Yao Z, Brennan CK, Scipioni L, Chen H, Ng KK, Tedeschi G, Parag-Sharma K, Amelio AL, Gratton E, Digman MA, Prescher JA.
Multiplexed bioluminescence microscopy via phasor analysis.
Nat Methods. 2022; Online ahead of print.Bioluminescence imaging with luciferase-luciferin pairs is a well-established technique for visualizing biological processes across tissues and whole organisms. Applications at the microscale, by contrast, have been hindered by a lack of detection platforms and easily resolved probes. We addressed this limitation by combining bioluminescence with phasor analysis, a method commonly used to distinguish spectrally similar fluorophores. We built a camera-based microscope equipped with special optical filters to directly assign phasor locations to unique luciferase-luciferin pairs. Six bioluminescent reporters were easily resolved in live cells, and the readouts were quantitative and instantaneous. Multiplexed imaging was also performed over extended time periods. Bioluminescent phasor further provided direct measures of resonance energy transfer in single cells, setting the stage for dynamic measures of cellular and molecular features. The merger of bioluminescence with phasor analysis fills a long-standing void in imaging capabilities, and will bolster future efforts to visualize biological events in real time ... [truncated at 150 words]
Tentori P, Signore G, Camposeo A, Carretta A, Ferri G, Pingue P, Luin S, Pozzi D, Gratton E, Beltram F, Caracciolo G, Cardarelli F.
Fluorescence lifetime microscopy unveils the supramolecular organization of liposomal Doxorubicin.
Nanoscale. 2022; Online ahead of print.The supramolecular organization of Doxorubicin (DOX) within the standard Doxoves® liposomal formulation (DOX®) is investigated using visible light and phasor approach to fluorescence lifetime imaging (phasor-FLIM). First, the phasor-FLIM signature of DOX® is resolved into the contribution of three co-existing fluorescent species, each with its characteristic mono-exponential lifetime, namely: crystallized DOX (DOXc, 0.2 ns), free DOX (DOXf, 1.0 ns), and DOX bound to the liposomal membrane (DOXb, 4.5 ns). Then, the exact molar fractions of the three species are determined by combining phasor-FLIM with quantitative absorption/fluorescence spectroscopy on DOXc, DOXf, and DOXb pure standards. The final picture on DOX® comprises most of the drug in the crystallized form (∼98%), with the remaining fractions divided between free (∼1.4%) and membrane-bound drug (∼0.7%). Finally, phasor-FLIM in the presence of a DOX dynamic quencher allows us to suggest that DOXf is both encapsulated and non-encapsulated, and that DOXb is present on both liposome-membrane ... [truncated at 150 words]
Solano A, Lou J, Scipioni L, Gratton E, Hinde E.
Radial pair correlation of molecular brightness fluctuations maps protein diffusion as a function of oligomeric state within live cell nuclear architecture.
Biophys J. 2022; 121(11): 2152-2167.Nuclear proteins can modulate their DNA binding activity and the exploration volume available during DNA target search by self-associating into higher order oligomers. Directly tracking this process in the nucleoplasm of a living cell is, however, a complex task. Thus, here we present a microscopy method based on radial pair correlation of molecular brightness fluctuations (radial pCOMB) that can extract the mobility of a fluorescently tagged nuclear protein as a function of its oligomeric state and spatiotemporally map the anisotropy of this parameter with respect to nuclear architecture. By simply performing a rapid frame scan acquisition, radial pCOMB has the capacity to detect within each pixel, protein oligomer formation and the size dependent obstruction nuclear architecture imparts on this complex's transport across sub-micron distances. From application of radial pCOMB to an oligomeric transcription factor and DNA repair protein, we demonstrate that homo-oligomer formation differentially regulates chromatin accessibility and interaction with ... [truncated at 150 words]
Fazel M, Jazani S, Scipioni L, Vallmitjana A, Gratton E, Digman MA, Pressé S.
High resolution fluorescence lifetime maps from minimal photon counts.
ACS Photonics. 2022; 2(3): 1015-1025.Fluorescence lifetime imaging microscopy (FLIM) may reveal subcellular spatial lifetime maps of key molecular species. Yet, such a quantitative picture of life necessarily demands high photon budgets at every pixel under the current analysis paradigm, thereby increasing acquisition time and photodamage to the sample. Motivated by recent developments in computational statistics, we provide a direct means to update our knowledge of the lifetime maps of species of different lifetimes from direct photon arrivals, while accounting for experimental features such as arbitrary forms of the instrument response function (IRF) and exploiting information from empty laser pulses not resulting in photon detection. Our ability to construct lifetime maps holds for arbitrary lifetimes, from short lifetimes (comparable to the IRF) to lifetimes exceeding interpulse times. As our method is highly data efficient, for the same amount of data normally used to determine lifetimes and photon ratios, working within the Bayesian paradigm, we report ... [truncated at 150 words]
Vorontsova I, Vallmitjana A, Torrado B, Schilling TF, Hall JE, Gratton E, Malacrida L.
In vivo macromolecular crowding is differentially modulated by aquaporin 0 in zebrafish lens: Insights from a nanoenvironment sensor and spectral imaging.
Sci Adv. 2022; 8(7). PMCID: PMC8849302Macromolecular crowding is crucial for cellular homeostasis. In vivo studies of macromolecular crowding and water dynamics are needed to understand their roles in cellular physiology and fate determination. Macromolecular crowding in the lens is essential for normal optics, and an understanding of its regulation will help prevent cataract and presbyopia. Here, we combine the use of the nanoenvironmental sensor [6-acetyl-2-dimethylaminonaphthalene (ACDAN)] to visualize lens macromolecular crowding with in vivo studies of aquaporin 0 zebrafish mutants that disrupt its regulation. Spectral phasor analysis of ACDAN fluorescence reveals water dipolar relaxation and demonstrates that mutations in two zebrafish aquaporin 0s, Aqp0a and Aqp0b, alter water state and macromolecular crowding in living lenses. Our results provide in vivo evidence that Aqp0a promotes fluid influx in the deeper lens cortex, whereas Aqp0b facilitates fluid efflux. This evidence reveals previously unidentified spatial regulation of macromolecular crowding and spatially distinct roles for Aqp0 in the lens.
Torrado B, Malacrida L, Ranjit S.
Linear combination properties of the phasor space in fluorescence imaging.
Sensors. 2022; 22(3): 999. PMCID: PMC8840623The phasor approach to fluorescence lifetime imaging, and more recently hyperspectral fluorescence imaging, has increased the use of these techniques, and improved the ease and intuitiveness of the data analysis. The fit-free nature of the phasor plots increases the speed of the analysis and reduces the dimensionality, optimization of data handling and storage. The reciprocity principle between the real and imaginary space—where the phasor and the pixel that the phasor originated from are linked and can be converted from one another—has helped the expansion of this method. The phasor coordinates calculated from a pixel, where multiple fluorescent species are present, depends on the phasor positions of those components. The relative positions are governed by the linear combination properties of the phasor space. According to this principle, the phasor position of a pixel with multiple components lies inside the polygon whose vertices are occupied by the phasor positions of these individual ... [truncated at 150 words]
Solano AN, Lou J, Scipioni L, Gratton E, Hinde E.
Brightness correlation spectroscopy tracks transcription factor diffusion as a function of oligomeric state within live cell nuclear architecture.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 302a, 1460-Plat.In the crowded environment of the cell nucleus, transcription factors form homo and hetero-oligomers that modulate the exploration volume available during DNA target search, as well as DNA binding affinity. To directly track this process in a living cell here we present brightness correlation spectroscopy as a method to extract oligomeric transcription factor dynamics within live-cell microscopy data. From correlation of brightness fluctuations that originate from a fluorescently tagged transcription factor this approach has the capacity to extract protein mobility as a function of oligomeric state, and spatiotemporally map the anisotropy of this parameter with respect to nuclear architecture within a rapid single-channel frame scan acquisition. Expanding this method, which can be applied to any oligomeric protein, to a dual-channel frame scan acquisition enables measurement of the spatiotemporal dynamics that underly hetero-oligomeric transcription factor complex formation. Application of one and two channel brightness correlation spectroscopy to the signal transducer and ... [truncated at 150 words]
Fazel M, Jazani S, LorenzoScipioni, Vallmitjana A, Gratton E, Digman MA, Pressé S.
High resolution fluorescence lifetime maps from minimal photon counts.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 141a, 688-Pos.Fluorescence lifetime imaging microscopy (FLIM) can be used to probe concentration profiles of key marco-molecules within sub-cellular environments. However, current analyses for FLIM require a large number of photons per pixel to provide such a quantitative picture of life. In order to acquire such large number of photons, we must either increase data acquisition time, which limits temporal resolution, or increase laser intensity, which causes greater photo damage to the sample, or both. Here, we propose to analyze FLIM data by leveraging tools from the Bayesian paradigm. Our framework also takes into account details of the experiment such as point spread function (PSF) and instrument response function (IRF) of arbitrary shapes. We show that our method is robust with respect to species lifetimes from below the IRF to exceeding inter-pulse times. Moreover, we achieve direct blind unmixing of lifetimes with sub-nanosecond resolution and sub-pixel spatial resolution using limited photon budgets. ... [truncated at 150 words]
Torrado B, Dvornikov A, Gratton E.
Using multi-channel detector for simultaneous hyperspectral and flim imaging in strongly scattering media.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 277a, 1342-Pos.We describe a method that allows imaging in scattering media, such as biological tissues, and perform multiplexed hyperspectral and FLIM analysis of these images. This method is based on a set of transmission filters placed in front of wide area 4-channel detector in the emission path of a fluorescence microscope. The special transmission filters with sin-cos or linear spectral profiles were used to resolve the emission wavelength in every pixel of an image. The set of filters are used to obtain the spectral phasor of the transmitted light, which is enough to perform spectral deconvolution over a broad wavelength range. With this method we can distinguish between free and protein-bound NADH, so it can be used in metabolic studies. In addition, a filter with transmission in the range of 400-500nm was used for simultaneous FLIM analysis of the NADH autofluorescence. The method can be applied to any type of imaging ... [truncated at 150 words]
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Using phasors.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 411a, 1989-Pos.We have designed and built a fluorescence microscope that can simultaneously acquire and process spectral emission and fluorescence lifetime of a fluorophore in real time (Phasor S-FLIM). We also developed an analysis pipeline capable of unmixing up to 7 different organelle-directed dyes over time by using only the spectral information. In particular, we are able to visualize dyes that report on lysosomes, lipid droplets, tubulin, chromatin, mitochondria and Golgi apparatus. This kind of data can reveal not only the position of cellular structures inside the cells but also their dynamics and interplay in living cells, providing invaluable data for the that can be used to model cell functions at a level of detail that is missing today. Furthermore, part of what is learned from the data obtained at the cellular level can be scaled up to clinical samples, where the effect of drugs can be followed at the tissue level. ... [truncated at 150 words]
Hinde E, Lou J, Liang Z, Solano A, Scipioni L, Gratton E.
The phasor approach to histone FLIM-FRET microscopy maps nuclear wide heterochromatin structure at the level of nucleosome proximity in a living cell.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 412a, 1997-Pos.Inside the nucleus of an intact cell, DNA is folded around histone proteins into nucleosomes and compacted into a multi-layered three-dimensional chromatin network. The nanometre spacing between nucleosomes positioned throughout this structural framework is known to locally modulate local DNA template access and regulate genome function. However, given that this structural feature occurs on a spatial scale well below the diffraction limit, real time observation of nucleosome proximity in live cells by optical microscopy has proven technically difficult, despite recent advances in live cell super resolution imaging. A promising alternative solution is to measure and spatially map Förster resonance energy transfer (FRET) between fluorescently labelled histones - the core protein of a nucleosome. In recent work we established that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) is a robust method for the detection of histone FRET which can quantify nuclear wide chromatin compaction at the level of nucleosome ... [truncated at 150 words]
Vallmitjana A, Vu TM, Gu J, La K, Flores J, Hedde PN, Zhao W, Gratton E.
Spectral and lifetime multiplexing with combinatorial labeling.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 139a, 681-Pos.It is currently of critical importance to enable technologies which permit the spatial analysis of multi-omics biomarkers, e.g. mRNA and proteins, by revealing their presence, counts, locations, dynamics, and interactions within each tissue and organ. Unfortunately, there is the lack of tools that can, in a single round of labelling and imaging, profile these biomarkers beyond 9 targets and scale from the molecular level to the tissue or organ level in large heterogeneous samples. Here, we introduce Multi-Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICATM), a new spatialomics technology that enables rapid, high-throughput, and highly multiplexed profiling of mRNA and protein targets while remaining cost-effective and easy to implement. MOSAICATM utilizes in situ labeling using combinatorial labeling of fluorescent species combined with spectral-lifetime microscopy and phasor-based image segmentation. The technology is highly scalable due to the orthogonality of the spectral and lifetime dimensions and the combinatorial approach to labelling. We ... [truncated at 150 words]
Scipioni L, Rossetta A, Tedeschi G, Anderson AJ, Gratton E.
Phasor S-FLIM for the physiological profiling of living cells.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 411a, 1990-Pos.Fluorescence Lifetime Imaging Microscopy (FLIM) and spectral imaging are two broadly applied methods for increasing dimensionality in microscopy. However, their combination is typically inefficient and slow in terms of both acquisition and processing. By integrating technological and computational advances, we developed a robust and unbiased Spectral FLIM (S-FLIM) system capable of extracting detailed and precise information about the photophysics of fluorescent specimens at optical resolution. The data are processed with the phasor approach to provide unbiased and fast analysis of S-FLIM data. We therefore named this system Phasor S-FLIM. Here, we exploit both spectral emission and fluorescence lifetime of environment-sensitive fluorescent dyes to report on multiple parameters related to cellular physiology. Environment-sensitive dyes are fluorophores that change their spectral emission and/or fluorescence lifetime according to changes in the nano-environment in which they localize. As an example, we can measure important physiological parameters such as mitochondrial membrane potential, lipid droplets composition ... [truncated at 150 words]
Jones B, Torrado B, Rosenberg A, Gratton E, Levi M, Ranjit S.
Determination of cardiac fibrosis using picrosirius red fluorescence.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 279a, 1349-Pos.Automatic measurement and quantification of collagen accumulation and fibrosis in the heart using multiphoton excitation methods is more difficult compared to other organs, as myosin and collagen both contribute to the second harmonic generation (SHG) signal, inhibiting any specificity of collagen only signals. Therefore, the most common fibrosis quantification using SHG often fails to quantify cardiac fibrosis. Picrosirius red (PSR) is a common dye that is used for the quantification of fibrosis using polarized light. In this work, we have used PSR stained rat heart samples, from the DOCA-salt (deoxycorticosterone acetate) model of chronic hypertension model, to identify and quantify the extent of fibrosis. The samples were excited with a 1040 nm excitation using a two photon excitation scheme and the red fluorescence emission was collected using a 575-645 nm filter. Phasor approach to fluorescence lifetime imaging (Phasor-FLIM) and spectra (spectral phasor) were used to identify the origin of this ... [truncated at 150 words]
Palomba F, Scipioni L, Truong T, Stukenberg TP, Jiang H, Gratton E, Digman MA.
Fluorescence methods for the biophysical characterization of protein phase separated condensates.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 280a, 1353-Pos.Protein condensates are self-assembled membrane-less organelles that can orchestrate complex functions through a controlled confinement or exclusion of biologically relevant molecules. The biophysical mechanisms on how condensates can orchestrate the activities is hampered by the lack of methods capable of assessing phenomena across large spatiotemporal scales. Here, we propose three novel fluorescence-based approaches for the characterization of protein phase separated condensates (PPSC) at optical resolution that can cover a spatiotemporal range from sub-nanoseconds to seconds and from nanometer to micrometers. 1) We developed an approach to characterize at nanoscale (10−12 s, 10−9 m) the water dynamics and environment polarity within the condensates by monitoring the changes of optical properties of polarity-sensitive probes (ACDAN and ACRYLODAN). We demonstrate that this method can be applied to assess the phase separation diagram of a model coacervate when the classical methods (FRAP, FCS) fail. 2) We found that the type of phase separation (liquid-like, ... [truncated at 150 words]
Zaccheo K, Benítez-Mata AB, Digman MA, Barbee KA.
Investigating the effects of alcohols and cholesterol manipulations on bovine aortic endothelial cell membrane properties using phasor analysis of laurdan fluorescence lifetime microscopy and spectral imaging.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 366a, 1777-Pos.Endothelial cell membranes are made up of a heterogenous mixture of lipids and proteins and include liquid ordered (Lo) domains called lipid rafts and caveolae. These compartments have higher concentrations of cholesterol and sphingolipids and are concentrated in signaling molecules. Perturbations in membrane fluidity, including changes in cholesterol content can lead to changes in the structure of the plasma membrane and impair signaling mechanisms. In order to study how changing membrane fluidity can lead to changes in cellular signaling, we must be able to characterize changes in fluidity. Here we utilize two different methods using the fluorescent dye Laurdan, which exhibits a red spectral shift in emission when in a more liquid disordered (Ld) domain, to examine the effects of alcohols and cholesterol manipulations on membrane fluidity and cholesterol content in bovine aortic endothelial cells (BAEC). Laurdan-labeled BAECs were treated with dodecanol and benzyl alcohol to fluidize the membrane, methyl-β-cyclodextrin ... [truncated at 150 words]
Benítez-Mata AB, Palomba F, Scipioni L, Digman MA.
Analysis of membrane fluidity disturbances by mutant huntingtin in different membranal compartments using phasor analysis of local ICS and spectral phasor analysis.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 138a, 676-Pos.Huntington's Disease (HD), caused by the expression of extended polyQ repeats in the protein huntingtin, is known to disturb cholesterol metabolism. The misfolded proteins alter biosynthesis and intracellular levels of cholesterol, leading to membranes deterioration. In this work, we aim to describe the effect of said disturbances in various membrane compartments caused by different polyQ expansions from the membrane fluidity point of view, an approach never applied before. To this end, we use the environment-sensitive fluorescent probe LAURDAN to study the membrane fluidity at different compartments of SHSY5Y cells modified to express the non-pathogenic (18QmCherry) and pathogenic (53QmCherry, 77QmCherry) protein. We focus on three categories of compartments: cellular plasma membrane, internal membranes, and lipid droplets, all known to be rich in cholesterol and other lipids. Our approach leverages LAURDAN's propensity to insert in hydrophobic regions, such as membranes, where its emission is the more blue-shifted the less water is accessible ... [truncated at 150 words]
Huang Y, Fang T, Wei J, Mena JEM, Lakey PSJ, Kleinman MT, Shiraiwa M, Digman MA.
Phasor approach FLIM as an indicator for NADPH oxidase during exposure to secondary organic aerosols.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 415a, 2012-Pos.Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a multi-subunit enzyme which is responsible for host defense and inflammation signaling against either bacterial infection or pollutants. During activation, free oxygen is turned into superoxide anions which further leads to cascade formation of reactive oxygen species (ROS). However, once accumulation of ROS exceeds cell capacity, it damages components including proteins, lipids, and DNA known as oxidative stress which contributes to various lethal impacts such as lipid peroxidation and cancer. Therefore, it gives rise to the importance of monitoring NOX activity to avoid ROS accumulation. While most measurements use fluorescent probes to detect ROS, it lacks the ability to differentiate readings between cellular and extracellular which does not directly relate to NOX activity only. Here we aim to detect the impact of secondary organic aerosols (SOAs) on macrophage metabolism using fluorescence lifetime imaging (FLIM) and correlate the changes in fluorescence lifetime of NAD(P)H ... [truncated at 150 words]
Tedeschi G, Scipioni L, Palomba F, Digman MA.
Multiparametric fluorescence imaging approaches to investigate metabolic signature, lipid droplets, and collagen remodeling in breast cancer spheroids.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 409a, 1980-Pos.Tumor spheroids are 3D cell cultures models to study the behavior of avascular solid tumors in a controlled environment. Cellular metabolism, lipid storage and their relationship with the extracellular matrix (ECM) have been shown to be important elements in the characterization of tumor cell proliferation and metastatic invasion, but their simultaneous assessment remains challenging. In order to obtain all the information simultaneously, we imaged tumor spheroid with the following three imaging approaches over a 24 hours period. The metabolic signature at the single cell level was obtained by analyzing NADH lifetime, using the Phasor Fluorescence Lifetime Imaging Microscopy (FLIM) approach. Differences in lipid droplets content were obtained by analyzing the emission signal of an environmental-sensitive (solvatochromic) dye, Nile Red, with Spectral Phasors. Finally, we evaluated the spheroids-ECM interaction with Second Harmonic Generation (SHG) with PLICS (Phasor analysis of Local Image Correlation Spectroscopy). The main advantage of the proposed techniques is ... [truncated at 150 words]
Zhu S, Scipioni L, Digman MA.
Phasor unmixing to reveal organelle organization and cellular response.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 278a, 1346-Pos.Organelles are a series of subcellular structures that perform various functions inside eukaryotic cells. The coordination between organelles play an important role in understanding the intricate biochemical processes within eukaryotic cells as organelles must work in concert to maintain cell function. Here, we perform simultaneous excitation of seven fluorophores and spectral phasor unmixing to achieve robust emission separation of all seven stained subcellular compartments and organelles including the Golgi apparatus, tubulin, lipid droplets, lysosomes, mitochondria, nuclear and mitochondrial DNA. Our imaging approach uses a single round of two-photon excitation at 780 nm coupled with multicolor organelle-directed staining to obtain multi-parametric physiological profiling of live MCF10A cells, a non-tumorigenic epithelial breast cancer cell line, under stress related treatments. We created an analysis pipeline to spectrally unmix the contribution of each organelle and obtain phenotypic signatures under each type of treatment. Ultimately, we characterized significant differences in organelle phenotypes upon application of ... [truncated at 150 words]
Lopez KL, Tedeschi G, Palomba F, Digman MA.
Metabolic fingerprinting of HUVEC cells during phenotypic shifts using fluorescence lifetime image microscopy (FLIM).
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 404a, 1954-Pos.Adult human vascular endothelial cells (ECs) change their phenotype in response to changes in vascular tumor microenvironments, oxygen concentration and during wound healing. During angiogenesis, the physiological process of creating new blood vessels, hundreds of genes are activated with one of the most important being VEGF (vascular endothelial growth factor). VEGF, the signaling protein, stimulates EC migration, proliferation, and vascularization. During angiogenesis, ECs undergo an ‘angiogenic switch’; they change from a resting (quiescent) phenotype to a more elongated and mobile (tip cells) or proliferative and mobile (stalk cells) phenotype. Differences between phenotypes are not easy to assess. Currently, the most common approaches include Seahorse assays, fluorescent labeling or transfections in an attempt to categorize the HUVEC phenotypes by their metabolic signature. Although these methods have shown the importance of metabolic profiling to separate the cells, they are invasive and may ultimately alter the cellular metabolism. Additionally, a non-invasive metabolic imaging ... [truncated at 150 words]
Brennan CK, Yao Z, Scipioni L, Chen H, Ng K, Tedeschi G, Digman MA, Prescher JA.
Multiplexed bioluminescence microscopy via phasor analysis.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 142a, 695-Pos.Imaging tools allow us to visualize biological processes, including single cell dynamics and metabolic fluxes. Tracking multiple cells or features over extended periods of time, though, remains challenging. Common approaches rely on fluorescent probes, which require intense excitation light that can damage cells and cause phototoxicity. Bioluminescence imaging (BLI) relies on light production from a chemical reaction, the luciferase-catalyzed oxidation of small molecule substrates (luciferins), and can offer a more bio-friendly alternative for monitoring cellular events in real time. Although luciferases and luciferins have been used for decades to image processes in complex tissues and preclinical models, microscopic BLI has been historically challenging due to a lack of detection methods and probes that can be easily resolved by their emission spectra. We addressed this limitation by combining bioluminescence with phasor analysis, a method commonly used to distinguish spectrally similar fluorophores. We applied the approach on a camera-based microscope by means ... [truncated at 150 words]
Palomba F, Digman MA, Luo X, Lee A.
NADH autofluorescence phasor FLIM for the metabolic characterization of T cell and leukemia cell in a droplet.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 359a, 1733-Pos.Immune cells are known to shift their metabolism when stimulated by the interaction with binding receptors. The study of metabolic activation can be very important for the development of immune therapies and for understanding the machinery of immune active cells. However, there are challenges for monitoring real-time cell to cell interactions. Here we report the combination of the autofluorescence NADH Phasor FLIM method with the metabolic fingerprint of Jurkat cells (T lymphocyte) co-encapsulated with K562 (leukemia cells) in a water-in-oil droplet microfluidic chamber. The NADH autofluorescence, produced by all cells, lifetime and percent fractional contribution is characterized by two states: i) the enzyme bound state,3.4 ns, representative of OXPHOS and ii) the free state, 0.4 ns, mainly attributed to Glycolysis. The cells are co-encapsulated in a 1:1 ratio in a water droplet. To measure the NADH autofluorescence FLIM fingerprints, we used two photon excitation at 740 nm, with a confocal ... [truncated at 150 words]
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Metabolic imaging using the phasor approach to FLIM and tracking phenotypic change of mitochondria in cancer cells with Mitometer.
Single Molecule Spectroscopy and Superresolution Imaging XV. San Francisco, California. January 22-27, 2022.
The hallmark of metabolic alteration of increase glycolysis, i.e. Warburg effect, in cancer cells together with atypical extracellular matrix structure may be responsible for tumor cell aggressiveness and drug resistance. Here we apply the phasor approach technique in fluorescence lifetime imaging microscopy (FLIM) as a novel method to measure metabolic alteration as a function of ECM mechanics. We imaged and compared triple-negative breast cancer (TNBC) cells to non-cancerous cells on various ECM stiffness. Dysregulation of mitochondrial motion may contribute to the fueling of bioenergy demands in metastatic cancer. To measure mitochondria motion and analyze their fusion and fission events, we developed a new algorithm called “mitometer” that is unbiased, and allows for automated segmentation and tracking of mitochondria in live cell 2D and 3D time-lapse images.
Vu T, Vallmitjana A, Gu J, La K, Xu Q, Flores J, Zimak J, Shiu J, Hosohama L, Wu J, Douglas C, Waterman ML, Ganesan A, Hedde PN, Gratton E, Zhao W.
Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis.
Nat Commun. 2022; 13(1), 169. PMCID: PMC8748653Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications. Unfortunately, existing spatial transcriptomics methods are limited due to either low multiplexing or complexity. Here, we introduce a spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, spectral and time-resolved fluorescence imaging, and machine learning-based decoding. We demonstrate MOSAICA's multiplexing scalability in detecting 10-plex targets in fixed colorectal cancer cells using combinatorial labeling of five fluorophores with facile error-detection and removal of autofluorescence. MOSAICA's analysis is strongly correlated with sequencing data (Pearson's r = 0.96) and was further benchmarked using RNAscopeTM and LGC StellarisTM. We further apply MOSAICA for multiplexed analysis of clinical melanoma Formalin-Fixed Paraffin-Embedded (FFPE) tissues. We finally demonstrate simultaneous co-detection of protein and mRNA in cancer ... [truncated at 150 words]
Sallaberry I, Luszczak A, Philipp N, Navarro GSC, Gabriel MV, Gratton E, Gamarnik AV, Estrada LC.
In vivo pair correlation microscopy reveals dengue virus capsid protein nucleocytoplasmic bidirectional movement in mammalian infected cells.
Sci Rep. 2021; 11(1), 24415. PMCID: PMC8709865Flaviviruses are major human disease-causing pathogens, including dengue virus (DENV), Zika virus, yellow fever virus and others. DENV infects hundreds of millions of people per year around the world, causing a tremendous social and economic burden. DENV capsid (C) protein plays an essential role during genome encapsidation and viral particle formation. It has been previously shown that DENV C enters the nucleus in infected cells. However, whether DENV C protein exhibits nuclear export remains unclear. By spatially cross-correlating different regions of the cell, we investigated DENV C movement across the nuclear envelope during the infection cycle. We observed that transport takes place in both directions and with similar translocation times (in the ms time scale) suggesting a bidirectional movement of both C protein import and export.Furthermore, from the pair cross-correlation functions in cytoplasmic or nuclear regions we found two populations of C molecules in each compartment with fast and slow ... [truncated at 150 words]
Ma D, Hernandez GA, Lefebvre AEYT, Alshetaiwi H, Blake K, Dave KR, Rauf M, Williams JW, Davis RT, Evans KT, Longworth A, Masoud MYG, Lee R, Edwards RA, Digman MA, Kessenbrock K, Lawson DA.
Patient-derived xenograft culture-transplant system for investigation of human breast cancer metastasis.
Commun Biol. 2021; 4(1): 1268. PMCID: PMC8571269Metastasis is a fatal disease where research progress has been hindered by a lack of authentic experimental models. Here, we develop a 3D tumor sphere culture-transplant system that facilitates the growth and engineering of patient-derived xenograft (PDX) tumor cells for functional metastasis assays in vivo. Orthotopic transplantation and RNA sequencing (RNA-seq) analyses show that PDX tumor spheres maintain tumorigenic potential, and the molecular marker and global transcriptome signatures of native tumor cells. Tumor spheres display robust capacity for lentiviral engineering and dissemination in spontaneous and experimental metastasis assays in vivo. Inhibition of pathways previously reported to attenuate metastasis also inhibit metastasis after sphere culture, validating our approach for authentic investigations of metastasis. Finally, we demonstrate a new role for the metabolic enzyme NME1 in promoting breast cancer metastasis, providing proof-of-principle that our culture-transplant system can be used for authentic propagation and engineering of patient tumor cells for functional studies of ... [truncated at 150 words]
Gabriel MV, Sallaberry I, Navarro GSC, Gratton E, Gamarnik AV, Estrada LC.
Dengue virus capsid-protein dynamics in live infected cells studied by pair correlation analysis.
Dengue Virus (Methods in Molecular Biology, Vol. 2409). By R Mohana-Borges (Editors). Humana, New York, NY, pp. 99-117, 2022. It has become increasingly evident that unveiling the mechanisms of virus entry, assembly, and virion release is fundamental for identifying means for preventing viral spread and controlling viral disease. Due to virus mobility and structural and/or functional heterogeneity among viral particles, high spatiotemporal resolution single-virus/single-particle techniques are required to capture the behavior of viral particles inside infected cells.In this chapter, we present fluorescence imaging analysis methods for studying the mobility of fluorescently labeled dengue virus (DENV) proteins in live infected cells. Some of the most recent Fluorescence Fluctuation Spectroscopy (FFS) methods will be presented and, in particular, the pair Correlation Functions (pCF) approach will be discussed. The pCF method does not require individual molecule isolation, as in a particle-tracking experiment, to capture single viral protein behavior. In this regard, image acquisition is followed by the spatiotemporal cross-correlation function at increasing time delays, yielding a quantitative view of single-particle mobility in ... [truncated at 150 words]
Nicolai E, Pieri M, Gratton E, Motolese G, Bernardini S.
Bacterial infection diagnosis and antibiotic prescription in 3 h as an answer to antibiotic resistance: the case of urinary tract infections.
Antibiotics (Basel). 2021; 10(10): 1168. PMCID: PMC8532666Current methods for the diagnosis of urinary tract infections with antimicrobial susceptibility testing take 2-3 days and require a clinical laboratory. The lack of a rapid, point-of-care antibiotic susceptibility test (AST) has contributed to the misuse of antibiotics when treating urinary tract infections (UTIs) and consequently to the rise of multi-drug-resistant organisms. The current clinical approach has led to reduced treatment options and increased costs of diagnosis and therapy. To address this issue, novel diagnostics are needed for the timely determination of antimicrobial susceptibility. We present a rapid, point-of-care, phenotypic AST device that can report the antibiotic susceptibility/resistance of a uropathogen to a panel of antibiotics in as few as 3 h by utilizing fluorescent-labelling chemistry and a highly sensitive particle-counting instrument. We analysed 744 urine samples from the outpatients and inpatients of two Italian hospitals. The 130 UTI-positive patient urine samples we found were measured using a panel of ... [truncated at 150 words]
Shi B, Li W, Song Y, Wang Z, Ju R, Ulman A, Hu J, Palomba F, Zhao Y, Le JP, Jarrard W, Dimoff D, Digman MA, Gratton E, Zang C, Jiang H.
UTX condensation underlies its tumour-suppressive activity.
Nature. 2021; 597(7878): 726-731. PMCID: PMC9008583UTX (also known as KDM6A) encodes a histone H3K27 demethylase and is an important tumour suppressor that is frequently mutated in human cancers1. However, as the demethylase activity of UTX is often dispensable for mediating tumour suppression and developmental regulation2-8, the underlying molecular activity of UTX remains unknown. Here we show that phase separation of UTX underlies its chromatin-regulatory activity in tumour suppression. A core intrinsically disordered region (cIDR) of UTX forms phase-separated liquid condensates, and cIDR loss caused by the most frequent cancer mutation of UTX is mainly responsible for abolishing tumour suppression. Deletion, mutagenesis and replacement assays of the intrinsically disordered region demonstrate a critical role of UTX condensation in tumour suppression and embryonic stem cell differentiation. As shown by reconstitution in vitro and engineered systems in cells, UTX recruits the histone methyltransferase MLL4 (also known as KMT2D) to the same condensates and enriches the H3K4 methylation activity ... [truncated at 150 words]
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The phenotypic investigation of mitochondria in cancer.
PhD in Biomedical Engineering, University of California, Irvine, 2021.
Advisor: Digman, Michelle
Metastasis 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 ... [truncated at 150 words]
Lefebvre AEYT, Ma D, Kessenbrock K, Lawson DA, Digman MA.
Automated segmentation and tracking of mitochondria in live-cell time-lapse images.
Nat Methods. 2021; 18(9): 1091-1102.Mitochondria display complex morphology and movements, which complicates their segmentation and tracking in time-lapse images. Here, we introduce 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 requires only the pixel size and the time between frames to identify mitochondrial motion and morphology, including fusion and fission events. The segmentation algorithm isolates individual mitochondria via a shape- and size-preserving background removal process. The tracking algorithm links mitochondria via differences in morphological features and displacement, followed by a gap-closing scheme. Using Mitometer, we show that mitochondria of triple-negative breast cancer cells are faster, more directional, and more elongated than those in their receptor-positive counterparts. Furthermore, we show that mitochondrial motility and morphology in breast cancer, but not in normal breast epithelia, correlate with metabolic activity. Mitometer is an unbiased and user-friendly tool that will help resolve fundamental questions regarding ... [truncated at 150 words]
