Murata MM, Kong X, Moncada E, Chen Y, Imamura H, Wang P, Berns MW, Yokomori K, Digman MA.
NAD+ consumption by PARP1 in response to DNA damage triggers metabolic shift critical for damaged cell survival.
Mol Biol Cell. 2019; 30(20): 2584-2597. PMCID: PMC6740200DNA damage signaling is critical for the maintenance of genome integrity and cell fate decision. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor rapidly activated in a damage dose- and complexity-dependent manner playing a critical role in the initial chromatin organization and DNA repair pathway choice at damage sites. However, our understanding of a cell-wide consequence of its activation in damaged cells is still limited. Using the phasor approach to fluorescence lifetime imaging microscopy (FLIM) and fluorescence-based biosensors in combination with laser microirradiation, we found a rapid cell-wide increase of the bound NADH fraction in response to nuclear DNA damage, which is triggered by PARP-dependent NAD+ depletion. This change is linked to the metabolic balance shift to oxidative phosphorylation (oxphos) over glycolysis. Inhibition of oxphos, but not glycolysis, resulted in parthanatos due to rapid PARP-dependent ATP deprivation, indicating that oxphos becomes critical for damaged cell survival. The results reveal ... [truncated at 150 words]
Ma N, de Mochel NR, Pham PD, Yoo TY, Cho KWY, Digman MA.
Label-free assessment of pre-implantation embryo quality by the Fluorescence Lifetime Imaging Microscopy (FLIM)-phasor approach.
Sci Rep. 2019; 9(1), 13206. PMCID: PMC6744410Development of quantitative, safe and rapid techniques for assessing embryo quality provides significant advances in Assisted Reproductive Technologies (ART). Instead of assessing the embryo quality by the standard morphologic evaluation, we apply the phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) method to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological caliber for embryo quality. Here, we identify, under hypoxic and non-hypoxic conditions, the unique spectroscopic trajectories at different stages of mouse pre-implantation development, which is referred to as the developmental, or "D-trajectory", that consists of fluorescence lifetime from different stages of mouse pre-implantation embryos. The D-trajectory correlates with intrinsic fluorescent species from a distinctive energy metabolism and oxidized lipids, as seen with Third Harmonic Generation (THG) that changes over time. In addition, we have defined a non-morphological Embryo Viability Index (EVI) to distinguish pre-implantation embryo quality using the Distance Analysis (DA), a machine learning algorithm to process the fluorescence lifetime ... [truncated at 150 words]
Trivedi P, Palomba F, Niedzialkowska E, Digman MA, Gratton E, Stukenberg PT.
The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex.
Nat Cell Biol. 2019; 21(9): 1127-1137.The inner centromere is a region on every mitotic chromosome that enables specific biochemical reactions that underlie properties, such as the maintenance of cohesion, the regulation of kinetochores and the assembly of specialized chromatin, that can resist microtubule pulling forces. The chromosomal passenger complex (CPC) is abundantly localized to the inner centromeres and it is unclear whether it is involved in non-kinase activities that contribute to the generation of these unique chromatin properties. We find that the borealin subunit of the CPC drives phase separation of the CPC in vitro at concentrations that are below those found on the inner centromere. We also provide strong evidence that the CPC exists in a phase-separated state at the inner centromere. CPC phase separation is required for its inner-centromere localization and function during mitosis. We suggest that the CPC combines phase separation, kinase and histone code-reading activities to enable the formation of a ... [truncated at 150 words]
Dong Y, Sameni S, Digman MA, Brewer GJ.
Reversibility of age-related oxidized free NADH redox states in Alzheimer's disease neurons by imposed external Cys/CySS redox shifts.
Sci Rep. 2019; 9(1), 11274. PMCID: PMC6677822Redox systems including extracellular cysteine/cystine (Cys/CySS), intracellular glutathione/oxidized glutathione (GSH/GSSG) and nicotinamide adenine dinucleotide reduced/oxidized forms (NADH/NAD+) are critical for maintaining redox homeostasis. Aging as a major risk factor for Alzheimer's disease (AD) is associated with oxidative shifts, decreases in anti-oxidant protection and dysfunction of mitochondria. Here, we examined the flexibility of mitochondrial-specific free NADH in live neurons from non-transgenic (NTg) or triple transgenic AD-like mice (3xTg-AD) of different ages under an imposed extracellular Cys/CySS oxidative or reductive condition. We used phasor fluorescence lifetime imaging microscopy (FLIM) to distinguish free and bound NADH in mitochondria, nuclei and cytoplasm. Under an external oxidative stress, a lower capacity for maintaining mitochondrial free NADH levels was found in old compared to young neurons and a further decline with genetic load. Remarkably, an imposed Cys/CySS reductive state rejuvenated the mitochondrial free NADH levels of old NTg neurons by 71% and old 3xTg-AD neurons by ... [truncated at 150 words]
Levi M, Gratton E, Forster IC, Hernando N, Wagner CA, Biber J, Sorribas V, Murer H.
Mechanisms of phosphate transport.
Nat Rev Nephrol. 2019; 15(8): 482-500.Over the past 25 years, successive cloning of SLC34A1, SLC34A2 and SLC34A3, which encode the sodium-dependent inorganic phosphate (Pi) cotransport proteins 2a-2c, has facilitated the identification of molecular mechanisms that underlie the regulation of renal and intestinal Pi transport. Pi and various hormones, including parathyroid hormone and phosphatonins, such as fibroblast growth factor 23, regulate the activity of these Pi transporters through transcriptional, translational and post-translational mechanisms involving interactions with PDZ domain-containing proteins, lipid microdomains and acute trafficking of the transporters via endocytosis and exocytosis. In humans and rodents, mutations in any of the three transporters lead to dysregulation of epithelial Pi transport with effects on serum Pi levels and can cause cardiovascular and musculoskeletal damage, illustrating the importance of these transporters in the maintenance of local and systemic Pi homeostasis. Functional and structural studies have provided insights into the mechanism by which these proteins transport Pi, whereas in vivo ... [truncated at 150 words]
Gabriel M, Anzalone A, Gratton E, Estrada LC.
A tracking-based nanoimaging method for fast detection of surfaces' inhomogeneities using gold nanoparticles.
Microsc Res Tech. 2019; [Epub ahead of print].The localization of surfaces inhomogeneities is central to many areas of technology, chemistry and biology, ranging from surface defects in industry to the identification and screening of early bio-defects inside cells. The development of methods that enable direct, sensitive, and rapid detection of those inhomogeneities is both relevant and timely. To address this challenge, we developed a far-field nanoimaging method to detect the presence of surface's nanodefects that modify the signal emitted by gold nanoparticles (AuNPs) under laser irradiation. Our technique is based on the formation of hot spots due to the confinement of light in the proximity of the AuNP, whose positions depend on the polarization direction of the incident beam. An inhomogeneity is detected as an increase in the intensity collected from the hot spots when a laser beam is orbiting the nanoparticle and the incident polarization direction of the laser beam is changed periodically.
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The spectral phasor approach as a potential diagnostic tool for breast cancer.
Master in Biomedical Engineering, University of California, Irvine, 2019.
Advisor: Enrico Gratton
Breast 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 ... [truncated at 150 words]
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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 Digman
Cellular 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 ... [truncated at 150 words]
Dvornikov A, Malacrida L, Gratton E.
The DIVER microscope for imaging in scattering media.
Methods Protoc. 2019; 2(2): 53. PMCID: PMC6632175We describe an advanced DIVER (Deep Imaging Via Emission Recovery) detection system for two-photon fluorescence microscopy that allows imaging in multiple scattering media, including biological tissues, up to a depth of a few mm with micron resolution. This detection system is more sensitive to low level light signals than conventional epi-detection used in two-photon fluorescence microscopes. The DIVER detector efficiently collects scattered emission photons from a wide area of turbid samples at almost any entrance angle in a 2π spherical angle. Using an epi-detection scheme only photons coming from a relatively small area of a sample and at narrow acceptance angle can be detected. The transmission geometry of the DIVER imaging system makes it exceptionally suitable for Second and Third Harmonic Generation (SHG, THG) signal detection. It also has in-depth fluorescence lifetime imaging (FLIM) capability. Using special optical filters with sin-cos spectral response, hyperspectral analysis of images acquired in-depth in ... [truncated at 150 words]
Ranjit S, Malacrida L, Stakic M, Gratton E.
Determination of the metabolic index using the fluorescence lifetime of free and bound NADH in the phasor approach.
J Biophotonics. 2019; [Epub ahead of print].The fluorescence lifetime of nicotinamide adenine dinucleotide (NADH) is commonly used in conjunction with the phasor approach as a molecular biomarker to provide information on cellular metabolism of autofluorescence imaging of cells and tissue. However in the phasor approach, the bound and free lifetime defining the phasor metabolic trajectory is a subject of debate. NADH increases the fluorescence lifetime when bound to an enzyme, in contrast to the short multiexponential lifetime displayed by NADH in solution. The extent of fluorescence lifetime increase depends on the enzyme to which NADH is bound. With proper preparation of lactate dehydrogenase (LDH) using oxalic acid as an allosteric factor, bound NADH to LDH has a lifetime of 3.4 ns and is positioned on the universal semi-circle of the phasor plot, inferring a mono-exponential lifetime for this species. Surprisingly, measurements in the cellular environments with different metabolic states show a linear trajectory between free NADH ... [truncated at 150 words]
Polyzos AA, Lee DY, Datta R, Hauser M, Budworth H, Holt A, Mihalik S, Goldschmidt P, Frankel K, Trego K, Bennett MJ, Vockley J, Xu K, Gratton E, McMurray CT.
Metabolic reprogramming in astrocytes distinguishes region-specific neuronal susceptibility in Huntington mice.
Cell Metab. 2019; 29(6): 1258-1273. PMCID: PMC6583797The basis for region-specific neuronal toxicity in Huntington disease is unknown. Here, we show that region-specific neuronal vulnerability is a substrate-driven response in astrocytes. Glucose is low in HdhQ(150/150) animals, and astrocytes in each brain region adapt by metabolically reprogramming their mitochondria to use endogenous, non-glycolytic metabolites as an alternative fuel. Each region is characterized by distinct metabolic pools, and astrocytes adapt accordingly. The vulnerable striatum is enriched in fatty acids, and mitochondria reprogram by oxidizing them as an energy source but at the cost of escalating reactive oxygen species (ROS)-induced damage. The cerebellum is replete with amino acids, which are precursors for glucose regeneration through the pentose phosphate shunt or gluconeogenesis pathways. ROS is not elevated, and this region sustains little damage. While mhtt expression imposes disease stress throughout the brain, sensitivity or resistance arises from an adaptive stress response, which is inherently region specific. Metabolic reprogramming may have ... [truncated at 150 words]
Rodriguez-Agudo D, Malacrida L, Kakiyama G, Sparrer T, Fortes C, Maceyka M, Sublerh MA, Windle JJ, Gratton E, Pandak WM, Gil G.
StarD5: an ER stress protein regulates plasma membrane and intracellular cholesterol homeostasis.
J Lipid Res. 2019; 60(6): 1087-1098. PMCID: PMC6547630How plasma membrane (PM) cholesterol is controlled is poorly understood. Ablation of the gene encoding the ER stress steroidogenic acute regulatory-related lipid transfer domain 5 (StarD5) leads to a decrease in PM cholesterol content, a decrease in cholesterol efflux, and an increase in intracellular neutral lipid accumulation in macrophages, the major cell type that expresses StarD5. ER stress increases StarD5 expression in mouse hepatocytes, which results in an increase in accessible PM cholesterol in wild type but not in StarD5-/- hepatocytes. StarD5-/- mice store higher levels of cholesterol and triglycerides and leads to altered expression of cholesterol-regulated genes. In vitro, a recombinant GST-StarD5 protein transfers cholesterol between synthetic liposomes. StarD5 overexpression leads to a marked increase in PM cholesterol. Phasor analysis of LAURDAN FLIM data revealed an increase in PM fluidity in StarD5-/- macrophages. Taken together, these studies show that StarD5 is a stress responsive protein that regulates PM cholesterol ... [truncated at 150 words]
Chen H, Ma N, Kagawa K, Kawahito S, Digman M, Gratton E.
Widefield multifrequency fluorescence lifetime imaging using a two-tap complementary metal-oxide semiconductor camera with lateral electric field charge modulators.
J Biophotonics. 2019; 12(5), e201800223.Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) measures the fluorescence lifetime of entire images in a fast and efficient manner. We report a widefield FD-FLIM system based on a complementary metal-oxide semiconductor camera equipped with two-tap true correlated double sampling lock-in pixels and lateral electric field charge modulators. Owing to the fast intrinsic response and modulation of the camera, our system allows parallel multifrequency FLIM in one measurement via fast Fourier transform. We demonstrate that at a fundamental frequency of 20 MHz, 31-harmonics can be measured with 64 phase images per laser repetition period. As a proof of principle, we analyzed cells transfected with Cerulean and with a construct of Cerulean-Venus that shows Förster Resonance Energy Transfer at different modulation frequencies. We also tracked the temperature change of living cells via the fluorescence lifetime of Rhodamine B at different frequencies. These results indicate that our widefield multifrequency FD-FLIM system is a ... [truncated at 150 words]
Planes N, Digman MA, Vanderheyden PPML, Gratton E, Caballero-George C.
Number and brightness analysis to study spatio-temporal distribution of the angiotensin II AT1 and the endothelin-1 ETA receptors: Influence of ligand binding.
Biochim Biophys Acta Gen Subj. 2019; 1863(5): 917-924.The angiotensin II AT1 and the endothelin 1 ETA receptors play a crucial role in the pathogenesis of cardiovascular diseases like hypertension, heart failure, stroke, pulmonary hypertension, and cardiac hypertrophy. Both receptors are members of the rhodopsion-like superfamily of G protein-coupled receptors which can exist as monomers, dimers, and higher order aggregates. Recently, oligomerization of these two receptors have been described by several biophysical methods based mainly on luminescence and fluorescence energy transfer. Since this oligomerization can occur either spontaneously or it can be induced by ligand-binding, the aim of this work was to address whether the oligomerization of these receptors occurs upon ligand-binding. For this purpose the Number and Brightness analysis, a method that allows the identification, localization, and quantification of protein aggregates in the plasma membrane of a single cell, was used. An advantage of this method is that it is not limited to certain dyes specially required ... [truncated at 150 words]
Lou J, Scipioni L, Wright BK, Bartolec TK, Zhang J, Masamsetti VP, Gaus K, Gratton E, Cesare AJ, Hinde E.
Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response.
Proc Natl Acad Sci USA. 2019; 116(15): 7323-7332. PMCID: PMC6462080To investigate how chromatin architecture is spatiotemporally organized at a double-strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image-correlation spectroscopy of histone fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) microscopy data acquired in live cells coexpressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that, when coupled with laser microirradiation-induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ataxia-telangiectasia mutated (ATM) and RNF8 regulate rapid chromatin decompaction at DSBs and formation of compact chromatin foci surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.
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Visualizing the regulation of SLC34 proteins at the apical membrane.
Pflugers Arch. 2019; 471(4): 533-542. PMCID: PMC6436987The cloning of the renal NaPi-2a (SLC34A1) and NaPi-2c (SLC34A3) phosphate transporters has made it possible to characterize the molecular and biophysical regulation of renal proximal tubular reabsorption of inorganic phosphate (Pi). Dietary factors, such as Pi and K, and several hormones and phosphatonins, including parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and glucocorticoids, regulate the transporters through various transcriptional, translational, and post-translational mechanisms that involve acute trafficking via endocytosis or exocytosis, interactions with PDZ domain proteins, lipid microdomains, and diffusion and clustering in the apical brush border membrane. The visualization of these trafficking events by means of novel microscopy techniques that includes fluorescence lifetime imaging microscopy (FLIM), Förster resonance energy transfer (FRET), fluctuation correlation spectroscopy (FCS), and modulation tracking (MT), is the primary focus of this review.
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3D orbital tracking under STED microscopy.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 440a, 2174-Pos.Orbital tracking is a well-established particle tracking technique in which the scanner of a conventional fluorescence scanning microscope is controlled to scan orbits around an object instead of forming a raster image. By recording the fluorescence along the orbits, a movement of the object inside the orbit is detected as an increase in the intensity measured at a particular point in the orbit which is in turn used to re-center the subsequent orbits in the appropriate position. Movements along the z direction can be detected by performing two orbits, one above and one below, using a piezo objective. This allows following the object in time and obtaining a 3D trajectory from which we can extract information about the diffusion and the structure of the biological system in which the object is moving. Moreover, the fluorescence intensity signal acquired at each orbit during a tracking experiment provides a space-time image of ... [truncated at 150 words]
Gratton E, Rossetta A, Chen H.
A new faster Flimbox with higher harmonic content and multi detector input.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 441a, 2179-Pos.The FLIMbox is an FPGA chip programed to perform frequency-domain FLIM acquisition. The original FLIMbox setup (Colyer et al.) was designed around Xilinx Spartan3 FPGA chip. The original design had a maximum internal frequency of operation of 320 MHz, which in our design, limits the spectral bandwidth to the first harmonic of a laser source at 80 MHz, typically used for FLIM images in 2-photon setups. The new Xilinx Kintex-7 FPGA KC705 Evaluation Kit has much higher internal operational frequencies allowing the acquisition of harmonic content in excess of 1GHz. Also this new FPGA chip can be used with a USB3 interface with a large increase in transmission speed. More importantly, the Kintex 7 board can operate on many inputs simultaneously allowing data acquisition of photomultiplier arrays (8x8). In this work we show the larger harmonic content obtained with this chip as well as the capability for multi detector acquisition. ... [truncated at 150 words]
Tedeschi G, Scipioni L, Trinh A, Lee KA, Malacrida LS, Digman MA, Gratton E.
Characterization of the metabolic state and molecular crowding in breast cancer spheroids.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 421a, 2080-Pos.Spheroids are 3D cultures of cells specifically grown in order to create a single spherical cell mass, which can be used as a model for studying the behavior of tumors in a controlled environment. Despite being a simplistic model, it allows for the rapid creation of the samples and displays a more complex physiology when compared to 2D cultures [1].
The spheroids grown for this study are derived from two different breast adenocarcinoma cell lines, MCF7 and MDA-MB231, which display less and more invasive behavior, respectively. From 20,000 cells, our protocol provides spheres with a diameter of approximately 400 μm. The spheres were then transferred to a collagen Type-1 gel matrix in order to mimic the extracellular matrix.
Once obtained and transferred in the collagen, the spheres were observed at different time points over 24 hours. We measured their single cell metabolic signature by analyzing the NADH lifetime with the Phasor-FLIM approach ... [truncated at 150 words]
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Adaptive optics in strongly scattering samples.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 441a, 2181-Pos.One of the challenges in deep tissue microscopy is the sharp decline in image quality as depth is increased. To improve image quality, we are developing an adaptive optical system in our multiphoton microscope, the DIVER (Deep Imaging Via Enhanced photon Recovery). The transmission geometry used in the DIVER allows us to ramp up laser power as needed to counteract the effects of scattering and absorption without blinding the detector with out of focus surface fluorescence, which is a constraint in epi-detection schemes. Although the photons contributing to the multiphoton excitation are mostly spatially and temporally coherent, phase delays introduced by the spatial variations in refractive index throughout the tissue distort the focal volume. We use a segmented hexagonal deformable mirror from Boston Micromachines Corporation with 111 actuators to cancel out the low spatial frequency aberrations introduced by the tissue as well as those inherent in the imaging system. To ... [truncated at 150 words]
Malacrida LS, Scipioni L, Gratton E.
In vivo chromatin compaction changes as detected by water dipolar relaxations: the molecular crowding role revealed by the ACDAN fluorescence.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 70a, 355-Pos.Chromatin occupies territories in the nucleus in regards to its different status, hetero and euchromatin. Chromatin dynamics responds to chemical and physical inputs, producing changes in the molecular crowding and thermodynamic parameters. Using ACDAN photophysics we demonstrate the correlation between the water dipolar relaxation and the chromatin status(Malacrida-2017). ACDAN was synthesized to sense polarity and dipolar relaxation in proteins(Weber-1979). Inside the cell one of the main dipolar relaxation active molecules is water. Using spectral phasors we analyzed the ACDAN spectral shift due to modification of chromatin compaction using chemical and physical stimuli. First, we produce double-strand DNA break (DSB) by irradiating a small ROI (780nm ∼25mW, 10x10pixels, ∼150nm/pixel) inside the nucleus. Using Trichostatin-A (TSA, a histone deacetylase inhibitor) we produced chromatin decompaction by the unfolding of the heterochromatin driven by the inhibition of removal of acetyl group from histones. To induce chromatin compaction we used Okadaic acid (OA, a protein ... [truncated at 150 words]
Hedde PN, Staaf E, Singh SB, Johansson S, Gratton E.
Dynamics of activating and inhibitory receptors in Murine Natural Killer cells revealed by 2D pair correlation function analysis.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 280a, 1380-Pos.Natural Killer (NK) cells are innate immune cells that mediate anti-viral immunity and protect against cancer. Their killing threshold is determined by a balance between inhibitory and activating membrane receptors. By expressing self MHC class I specific inhibitory receptors they become educated, i.e., they learn to recognize self from non-self. Educated NK cells are more efficient in killing aberrant cells. However, the mechanism for this functional difference between educated and uneducated NK cells is still unclear. In a previous study, we found that murine educated NK cells exhibited faster movement of the activating receptor NKp46 and confinement of the inhibitory receptor Ly49A by means of image mean square displacement analysis. Yet, these values represent a spatial average over a region of interest at the cell membrane. Here, we utilized the 2D pair correlation function approach in order to study the spatial distribution of obstacles to NK cell receptors at the ... [truncated at 150 words]
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Hyperspectral imaging in scattering media using two filters.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 441a, 2178-Pos.Hyperspectral imaging is a common technique in fluorescence microscopy to obtain the emission spectrum at each pixel of an image. However, methods to obtain spectral resolution based on diffraction gratings or integrated prisms work poorly when the sample is strongly scattering. We developed a microscope named the DIVER that collects the fluorescence emission over a very large angle and sample area. We have shown that this microscope, depending on the tissue thickness, can capture several orders of magnitude more light than conventional microscopes using epi detection, which in turn significantly increases imaging depth. Since the fluorescence light after passing through the multiple scattering sample is not collimated, the use of grating or prisms strongly limits the amount of light that can be used with available hyperspectral devices. Here we show that two filters that accept uncollimated light over a large aperture are sufficient to calculate the spectral phasor rather than ... [truncated at 150 words]
Begarani F, D'Autilia F, Signore G, Gratton E, Beltram F, Cardarelli F.
Capturing metabolism-dependent solvent polarity fluctuations in a trafficking Lysosome.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 307a, 1516-Plat.The eukaryotic cell compartmentalization acts as a master regulator of metabolism by arranging specific sets of molecular components in spatially confined, membrane-enclosed, intracellular structures (e.g. organelles, endosomes, vesicles). Here, peculiar physicochemical properties of the local environment do emerge and participate to the regulation of molecular processes. In spite of the huge amount of available environmental probes, experiments on sub-cellular structures are severely challenged by their restless 3D-movement. Classical optical microscopy tools fail to subtract the 3D evolution of the system while preserving the temporal resolution required to probe the physicochemical properties of the local environment. This bottleneck is tackled by focusing an excitation light-beam in a periodic orbit around the structure of interest. The recorded signal is used as feedback to localize the structure position with unprecedented temporal resolution: microseconds along the orbit, milliseconds between orbits. The lysosome is proposed as paradigmatic intracellular target. 6-acetyl-2-dimethylaminonaphthalene (ACDAN) is used as a ... [truncated at 150 words]
Scipioni L, Lanzanò L, Diaspro A, Gratton E.
Airyscan CCA provides structural and dynamics fingerprinting of subcellular compartments in living cells.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 282a, 1389-Pos.In this work we will present our current advances in the development of the CCA (Comprehensive Correlation Analysis) technique using the Zeiss Airyscan detector. This detector consists of 32 GaAsP PMT arranged in a hexagonal pattern, featuring a fast temporal sampling (down to 1.28 μs per frame). It can also be used in a super-resolution configuration as per the ISM [1] principle, while the arrangement of the detectors allows for the implementation of advanced state-of-the-art spatiotemporal correlation techniques. We will present a novel application of the spot-variation FCS technique [2] in super-resolution, as well as the implementation of the 2D-pCF [3], iMSD [4] and number and brightness [5] analysis using this fast detector array. This simultaneous analysis of the same dataset provides biophysical information regarding the fluorescent probe and the surrounding environment, such as diffusion coefficient, concentration, diffusion modality, environment organization, direction and anisotropy of molecular flows, diffusion connectivity and ... [truncated at 150 words]
Ranjit S, Malacrida LS, Gratton E.
The fluorescence lifetime of bound NADH: clues from the phasor plots.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 565a, 2804-Pos.Changes in metabolism in cells are often studied by the ratio of NAD+ to NADH. This ratio is correlated to free and enzyme bound NADH, which can be used as a measurement of metabolic states in cells using fluorescence lifetime imaging (FLIM). Binding of NADH to the enzyme causes separation between nicotinamide and adenine moieties of Nicotinamide adenine dinucleotide (NADH) from their collapsed structure in solution and results in increase of fluorescence quantum yield and lifetime. The extent of increase in fluorescence lifetime is dependent on the apoenzyme and presence of auxiliary ions. Reports from past studies show distinctive discrepancies in calculation of the bound NADH lifetime, often related to complications in sample preparation and limitations in data acquisition. In this work, we show that in presence of oxalic ion, proper preparation of lactate dehydrogenase (LDH) bound NADH has a lifetime of 3.4 ns and is positioned on the universal ... [truncated at 150 words]
Chen H, Ma N, Kagawa K, Kawahito S, Digman MA, Gratton E.
Widefield multi-frequency fluorescence lifetime imaging using a two-tap CMOS camera with lateral electric field charge modulators.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 567a, 2812-Pos.Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) measures fluorescence lifetime of entire images in a fast and efficient manner. We report a widefield FD-FLIM system based on a CMOS camera equipped with two-tap true correlated double sampling (CDS) lock-in pixels and lateral electric field charge modulators. Due to the fast intrinsic response and modulation of the camera, our system allows parallel multi-frequency FLIM in one measurement via Fast Fourier Transform. We demonstrate that at a fundamental frequency of 20MHz, 31-harmonics (620MHz) can be measured with 64 phase images per laser repetition period. As a proof of principle, we analyzed cells transfected with Cerulean and with a construct of Cerulean-Venus that shows Förster resonance energy transfer at different modulation frequencies. We also tracked the temperature change of living cells via the fluorescence lifetime of Rhodamine B at different frequencies. Furthermore, we can rapidly distinguish the pre-implantation embryo development stages at the ... [truncated at 150 words]
Cinco R, Hedde PN, Malacrida L, Digman MA, Gratton E.
Multi-modal fluorescence characterization of cell cycle progression and cytokinesis.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 24a, 125-Plat.Cell division is central to both the creation of life and the propagation of diseases such as cancer and neurodegenerative disorders. While many of the genes and enzymes responsible for regulating cell cycle progression have been extensively characterized, much of the cellular bioenergetics responsible for fueling cell cycle progression and biophysics of cell division is poorly defined. Here, we characterize cell cycle progression in human HeLa cells using Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) with a variety of advanced fluorescence optical approaches. Application of NADH phasor Fluorescence Lifetime Imaging Microscopy (FLIM) identified significant changes both in NADH concentration and free/bound NADH between several stages of the cell cycle defined by FUCCI. We also present detailed timelapse images of human HeLa cells undergoing cytokinesis using NADH Phasor FLIM, Laurdan Phasor FLIM, and timelapse 740nm 2-Photon spectral phasor to spatially characterize and quantify metabolism, membrane fluidity, and unlabeled fluorescence emission during the ... [truncated at 150 words]
Digman MA, Lefebvre AEYT, Adame FA, Fong E.
The phasor FLIM analysis monitors metabolic changes at the leading edge in response to Rac photo-activation and mitochondrial transport in MDA MB231 cells.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 442a, 2185-Pos.Subcellular enrichments can promote metabolic shifts in cancer cells that may in turn increase invasiveness. There is a direct relationship between mitochondrial transport at the leading edge and increased invasion within various cancer types including prostate, breast, lung and glia. This recruitment is increased when PI3K inhibitors are added as cancer treatment but instead of reducing cancer invasion, it stimulates the reactivation of Akt2 or mTOR that causes the contradictory increase in tumor cell invasion. Given that mTOR controls actin cytoskeleton dynamics through Rac GTPase activation, we reasoned that activating Rac may increase mitochondria recruitment. We investigated mitochondrial recruitment by photo-activating the Rac GTPAse photoactivatable protein in living cells and monitored local energy metabolism at the leading edges of the cell membrane using the phasor approach to Fluorescence Lifetime imaging Microscopy (FLIM). We also hypothesis that matrix density may regulate intracellular tension at cell's leading edge. This tension may trigger ... [truncated at 150 words]
Lefebvre AEYT, Adame FA, Digman MA.
A non-invasive metabolic investigation of breast cancer invasion.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 550a, 2727-Pos.Over 90% of cancer related deaths are the result of complications arising due to cancer metastasis. The reconfiguration of an invasive cancer cell's metabolic signature due to its mechanosensing ability is well acknowledged yet remains poorly understood. By probing the metabolic response of cells of differing invasivity using non-invasive imaging techniques, novel insights into the mechanisms of metastatic disease may be elicited.In this work, we used fluorescence lifetime imaging microscopy (FLIM) together with phasor analysis, to determine the free to bound NADH ratio present within the different cells. This free to bound NADH ratio analysis can determine whether a cell is relying relatively more on glycolytic metabolism or on oxidative phosphorylation. Additionally, we used spectral imaging with phasor analysis on LAURDAN stained cells, allowing us to determine the cell's membrane fluidity under varying conditions. We probed the metabolic and fluidity profiles of three breast cells of varying invasivity. These cells ... [truncated at 150 words]
Ou CY, Vu T, Grunwald JT, Toledano M, Zimak J, Toosky M, Shen B, Zell JA, Gratton E, Abram TJ, Zhao W.
An ultrasensitive test for profiling circulating tumor DNA using integrated comprehensive droplet digital detection.
Lab Chip. 2019; 19(6): 993-1005. PMCID: PMC6559803Current cancer detection systems lack the required sensitivity to reliably detect minimal residual disease (MRD) and recurrence at the earliest stages when treatment would be most effective. To address this issue, we present a novel liquid biopsy approach that utilizes an integrated comprehensive droplet digital detection (IC3D) digital PCR system which combines microfluidic droplet partitioning, fluorescent multiplex PCR chemistry, and our rapid 3D, large-volume droplet counting technology. The IC3D ddPCR assay can detect cancer-specific, ultra-rare genomic targets due to large sample input and high degree of partitioning. We first demonstrate our droplet digital PCR assay can robustly detect common cancer mutants including KRAS G12D spiked in wild-type genomic background or isolated from patient samples with 100% specificity. We then demonstrate that the IC3D ddPCR system can detect oncogenic KRAS G12D mutant alleles against a background of wild-type genomes at a sensitivity of 0.00125-0.005% with a false positive rate of 0% ... [truncated at 150 words]
Stringari C, Gratton E, Digman MA, Donovan P.
Phasor method to fluorescence lifetime microscopy to discriminate metabolic state of cells in living tissue.
Patent US10222335B2, 05 Mar 2019.A label-free imaging method to monitor stem cell metabolism discriminates different states of stem cell as they differentiate in a living tissues. We use intrinsic fluorescence biomarkers and the phasor approach to Fluorescence Lifetime Imaging Microscopy (FLIM). We identify and map intrinsic fluorophores such as collagen, retinol, retinoic acid, flavins, nicotinamide adenine dinucleotide (NADH) and porphyrin. We measure the phasor values of germ cells in C. Elegans germ line. Their metabolic fingerprint cluster according to their differentiation state, reflecting changes in FAD concentration and NADH binding during the differentiation pathway. The phasor approach to lifetime imaging provides a label-free, fit-free and sensitive method to identify different metabolic state of cells during differentiation, to sense small changes in the redox state of cells and may identify symmetric and asymmetric divisions and predict cell fate.
