test this technique and see if it produces results that were comparable to the well established Fd-NIRS in distinguishing physiological processes. Secondly, we wanted to prove that this technique was light transport regime independent which is not the case for the Fd-NIRS. The cat was chosen as an ideal test subject as its anatomy is such that photons are not fully diffusive before being detected as the total size of the grey matter in the cat is roughly 3mm thick. Additionally, we had a priori information about the activation of the visual cortex as a response to specific stimuli. - Spatial correlation of lipid phase fluctuations in model membranes studied with two-photon fluorescence microscopy.PhD in Physics, University of Illinois at Urbana-Champaign, 2006.
Advisor: Enrico GrattonThe temporal dynamics of lipid phase fluctuations and the spatial organization of model lipid membranes was studied using Two-photon excitation fluorescence microscopy. Lipid membranes of different composition were reconstituted in Giant Unilamellar Vesicles (GUVs) and labeled with the environment sensitive dye LAURDAN whose spectral properties are sensitive to the lipid phase. The Generalized Polarization (GP) function was used to quantify the lipid phase state as detected by LAURDAN. Long lived micron sized structures were detected on membranes composed of a single lipid specie as the temperature was decreases towards the main (Liquid-crystalline to gel) phase transition by calculating the GP autocorrelation function of circular scanning measurements. In binary mixtures membranes, the influence of coexisting lipid species on the phase behavior of each other was detected and characterized comparing the experimental results with a simple two state approximation. High hydrostatic pressure was used to induce morphological transformation of the GUVs and study the coupling between mechanical stress and phase behavior of the membrane. The ability to visualize the morphological and phase behavior of single GUVs under the microscope has allowed studying the coupling between lipid phase and membrane curvature.
2005
- Measurement of internal movements of the Escherichia coli ribosome using Förster resonance energy transfer and microsecond, continuous-flow turbulent mixing in micro-fabricated devices.PhD in Physics, University of Illinois at Urbana-Champaign, 2005.
Advisor: Robert M CleggWe have studied internal movements of the Eschericia coli ribosome with Förster Resonance Energy Transfer (FRET) using multiple donor-acceptor pairs labeled at specific ribosomal protein residues. We have developed a novel methodology that allows a more quantitative interpretation of distance data from FRET measurements, accounting for specific effects when using fluorescent probes, such as: non-stoichiometric labeling when biochemical separation is not possible, quantification of static and dynamic quenching, changes in extinction coefficients, effects of the orientation factor and the presence of random and systematic errors. From the obtained distance data, 13 donor-acceptor positions (from 18 independent FRET pairs) are used to model internal movements within the 30S subunit upon 70S association. These measurements are also applied to monitoring inter-subunit movements in functional states of the ribosome that are associated with the translocation cycle of the ribosome. This work reveals internal movements of the ribosome observed for the first time in solution, and presents in vitro evidence for large concerted inter-subunit motions associated with ribosome translocation.
The second half of this thesis is independent of the above. We present the design, construction and implementation of micro-fabricated, continuous-flow, turbulent mixing devices that can mix two or three fluids to complete homogeneity on the molecular scale in the microsecond range. The prototypical designs are compact, portable, simple to fabricate and consume smaller sample volumes than current technology. We characterize the turbulent mixing process in microfluidic channels with fluorescence intensity and lifetime imaging and show that both the dependence of mixing times and pressure drop on the flow velocity and Reynolds number agree well with theoretical expectations for turbulent pipe flow. The novelties in this work are: the new methods of fabrication which enable production of three-dimensional geometries in glass with micro-fabrication methods, the implementation of turbulent mixing and measurement at microsecond timescales and showing the ability to generalize theoretical expectations for turbulence in pipes with micrometer scale dimensions. - Fluorescence correlation spectroscopy: ultrasensitive detection in clear and turbid media.PhD in Physics, University of Illinois at Urbana-Champaign, 2005.
Advisor: Enrico GrattonIn this work, I describe the development of a simple, inexpensive, and powerful alternative technique to detect and analyze, without enrichment, extremely low concentrations of cells, bacteria, viruses, and protein aggregates in turbid fluids for clinical and biotechnological applications. The anticipated applications of this technique are many. They range from the determination of the somatic cell count in milk for the dairy industry, to the enumeration and characterization of microorganisms in environmental microbiology and the food industry, and to the fast and ultrasensitive detection of protein aggregates for the diagnosis of Alzheimer’s and other neurodegenerative diseases in clinical medicine. A prototype instrument has been built and allowed the detection and quantification of particles down to a few per milliliter in short scanning times. It consists of a small microscope that has a horizontal geometry and a mechanical instrument that holds a cylindrical cuvette (1 cm in diameter) with two motors that provide a rotational and a slower vertical inversion motions. The illumination focus is centered about 200 μm from the wall of the cuvette inside the sample. The total volume that is explored is large (~ 1ml/min for bright particles). The data is analyzed with a correlation filter program based on particle passage pattern recognition. I will also describe further work on improving the sensitivity of the technique, expanding it for multiple-species discrimination and enumeration, and testing the prototype device in actual clinical and biotechnological applications. The main clinical application of this project seeks to establish conditions and use this new technique to quantify and size-analyze oligomeric complexes of the Alzheimer’s disease β-peptide in cerebrospinal fluid and other body fluids as a molecular biomarker for persons at risk of Alzheimer’s disease dementia. The technology could potentially be extended to the diagnosis and therapeutic monitoring of other protein neurologic diseases where oligomeric forms may play a role. - Fast fluorescence lifetime imaging using a full-field homodyne system with applications in biology.PhD in Physics, University of Illinois at Urbana-Champaign, 2005.
Advisor: Robert M CleggA fast, full-field fluorescence lifetime imaging system based on the homodyne technique was built and used for several applications. Also contained herein is the basic theory and background for fluorescence lifetime imaging, as well as data analysis of lifetime distributions. The fast, easy-to-use lifetime imager has proven useful for many applications and essential for several. Examples of applications where such an instrument is needed (and where a conventional system is inadequate) include: any system with large photo-bleaching effects, systems where the intensity changes on the timescale of the measurement, and situations that require real-time display of lifetime images. Measuring photo-porphyrin IX in cells is an example of a system where photo-bleaching makes it difficult to measure. Measuring the lifetime changes in dark adapted plant leaves upon exposure to light is an example of a system with complicated intensity changes. Real-time display of lifetime images is required for clinical work. This paper includes details of the hardware and software for the instrument as well as data taken with the instrument. - Near-infrared spectroscopy and the swallowing event.Master in Speech and Hearing Science, University of Illinois at Urbana-Champaign, 2005.
Advisor: Adrienne L Perlman, Enrico GrattonThis study is an introduction of non-invasive near-infrared technique to the field of speech and hearing science. Since new technologies are introduced at a fast pace in medicine, it is important to rapidly evaluate their applicability to different clinical areas. In this thesis, I applied near infrared spectroscopy (NIRS) to observe brain hemodynamics in the frontal lobes during the swallowing event. NIRS has been a paradigm of new technology and medicine for more than 20 years, covering many fields of study. However, this promising technology has not been used before in the speech language pathologyst field to study the important event of swallowing. The swallowing hemodynamic response was correlated with the two major swallowing events that occur during the oral transport phase: submental muscle group activity and breathing. Important to the latter were the breathing rate and air volume. The interplay of these two events with the swallowing apnea was the core result of this investigation. Data analysis and interpretation were based on cross and time correlations due to the physiology of the swallowing. Since swallowing is a process in which the synchronization and synergy among structures is necessary to successful and healthy deglutition the correlation concept captures many important features relevant to clinical studies. The results were based on observations in a healthy population and served as a guide for future research in disordered deglutition or dysphagia. - Characterization of membrane organization and dynamics in giant unilamellar vesicles assembled from rat kidney membrane extracts and fragments.PhD in Biochemistry, University of Illinois at Urbana-Champaign, 2005.
Advisor: William W MantulinIn renal proximal tubular cells, the plasma membrane is polarized in terms of composition, structure and function into distinct regions: the brushborder (BBM) and the basolateral (BLM) membrane. The purpose of our work is to gain insight into the complex structure of biological membranes by applying the techniques that have been used in characterizing artificial membrane systems to natural membranes reconstituted into giant unilamellar vesicles (GUVs). In GUVs made from natural lipid extracts from BBM and BLM, we observed phase separation through the formation of non-fluorescent domains at physiological temperatures, implying that domain formation may be driven by lipid-lipid interactions. In intact membrane GUVs, the morphology and membrane fluidity of the vesicles demonstrate that membrane proteins also play an important role in the organization of natural membranes. The presence of domains in these membrane preparations gives support to the presence of 'rafts' or raft-like membrane domains in natural membranes. GUV studies of natural membranes would be incomplete without the determination that the membrane in the vesicles closely approximates the structure and composition of the plasma membrane. Thus the incorporation of proteins in the GUVs assembled from intact membrane fragments was confirmed through primary and secondary antibody detection of various membrane proteins: type IIa Na/Pi cotransporter (NaPi-IIa), ß-actin and Na+/K+ ATPase. Fluctuation correlation spectroscopy (FCS) measurements performed on the intact BBM reveal that the protein-lipid dynamics in renal BBM is complex with a large range of diffusing species. In particular, the dynamics of NaPi-IIa was related to the perturbations in the BBM as a result of dietary potassium deficiency. By calculating the diffusion coefficient of NaPi-IIa and using photon counting histogram (PCH) analysis to measure protein aggregation, we determined that the lateral diffusion of NaPi-IIa is slowed down and its aggregation is increased in potassium deficiency, both of which are associated with the decreased Na/Pi cotransport activity. This model was proposed as a means for posttranslational regulation of Na/Pi cotransport activity that is mediated by the increased sphingomyelin and glycosphingolipid content in potassium-deficient BBM, although the mechanisms by which these lipids may modulate NaPi-IIa activity have yet to be determined.
2003
- Cerebral hemodynamics and its oxygenation: study of their baseline and spontaneous oscillation.PhD in Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 2003.
Advisor: Enrico GrattonNear infrared spectroscopy (NIBS) has been used for determination of tissue optical properties. This technique has been especially useful for in vivo monitoring of hemodynamics and oxygen saturation in tissue. For this work, I have developed a large-ranged multidistance probe for NIBS, which allows measurements of cerebral hemodynamics and oxygen saturation. The measurements on the adult forehead were analyzed using a two-layer model of the adult head. The average values of the hemodynamic parameters and their fluctuations were determined for a group of 30 subjects. In addition to base line, we also investigated cerebral vasomotion which results in spontaneous oscillation of the hemoglobin content in the brain. The spatio-temporal structural of the cerebral vasomotion was investigated by use of phase portrait, cross-correlation, and nonlinear dynamics analytical methods. We found that the cerebral vasomotion is temporally coherent as well as spatially coherent. The correlation lengths for temporal and spatial coherence are about 3 minutes and 1-2 cm in our measurements. Cerebral vasomotion couples to brain activity by changing the phase of the oxy and deoxy-hemoglobin concentration waves to be in phase with a periodic task. - Fluorescence lifetime imaging at video rate - a new technique in photosynthesis research.PhD in Mathematics and Natural Sciences, Technical University of Berlin, 2003.
Advisor: Robert M Clegg, Gernot RengerThe achievements of this work are twofold: (1) An instrument was designed and constructed for fast Fluorescence Lifetime-resolved Imaging (FLI) - it can provide fluorescence lifetime images at video-rate. (2) The rapid image acquisition capabilities afforded by the new instrument were used to answer important questions concerning the photoprotection mechanisms of photosynthetic samples. The first of its kind, the FLI instrument described in this work enables continuous image acquisition with concurrent data analysis and visualization of fluorescence lifetime images at video-rate. Sustained rates of up to 26 fluorescence lifetime images per second can be obtained for images of 320 × 220 pixels. This was achieved by a combination of new and conceptually different hardware and software design, which integrates modern opto-electronic components and custom built software for personal computers. The instrument is based on the principle of the phase and modulation method in homodyne operation mode: In a fluorescent sample, high frequency modulated light excites an emission signal that is modulated at the same frequency, but that exhibits a lifetime dependent phase shift and demodulation. Using a modulatable image intensifier, it is possible to detect both measurement parameters with a fast charge-coupled device (CCD)-camera. Under intense irradiation, plants and photosynthetic algae activate photoprotection mechanisms to thermally dissipate potentially harmful energy that exceeds their photosynthetic capacity. Photoprotection of this kind can be monitored by non-photochemical quenching (npq) of the chlorophyll (Chl) a fluorescence produced in photosystem II. The so-called xanthophyll-cycle is known to be involved in photoprotection by conversion of violaxanthin to zeaxanthin under intense irradiation. The new capabilities for rapid fluorescence lifetime-resolved imaging microscopy were used to investigate differences between the Chl a fluorescence of wild type (WT) and xanthophyll-cycle npq mutants, npq1 and npq2 of the photosynthetic alga Chlamydomonas reinhardtii. When photosynthetic samples are brought from darkness to constant irradiation, fluorescence intensity undergoes large changes with time. Rapid measurements are necessary to determine the fluorescence lifetimes during this so-called Chl a fluorescence transient (induction). WT and the mutant npq1, (which accumulates violaxanthin), show similar fluorescence intensities and lifetimes while the npq2 mutant, (which accumulates zeaxanthin), displays reduced fluorescence intensity, (about 25-35% at P-level), compared to WT/npq1 cells. This reduction in fluorescence intensity is correlated with 20-30% shorter apparent single lifetimes from phase and modulation. These results support the important function of zeaxanthin in the photoprotection mechanism of photosynthetic samples. Similar reductions at P-level and P-to-S decay in fluorescence intensities and lifetimes for the npq2 mutant compared to WT/npq1 are also found for cells that were treated with the electron inhibitor DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), the protonophore nigericin and the efficient PSI electron acceptor methyl viologen. These results might imply that the quenching process includes contributions from additional processes not dependent on the npq mechanisms. We present the first fluorescence lifetime transients: the simultaneous measurements of transient changes of fluorescence intensity and lifetime during the fluorescence induction for the WT as well as for the mutants. Lifetime heterogeneities were observed in ensemble experiments (averaged over multiple cells) as well as at the single cell level. Cells in the state of negative geotaxis, (induced by several hours of darkness), show shortened lifetimes.
2002
- Two-photon dual channel fluctuation correlation spectroscopy: theory and application.PhD in Physics, University of Illinois at Urbana-Champaign, 2002.
Advisor: Enrico GrattonFluctuation correlation spectroscopy (FCS) is a non-perturbative method used to gain molecular information from stochastic processes such as Brownian motion (Magde, Elson et al. 1972). Dual channel FCS allows for the investigation of static (molecular associations) and kinetic (protein dynamics) parameters of interaction between two species. This technique is able to quantitate in vivo molecular interactions that are critical for cellular study. Data acquisition hardware was built which allows 25 ns time resolution and records the entire photon sequence using a hardware data compression technique. This allows multiple analysis to be performed (i.e. photon counting histogram (Müller, Chen et al. 1999), moment analysis, etc.) The basic theory and simulation of oligomer size effect on dual channel FCS data was done to allow interpretation of an in vivo study involving the amount of aggregation between different somatostatin membrane receptors. The cross-correlation, in combination with Förster resonance energy transfer (FRET), provides a more elegant method to study protein dynamics than the equivalent quenching experiment involving only one channel of detection (Haas and Steinberg 1984). The extra relaxation is present as an extra hump in the autocorrelation and as an anti-correlation for the cross-correlation. This signature anti-correlation differentiates this process from others such as triplet state, rotational diffusion, quenching, etc. In vitro confirmation is provided through studies of ribosome and cameleon proteins. - Three-dimensional single particle tracking on the two-photon microscope.PhD in Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 2002.
Advisor: Enrico GrattonWe have developed a 3D single-particle-tracking (SPT) system based around the two-photon laser-scanning fluorescence microscope that can track particles in all three dimensions and at a high frequency response. We have implemented two different techniques to achieve this goal. Both techniques employ feedback control in order to track the particle but differ in the approach they use to ascertain the particle's 3D position. The first technique scans a small volume around a particle to build up a volumetric image that is then used to determine the particle's position. The second technique scans only a single plane but utilizes optical aberrations which have been introduced into the optical system that break the axial symmetry of the point spread function and serves as an indicator of the particle's axial position. We identified several different modes of motion in sucrose solutions and agarose gels, including the transient trapping of particles in the microdomains of agarose gels. As an application of this technique, we have investigated the viscoelastic mechanical response of biological cells. The study involves attaching magnetic spheres to the surface of a cell, and then applying a magnetic field and monitoring the response of nearby fluorescent polystyrene spheres. We have observed significant motion of the cell in all three dimensions and have strong evidence that in order to adequately model the mechanical response of the cell it is important to monitor the motion in all three dimensions. - Dynamics of hairpin ribozyme.PhD in Physics, University of Illinois at Urbana-Champaign, 2002.
Advisor: Robert M CleggThis thesis research uses single molecule spectroscopy to study the dynamics of hairpin ribozyme - an enzymatic RNA molecule capable of cleaving phosphodiester linkage. RNA controls key processes of gene expression. Ribozymes can catalytically edit precursor RNA message transcribed from DNA, the edited RNA then carries the genetic information to the translation machinery and participates in the process of protein synthesis. Using a set of four nucleotides, RNA is able to fold into complex structures with enzymatic power. This has led us to the speculation that RNA catalytic functions preceded the DNA-protein partnership. The experiments described here are aimed at better understanding how RNA organizes itself into an active conformation that performs its catalytic function. As a small nucleolytic ribozyme, the hairpin ribozyme provides an excellent opportunity to dissect the enzymatic origin of RNA systems. In our experiments we use RNA molecules that have been chemically synthesized. The in vitro solid state chemical synthesis procedure is amenable to site-directed modification, and this provides a straightforward method to check for functional blocks critical for biocatalysis. The high resolution crystal structure of the hairpin ribozyme guides us in carrying out sophisticated experiments to discover structure-function relationships, it aids our investigation of dynamic processes using fluorescence resonance energy transfer (FRET) techniques on single molecules. The functional blocks and dynamic mechanisms when unveiled would help us understand RNA catalysis. - In vivo investigation of protein interactions in Caenorhabditis elegans by Förster resonance energy transfer microscopy.PhD in Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 2002.
Advisor: Robert M CleggIn this thesis Förster resonance energy transfer (FRET) microscopy was applied in living C. elegans to visualize protein interactions in the structures that attach the body wall muscle cells of the nematode to its hypodermis and cuticle. FRET is a non-radiative transfer of energy from a fluorescent donor molecule in the excited state to an acceptor molecule in close proximity. Using genetically encoded fluorescent protein labeling, FRET microscopy can be used to visualize protein conformational changes or interactions as they occur in vivo. In this study a cyan fluorescent protein (CFP) was used as the donor and a yellow fluorescent protein (YFP) as the acceptor. In the first part of the thesis the quantification of FRET between CFP and YFP by different microscopy methods was assessed by measurements of a solution of cameleon, a protein construct exhibiting varying degrees of intramolecular FRET between CFP and YFP depending on the calcium concentration in its environment. Different FRET microscopy methods reproducibly measured the degree of FRET in this sample. These methods include measurements of the sensitized YFP emission and of the CFP intensity before and after YFP photobleaching using one-photon full-field or two-photon excitation, and two-photon excitation time- and frequency-domain CFP lifetime measurements. In the second part of the thesis interactions of PAT-4, the C. elegans homologue of integrin-linked kinase (ILK), were investigated by FRET microscopy in living transgenic animals expressing CFP- and YFP-fusion proteins. Quantitative FRET microscopy in living C. elegans was most complicated by large autofluorescence contributions to the measured signals. Heterogeneous absolute and relative fusion protein expression levels were observed, and photobleaching of CFP during the measurements had to be accounted for. The CFP photobleaching kinetics were also measured to quantify the FRET efficiency. At high signal-to-noise, different FRET microscopy methods were reliable. The measurements indicate inter-PAT-4/ILK interactions in the muscle attachment structures in vivo, as well as interactions between PAT-4/ILK and PAT-6/actopaxin and between PAT-4/ILK and ßPAT-3 integrin. The thesis discusses advantages and disadvantages of the different measurement methods, and future strategies for the elucidation of the three-dimensional organization of the nematode's muscle attachment structures by in vivo FRET microscopy. - Protein dynamics: studies of adenylate kinase mutants from Escherichia coli and characterization of adenylate kinase isoforms from murine cells. Applications of fluorescence spectroscopy and microscopy.PhD in Biochemistry, University of Illinois at Urbana-Champaign, 2002.
Advisor: William W Mantulin, Michael GlaserThe first section of the thesis is focused on the fluorescence spectroscopic studies on the folding and dynamics of adenylate kinase (AKe) mutants from E. coli. We have constructed a series of single trp mutants with the mutations distributed in various regions of the enzyme. Careful characterization of each trp mutant suggested the trp replacement had little effect on its overall structure and enzymatic function. The trp residue of each AKe mutant acted as a fluorescence reporter which allowed us to follow the local environmental changes during its folding process. A model of protein folding was suggested based on the correlation of the folding kinetics of AKe and the recovery of the enzymatic activity. The second section of the thesis is focused on the characterization of two adenylate kinase isoforms (AK1 & AK1ß) from murine cells using a series of fluorescence microscopic techniques (two-photon excitation fluorescence imaging, fluorescence correlation spectroscopy (FCS), fluorescence lifetime imaging (FLIM)). In the in vivo studies, the genes of AK1 and AK1ß were fused with the gene of enhanced green fluorescence protein (EGFP) and then expressed in HeLa cells. Fluorescence images of the transfected cell showed that the AK1-EGFP was homogeneously distributed throughout the cell, while AK1ß-EGFP was mainly localized on the plasma membrane. The diffusion rates of AK1-EGFP and AK1ß-EGFP obtained from FCS measurements suggested that AK1 diffused freely in the cytosol of the cell, while AK1ß was bound to the plasma membrane. It has been suggested that AK1ß interacts with the plasma membrane through a myristoyl chain. To test the hypothesis, in the in vitro studies, AK1ß were over-expressed in E. coli, purified and tested for its interaction with a model membrane system (giant unilamellar vesicles (GUVs)). AK1ß showed no binding affinity to GUVs made from POPC in the fluorescence imaging study. However, when AK1ß was co-expressed with N-myristoyl-transferase (NMT), an enzyme that can myristoylate proteins with the proper signal in E. coli, the purified protein named as AK1ßmyr showed binding affinity to the GUVs. This result suggested that myristoylation of AK1ß played an important role in its binding to the GUVs.
2001
- Silicon nanoparticle characterization by fluorescence correlation spectroscopy.PhD in Physics, University of Illinois at Urbana-Champaign, 2001.
Advisor: Enrico GrattonThis thesis aims to characterize the fluorescence brightness and size of silicon nanocrystals in solution by measuring equilibrium fluctuations through the techniques of Fluorescence Correlation Spectroscopy (FCS) as well as Photon Counting Histogram (PCH). It was found that Si nanocrystals are comparably bright to fluorescein, a standard organic fluorophore, as well as comparably small (~1.1nm in diameter). Imaging results on single Si nanocrystals show that individual nanocrystals are photostable for over 150s of continuous illumination, orders of magnitude longer than possible with traditional organic fluorophores under similar conditions. Due to the poorly controlled sonication step in the preparation of the Si nanocrystal colloid from the porous Si precursor, it was desirable to quantify the heterogeneity of the Si nanocrystal colloid. This was achieved by extending the techniques of FCS and PCH by scanning the excitation energy. In this way each fraction excited at a given wavelength could be counted, and a spectrum of number density versus excitation wavelength could be built up. By directly measuring the molecular heterogeneity in this way it was found that there exists significant heterogeneity in the Si nanocrystal preparations (i.e. the number density changes as a function of excitation wavelength). This important new observable (number density spectrum) can now be used as a control variable in refining the production of Si nanocrystal colloids in the effort to produce homogenous samples, which would be a necessary condition for applications. Traditional ensemble techniques (fluorescence emission/excitation spectra, fluorescence lifetime) are also performed, corroborating the conclusion of heterogeneity, though such techniques are not able to quantify it at the molecular level. - Tissue blood flow and oxygen consumption measured with near-infrared frequency-domain spectroscopy.PhD in Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 2001.
Advisor: Enrico GrattonFor decades, researchers have contributed with new ways of applying physics' principles to medicine. Moreover, researchers were involved in developing new, non-invasive instrumentation for medical applications. Recently, application of optical techniques in biology and medicine became an important field. Researchers found a non-invasive approach of using visible and near-infrared light as a probe for tissue investigation. Optical methods can contribute to medicine by offering the possibility of rapid, low-resolution, functional images and real-time devices. Near-infrared spectroscopy (NIRS) is a useful technique for the investigation of biological tissues because of the relatively low absorption of water and high absorption of oxy- and deoxy-hemoglobin in the near-infrared region of 750-900 nm. Due to these properties, the near-infrared light can penetrate biological tissues in the range of 0.5-2 cm, offering investigation possibility of deep tissues and differentiate among healthy and diseased tissues. This work represents the initial steps towards understanding and improving of the promising near-infrared frequency-domain technique. This instrument has a very important advantage: it can be used non-invasively to investigate many parts of the human body, including the brain. My research consists primarily of in vivo measurements of optical parameters such as absorption and reduced scattering coefficients and consequently, blood parameters such as oxy, deoxy, and total hemoglobin concentrations, tissue oxygen saturation, blood flow and oxygen consumption of skeletal muscle of healthy and diseased subjects. This research gives a solid background towards a ready-to-use instrument that can continuously, in real-time, measure blood parameters and especially blood oxygenation. This is a very important information in emergency medicine, for persons under intensive care, or undergoing surgery, organ transplant or other interventions.
2000
- Spatial localization and temporal analysis of optical property fluctuations by multiplexed near-infrared photon density waves in turbid media: in vitro and in vivo studies.PhD in Physics, University of Illinois at Urbana-Champaign, 2000.
Advisor: Enrico GrattonIn recent years the application of near infrared non-invasive methods for medical diagnostics and clinical studies has grown rapidly. The ease of use, low cost and portability of these methods is a clear advantage over other techniques such as MRI. The limitations in detection of optical property inhomogeneities in tissues, such as tumors or hematomas, is due to the diffusive, highly scattering nature of near infrared light propagation. I have studied and developed methods to improve the spatial localization of these inhomogeneities in biological tissues, especially for the application of functional studies of the human brain in vivo. Recently much attention has been given to the study of the processes in the human brain that lead to the changing of the optical parameters that characterize the tissue, measured by our frequency-domain instrumentation. These processes have been divided into two main categories with different time-scales. The slower one is mostly due to the fluctuations in the absorption coefficient caused by oxy- and deoxy-hemoglobin changes in the tissue. The temporal analysis of the signal resulting from this process is studied in detail, and I also introduce a time-series data analysis technique that has not been applied to this field before but was introduced in another area very recently. The faster time-scale process has been attributed to the electrochemical excitation of the individual neurons in the brain that have been observed to cause a change in the scattering coefficient of the tissue. This is the other primary parameter that is measured by our frequency domain instrument. However, before this work it has not been clear how to go about to better localize these smaller fluctuations. I present a novel idea for improving spatial localization of macroscopic optical parameter fluctuations, and study the characteristics of this optical probe design using analytical solutions to the diffusion equation and Monte Carlo simulations that more appropriately represent the volume of excitation of the cortex neurons.
1998
- Analysis and applications of fluorescence fluctuation spectroscopy.PhD in Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 1998.
Advisor: Enrico GrattonFluorescence correlation spectroscopy (FCS) exploits the fluctuations of particles to determine kinetic processes from the autocorrelation function and is used to obtain information about the number of fluorescent particles in a small observation volume. We have developed a new technique, the photon counting histogram (PCH) method, to analyze fluctuation data. PCH and autocorrelation analysis focus on different properties of the fluctuation process: the autocorrelation function is a measure of the time-dependent decay of the fluctuations to their equilibrium value, while the photon counting histogram describes the amplitude distribution of these fluctuations. We derived the theory of the PCH for fluorescence fluctuation experiments and constructed an algorithm to calculate the histogram numerically. Measured photon counting distributions obtained with a two-photon excitation source agree within experimental error with the theoretical PCH. We revived another analysis method, moment analysis, which was introduced almost ten years ago. This method calculates G(0) from the fluctuation moments of the photon counts and corrects for their shot noise contribution. The main advantage of moment analysis is its simplicity and model independent determination of G(0). Binary dye mixtures were used to demonstrate the influence of the molecular brightness on the fluctuation amplitude G(0). This dependence allows to resolve multiple species based on the difference in their molecular brightness, which we applied to a titration study of an antibody with its ligand. The global fit of the average photon counts (k) and the fluctuation amplitude G(0) as a function of the protein concentration determined the number of binding sites and the dissociation coefficient KD. The analysis further demonstrated heterogeneity of the antibody complex on a time scale not easily accessible by other techniques. - Quantitative frequency-domain fluorescence spectroscopy in tissues and tissue-like media.PhD in Physics, University of Illinois at Urbana-Champaign, 1998.
Advisor: Enrico GrattonIn the never-ending quest for improved medical technology at lower cost, modern near-infrared optical spectroscopy offers the possibility of inexpensive technology for quantitative and non-invasive diagnoses. Hemoglobin is the dominant chromophore in the 700-900 nm spectral region and as such it allows for the optical assessment of hemoglobin concentration and tissue oxygenation by absorption spectroscopy. However, there are many other important physiologically relevant compounds or physiological states that cannot be effectively sensed via optical methods because of poor optical contrast. In such cases, contrast enhancements are required. Fluorescence spectroscopy is an attractive component of optical tissue spectroscopy. Exogenous fluorophores, as well as some endogenous ones, may furnish the desperately needed sensitivity and specificity that is lacking in near-infrared optical tissue spectroscopy. The main focus of this thesis was to investigate the generation and propagation of fluorescence photons inside tissues and tissue-like media (i.e., scattering dominated media). The standard concepts of fluorescence spectroscopy have been incorporated into a diffusion-based picture that is sometimes referred to as photon migration. The novelty of this work lies in the successful quantitative recovery of fluorescence lifetimes, absolute fluorescence quantum yields, fluorophore concentrations, emission spectra, and both scattering and absorption coefficients at the emission wavelength from a tissue-like medium. All of these parameters are sensitive to the fluorophore local environment and hence are indicators of the tissue's physiological state. One application demonstrating the capabilities of frequency-domain lifetime spectroscopy in tissue-like media is a study of the binding of ethidium bromide to bovine leukocytes in fresh milk. Ethidium bromide is a fluorescent dye that is commonly used to label DNA, and hence visualize chromosomes in cells. The lifetime of ethidium bromide increases by an order of magnitude upon binding to DNA. In this thesis, I demonstrated that the fluorescence photon migration model is capable of accurately determining the somatic cell count (SCC) in a milk sample. Although meant as a demonstration of fluorescence tissue spectroscopy, this specific problem has important implications for the dairy industry's warfare against subclinical mastitis (i.e., mammary gland inflammation), since the SCC is often used as an indication of bovine infection. - Image and optical property reconstruction from noninvasive measurements of turbid media, in vitro and in vivo: a promising method for optical mammography.PhD in Physics, University of Illinois at Urbana-Champaign, 1998.
Advisor: Enrico GrattonDue to their multiple scattering, near infrared photons diffuse in human tissues providing interesting physiological information over relatively large volumes. By employing frequency-domain techniques along with a diffusion model for photon transport, one can separate contributions to light attenuation from absorption and scattering processes. Both the absorption and reduced scattering parameters are sensitive to physiological characteristics of tissues. I have employed the diffusion model, which assumes that tissue is homogeneous, to create an efficient reconstruction routine based on the back-projection technique to detect and locate inhomogeneities qualitatively. Before my work in this field, researchers were uncertain whether the diffusion model would be valid for a medium containing inhomogeneities. I, along with a handful of other researchers demonstrated that the diffusion model is accurate enough to include well behaved heterogeneous tissue models in which a number of macroscopic objects of different size and shape are embedded in a homogeneous background medium. I have employed a fitting of the experimental data to the diffusion model to accurately reconstruct not only the position and size, but also the optical properties including the relative index of refraction, absorption and scattering coefficients of objects immersed in highly scattering media. In this thesis I describe a model for locating and characterizing optical inhomogeneities in a turbid medium. I will go on to discuss limits of the technique and present data collected in vivo from a 55 year old female subject with a malignant breast cancer that was detected and optically characterized by these techniques. - Applications of pump-probe and two-photon techniques in time-resolved fluorescence microscopy and spectroscopy.PhD in Physics, University of Illinois at Urbana-Champaign, 1998.
Advisor: Enrico GrattonPump-probe spectroscopy has long been useful in the elucidating ultrafast molecular phenomena. We developed a variation of time-resolved, pump-probe methodology using cross-correlation principles of the frequency-domain. An unique feature of our technique is that the low-frequency, cross-correlation signal and its harmonics which contain lifetime information at high excitation frequencies can be simultaneously acquired and analyzed by a frequency spectrum analyzer. The result is that high frequency spectral information can be acquired without a fast optical detector and simultaneous acquisition of multiple harmonics reduce data acquisition time. A second key feature of this technique is that the cross-correlation signal comes primarily from the focal volume and hence microscopic imaging using this technique results in axial depth discrimination and improved spatial resolution similar in two-photon excitation microscopy. Using this technique, spectroscopic data measuring the lifetime and rotational correlation time of aqueous rhodamine B are presented. Furthermore, time-resolved, microscopic images demonstrate this technique's capabilities in obtaining lifetimes and time-resolved polarization information of fluorescent molecules in mouse fibroblast (STO) cells. Two-photon fluorescence microscopy has proven to be an useful technique for imaging biological systems. Good spatial resolution, reduced photobleaching, and efficient rejection of excitation wavelength from emitted fluorescence are the main characteristics of this technique. As part of this work, extension of two-photon microscopy to time-resolved polarization imaging of probe molecules inside STO cells are shown. Depending on the type of probe molecules and the nature of their interaction with cellular constituents, different degrees of rotational correlation times are obtained.
1997
- Diffuse photon transport in tissue-like media: resolution limit for near-infrared imaging and an instrument for clinical spectroscopy of tissues.PhD in Physics, University of Illinois at Urbana-Champaign, 1997.
Advisor: Enrico GrattonIn this work I present the model for diffusive photon transport in tissue-like media along with solutions for a point source in infinite and semi-infinite media. Based on these solutions and experimental measurements, I consider the ultimate resolution of near-infrared based imaging of tissues using a simple model to describe resolution. I conclude that for a 5 cm thick tissue, the closest two objects buried in the center can be, and still be resolved is roughly 1.5 centimeters. I also show that light applied to the surface of a tissue-like medium, and detected on the same surface some distance away, penetrates into the medium. The depth of penetration is on the order of centimeters depending on the transport properties of the medium and source-detector separation. Since this light penetrates into tissue, and there is a model which describes how the transport of light through tissues depends on the tissue optical properties with sufficient accuracy, non-invasive tissue spectroscopy in the near-infrared is possible. During my thesis work a functioning tissue spectrometer was developed to demonstrate this point. I conclude by discussing measurements I made with this spectrometer on both laboratory samples and live patients. My experiments with the tissue spectrometer were instrumental in both demonstrating its capabilities and understanding advances to be made in the next generation of the device. - Spectroscopic studies of protein folding and protein dynamics using single-tryptophan mutants of E. coli adenylate kinase.PhD in Biochemistry, University of Illinois at Urbana-Champaign, 1997.
Advisor: William W Mantulin Michael GlaserComprehensive studies of protein dynamics and equilibrium protein denaturation are undertaken utilizing a combined approach of site-directed mutagenesis and optical spectroscopic techniques. The enzyme adenylate kinase from the organism E. coli serves as the system to be studied, having been chosen as an ideal candidate for site-directed, single-tryptophan-substitution mutations. A set of six such single-tryptophan mutants of adenylate kinase are studied in depth, using steady-state and time-resolved fluorescence techniques, as well as circular dichroism spectroscopy. Particular mutants are chosen for investigation only if their global secondary structure and enzymatic activities do not differ significantly from those of the wild-type protein. Following the initial spectroscopic characterization of the six single-tryptophan mutants at room temperature and neutral pH (both in the absence and presence of ligand), the mutants are studied exhaustively under conditions of high concentration of chemical denaturant, acidic pH, high temperature, and finally, high hydrostatic pressure–all at thermodynamic equilibrium. The structure and dynamics of the individual mutants under these various conditions are investigated using fluorescence spectroscopic techniques. Also, and of most importance, the interpretation of the results assumes that the tryptophans act as local reporter groups of the various tertiary micoenvironments of the wild-type enzyme. This provides a justification for modeling the non-native equilibrium states of the wild-type adenylate kinase. Finally, native states of the mutants are studied at high concentrations of stabilizing cosolvent, both at room temperature and liquid-nitrogen temperatures. These studies provide insight into the native-state dynamics of the protein which are impossible to observe with the given techniques at room temperature in buffer. Additionally, the stabilizing cosolvents are observed to modify the tertiary structure of the enzyme even while leaving its secondary structure and ligand-binding capacity unaffected. An 'atlas' of the various equilibrium states of E. coli adenylate kinase is presented in the final summary chapter.
1996
- The development of fluorescence lifetime imaging and an application in immunology.PhD in Physics, University of Illinois at Urbana-Champaign, 1996.
Advisor: Enrico GrattonThe development of two different methods for fluorescence lifetime imaging in microscopy are presented in addition to an application to an important immunological problem. The first method uses a fast CCD camera and a gated image intensifier to collect lifetime data with the frequency domain heterodyning technique. The camera system is capable of generating lifetime images in a few seconds, has a frame rate of up to 200 Hz, and a time resolution of 200 ps. The instrument I built has several advantages over previously reported systems. Because of the data collection technique, this instrument can measure the correct lifetime even when the sample undergoes strong photobleaching which is the major problem in the other systems using a camera. The technique used to acquire lifetime images, demonstrations of the capabilities of a fast camera system, and sample images of biological specimens are presented. The second fluorescence lifetime imaging method uses the relatively new technique of two photon excitation in microscopy. This instrument has a spatial point spread function of 0.3 micrometer (FWHM) radially and 0.9 micrometer (FWHM) axially for a 1.25 N.A. objective at 960 nm. The time resolution is 400 ps with common chromophores used in microscopy. Time resolution is obtained with heterodyning at a high cross-correlation frequency (12.5 kHz). Using this technique, lifetime information is collected on each pixel independently. Fluorescence lifetime resolved two photon microscopy was used to (non-invasively) monitor a fluorescent hapten-immunogen during intracellular vacuolar encapsulation and enzymatic processing. Fluorescein conjugated bovine serum albumin served as the soluble exogenous antigen. As a relatively non-fluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time dependent changes in the fluorescence properties. The fluorescence was limited to intracellular vacuoles. Three dimensionally resolved fluorescence lifetime images of the fluorescent probe in macrophages show that the initial lifetime of 0.5 ns increased to 2.2 ns after 24 hours of incubation indicating processing of the antigen.
1995
- Two-photon fluctuation correlation spectroscopy: method and applications to protein aggregation and intracellular diffusion.PhD in Physics, University of Illinois at Urbana-Champaign, 1995.
Advisor: Enrico GrattonFluctuation correlation spectroscopy (FCS) is an experimental technique for the study of hydrodynamics and molecular interactions of microscopic systems. The method monitors the statistical behavior of spontaneous equilibrium fluctuations in number occupation of a fluorescent species in a subvolume of a sample. Microscopic models have been developed which relate observed statistical fluctuations in fluorescence observables to physical quantities of interest. This thesis presents the first application of two-photon molecular excitation to FCS. Two-photon excitation offers several advantages over one-photon excitation for FCS experiments. These advantages are discussed, and model systems are studied to calibrate and characterize the capabilities of the technique. The theoretical framework necessary to recover physical parameters from observed fluctuation spectra is also presented. There are two biophysical applications proposed and discussed in this thesis. The first is the study of particle mobilities and intracellular diffusion. Measurements of particle diffusion in the cytoplasm of live cells are presented. The second application is the detection of protein aggregation in solution using two-photon scanning FCS. Measurements are shown demonstrating the potential to study aggregation equilibria at low protein concentrations, and the dissociation reactions of several oligomeric proteins are studied. The possibility to measure dissociation kinetics is also demonstrated. - Dynamics of oxygen penetration and diffusion into horse skeletal myoglobin revealed by quenching of zinc protoporphyrin IX fluorescence.PhD in Physics, University of Illinois at Urbana-Champaign, 1995.
Advisor: Enrico GrattonOxygen quenching of tryptophan fluorescence in a number of globular proteins provided some of the early experimental evidence for conformational fluctuations in proteins. Gratton et al. (Biophysical J. 1984. 45:789-794) proposed a sequential two step model called the dynamic model for oxygen quenching of internally buried fluorophores in proteins. For this study, that model is utilized to analyze the dynamics associated with conformational fluctuations of oxygen entry into the protein and to obtain thermodynamic parameters associated with these fluctuations. We used zinc protoporphyrin IX reconstituted myoglobin and performed oxygen quenching experiments at different temperatures. The data was fit to the above model and k+ (oxygen entry rate), k- (oxygen exit rate), and chi (oxygen migration rate) in horse skeletal myoglobin were obtained at each temperature. The activation energies were calculated using the value of k+ and k- at each temperature. The protein-to-solvent partition coefficient for oxygen, alpha, was also obtained at each temperature along with the thermodynamic variable, DeltaG, associated with this partition. The parameters k+, k-, chi, and alpha have also been determined for the quenching of zinc protoporphyrin IX in 40% sucrose to assess the effect of viscosity on these parameters. The agreement of the steady state Stern-Volmer plot, calculated using the kinetic constants derived from the time resolved measurements with the experimental data, indicates that the dynamic model accurately represents the oxygen quenching process in horse skeletal myoglobin in all the temperature and viscosity range investigated.
1994
- Imaging and spectroscopy of tissue-like phantoms using photon density waves: theory and experiments.PhD in Physics, University of Illinois at Urbana-Champaign, 1994.
Advisor: Enrico GrattonThe determination of the optical properties of turbid biological media is of primary importance in several areas of medicine and biotechnology. In particular, the quantitative determination and spatial localization of the optical scattering and absorbing properties of biological tissue would allow for the non-invasive and non-ionizing imaging of tissue structure and the monitoring of physiology. Until recently, such characterization of thick, highly scattering biological tissues using visible and near infrared light has been thwarted because of the inability to determine the absolute optical properties of thick tissues. This thesis presents the development of the concept, physical model, and experimental study of diffuse photon density waves in thick turbid media. The goal of this work is to determine the applicability of photon density waves to the optical tomography and spectroscopy of thick, multiply scattering media. Toward this end, analytic expressions based on the diffusion approximation to the Boltzmann transport equation are derived for the case of an isotropically emitting, sinusoidally intensity-modulated point source of light immersed in an infinite, macroscopically uniform, multiply scattering medium. These frequency-domain expressions are given in terms of the optical properties of the medium, and they predict that the photon density propagates outward from the light source as a spherical wave of constant phase velocity. Experiments are performed which support the validity of these frequency-domain expressions, and provide a basis for the understanding of photon transport in turbid media containing absorbing and/or reflecting objects. Further experiments demonstrate the feasibility of using frequency-domain data in conjunction with a frequency-domain diffusion model to determine the absolute optical parameters of thick, multiply scattering media.
1993
- Digital frequency domain fluorometry and the study of Hoechst 33258 dye-DNA interactions.PhD in Physics, University of Illinois at Urbana-Champaign, 1993.
Advisor: Enrico GrattonFluorescence is a powerful tool for the study of chemical and biological processes. The typical decay times of fluorescence are ideal to study events in the pico to nanosecond range. On these time scales, the motions of many biological processes can be studied. The use of frequency domain fluorometry to measure the lifetime of the excited state has been used for many years. However, the development of an acquisition system based on modern digital techniques, presented in this thesis, has opened the door to different types of experiments that previously were either too time consuming or could not be done. The use of digital techniques and the development of a method to modulate an image intensifier have made it possible to incorporate linear and matrix detectors in frequency domain fluorometry. The extension of time-resolved fluorescence measurements to linear arrays has made it possible to follow the time evolution of the emission spectra while the use of matrix detectors has permitted the measurement of the lifetime at every 'pixel' of an image. The dye Hoechst 33258 has been used for many years in the study of DNA and DNA binding. However, the fluorescent properties of Hoechst 33258 are not well understood. The dye is highly quenched in aqueous solutions and becomes brightly fluorescent when bound to A.T rich sequences of DNA or placed in non-aqueous solutions. The fluorescence of Hoechst 33258 seems to arise from two different solvation states of the molecule. When Hoechst 33258 binds to calf thymus DNA or poly(d(A.T)), the molecule becomes highly fluorescent, yet the two states can still be distinguished. The two states are attributed to different binding modes of the dye. The loose binding allows access of water molecules which results in different emission properties. On the other hand, when Hoechst binds onto d(CGCGAATTCGCG) only one lifetime is observed. The single lifetime has been attributed to strong binding of the Hoechst molecule onto the AATT site. The tight binding of Hoechst 33258 to AATT sites excludes water molecules from interacting with the dye, resulting in only one lifetime component. - Thermal and denaturation studies of the time-resolved fluorescence decay of human superoxide dismutase.PhD in Physics, University of Illinois at Urbana-Champaign, 1993.
Advisor: Enrico GrattonPrevious studies have shown that time-resolved fluorescence decay of various single tryptophan proteins is best described by a distribution of fluorescence lifetimes rather than one or two lifetimes. The thermal dependence of the lifetime distributions is consistent with the hypothesis that proteins fluctuate between a hierarchy of many conformational substates. With this scenario as a theoretical framework, the correlations between protein dynamic and structure are investigated by studying the time-resolved fluorescence and anisotropy decay of the single tryptophan (Trp) residue of human superoxide dismutase (HSOD) over a wide range of temperatures and at different denaturant concentrations. First, it is demonstrated that the center of the lifetime distribution can characterize the average deactivation environment of the excited Trp-protein system. A qualitative model is introduced to explain the time-resolved fluorescence decay of HSOD in 80% glycerol over a wide range of temperatures. The dynamical model features isoenergetic conformational substates separated by a hierarchy of energy barriers. The HSOD system is also investigated as a function of denaturant concentration in aqueous solution. As a function of guanidine hydrochloride (GdHCl), the width of the fluorescence lifetime distribution of HSOD displays a maximum which is not coincident with the fully denatured form of HSOD at 6.5M GdHCl. Furthermore, the width for the fully denatured form of HSOD is greater than that of the native form. This is consistent with the scenario that more conformational substates are being created upon denaturation of HSOD. HSOD is a dimeric protein and it was observed that the width of the lifetime distribution of HSOD at intermediate GdHCl concentrations increased with decreasing protein concentration. In addition, the secondary structure of HSOD at intermediate GdHCl concentration does not change with protein concentration. These results suggest that HSOD display structural microheterogeneity which is consistent with the hypothesis of conformational substates. Further analysis show that, during denaturation, the monomeric form of HSOD is an intermediate which displays native-like secondary structure and fluctuating tertiary structure; i.e., the monomeric form of HSOD is a molten globule.
1989
- Infrared-monitored flash-photolysis of carboxymyoglobin.PhD in Physics, University of Illinois at Urbana-Champaign, 1989.
Advisor: Enrico GrattonAn infrared-monitored flash-photolysis apparatus capable of measuring absorbance changes to 10^-3 OD over a time range from 10μs to 10s is described. Measurements made on this apparatus, combined with slower measurements made on a Fourier-transform infrared spectrometer, of the CO-rebinding kinetics of the A0, A1, and A3 conformations of sperm-whale myoglobin (Mb) at atmospheric pressure and neutral pH are reported. The A0, A1, and A3 rebinding kinetics are shown to be non-exponential and parameterized by activation-enthalpy distributions differing in prefactor k0/ (log(k0/s) = 10.8, 9.3, and 9.8 for the A0, A1, and A3 conformations, respectively) and peak activation-enthalpy Hp (Hp = 10.4, 9.6, and 17.6 kJ/M, respectively). CO-rebinding kinetics of MbCO ensembles prepared along different paths in the pressure-temperature plane ('freeze' and 'squeeze-freeze-release' ensembles) monitored at two frequencies within the A1 band are also reported. No differences between kinetics monitored at 1943.0cm^-1 and 1947.5cm^-1, and none between the kinetics of the freeze and squeeze-freeze-release ensembles, are resolved. The large uncertainty in the determination of the peak Hp of the activation-enthalpy distribution (Hp = 15 (+8,-7)kJ/M for the squeeze-freeze-release ensemble monitored at 1943.0cm^-1) suggests limits of the capability of the apparatus, which are discussed in terms of time resolution. - Dynamics of biological macromolecules investigated by fluorescence depolarization.PhD in Physics, University of Illinois at Urbana-Champaign, 1989.
Advisor: Enrico GrattonThe fast dynamics of biomolecular systems are believed to be important to their physiological function. Fluorescence depolarization is a powerful technique for the investigation of these dynamics. A general overview of the use of time-resolved fluorescence to investigate nanosecond dynamics is presented. The basic theory and experimental techniques for measuring time-resolved fluorescence properties in the frequency domain are outlined. Data obtained in fluorescence depolarization experiments is highly complex. Mathematical models for analyzing data from depolarization due to rotational motion have been largely based on the diffusion equation. It has been implicitly stated that a 'jump' model should give the same result for the anisotropy decay as the diffusion equation. In this work, we derive the general result from the jump model, where the excitation and emission dipoles are not necessarily coincident with any of the principal rotational axes of the fluorophore. This result is found to be different from that of the diffusion equation. This difference is significant since, for systems where the fluorophore is not much larger than the solvent molecules or where the molecule may be limited to a few preferred orientations (for example, residues in proteins), the actual physical mechanism of rotation may not be accurately represented by continuous diffusion. Since there are cases where symmetry causes the two models to agree, it is proposed that both models are only limiting cases of the underlying physical process of rotational motion. The physical assumptions behind the two models and the limits of applicability of each approach are discussed, and some of the thermodynamic properties are considered. Finally, several applications of the compartmental jump approach are presented: a free disk-like molecule, a rod-like molecule in a phospholipid bilayer, and a tryptophan residue in a protein matrix.
1987
- Fluorescence lifetime distributions in proteins.PhD in Physics, University of Illinois at Urbana-Champaign, 1987.
Advisor: Enrico GrattonState of the art frequency domain instrumentation was developed to measure the natural fluorescence decay of single tryptophan residue proteins. The instrumentation uses mode-locked, synchronously pumped, cavity dumped and frequency doubled dye laser excitation. The detectors are modulated phase coherently with the mode-locked laser pulses to accurately determine the phase delay and modulation of the fluorescence response. The instrument achieves 1 ps time resolution. The resolvability provided by the experimental data obtained with such instrumentation is determined. It is shown that the discrete exponential analysis of the fluorescence decay overestimates the resolvability of the data. It is shown that large sets of exponentially decaying components with lifetimes distributed continuously in lifetime space can be recovered, within the limits of resolvability provided by the frequency domain data, using probability density functions. It is also shown based on the dynamic nature of the macromolecules that the fluorescence decay of proteins can arise from distributions of exponentially decaying components. Lifetime distribution functions are derived based on (i) conformational interconverting models with distributions of activation energies and (ii) statistical mechanical models of the protein. The fluorescence lifetime distribution analysis of data from four single tryptophan residue proteins show that the shape of the distribution is determined by the structure of the protein. Such distributions become narrower and shifted to shorter lifetime values with increasing temperature. These results can be explained based on the dynamic origin of the fluorescence lifetime distribution in macromolecules. The inadequacy of single conformational model to account for the fluorescence lifetime distribution suggests that proteins fluctuate in a multitude of conformational sub-states.