Fazel M, Vallmitjana A, Scipioni L, Gratton E, Digman MA, Pressé S.
Fluorescence lifetime: Beating the IRF and interpulse window.
Biophys J. 2023; 122(4): 672-683. PMCID: PMC9989884Fluorescence lifetime imaging captures the spatial distribution of chemical species across cellular environments employing pulsed illumination confocal setups. However, quantitative interpretation of lifetime data continues to face critical challenges. For instance, fluorescent species with known in vitro excited-state lifetimes may split into multiple species with unique lifetimes when introduced into complex living environments. What is more, mixtures of species, which may be both endogenous and introduced into the sample, may exhibit 1) very similar lifetimes as well as 2) wide ranges of lifetimes including lifetimes shorter than the instrumental response function or whose duration may be long enough to be comparable to the interpulse window. By contrast, existing methods of analysis are optimized for well-separated and intermediate lifetimes. Here, we broaden the applicability of fluorescence lifetime analysis by simultaneously treating unknown mixtures of arbitrary lifetimes-outside the intermediate, Goldilocks, zone-for data drawn from a single confocal spot leveraging the tools of ... [truncated at 150 words]
Lopez KL, Palomba F, Digman MA.
Fluorescent single particle tracking enables high sensitivity viscometry measurements of micro-volume samples.
67th Annual Meeting of the Biophysical Society. San Diego, California. February 18-22.
Biophys J. 2023; 122(3, Suppl 1): 140a, 683-Pos.Measuring the viscosities of high-value materials at the microscale with minimal waste is essential for increasing turnaround times during drug development in the pharmaceutical industry. Currently, viscometers on the market fail to meet these needs as they can only process a limited number of samples at a time and still require large (>100 μL) loading volumes for accurate readings. Previously, researchers have parameterized fluorescence correlation spectroscopy (FCS) at low volumes to predict antibody behaviors at higher concentrations using the generalized Stokes-Einstein equation. However, these methods lack the ability to produce measurements at a fast turnaround rate. Here, we use a wide field camera-based fluorescence microscope modality and particle tracking system in parallel with a Python PT software to measure the viscosities of multiple solutions using a 10-μL working volume. We applied the generalized Stokes-Einstein equation to the coefficient output of the PT Python software to obtain our final viscosity values. ... [truncated at 150 words]
Tedeschi G, Zhu S, Aleghe G, Aleman E, Finch T, Ly C, Morenkov P, Scipioni L, Digman MA, Gratton E.
Investigation of breast cancer metabolic plasticity in the tumor microenvironment by single-cell organelle phenotyping.
67th Annual Meeting of the Biophysical Society. San Diego, California. February 18-22.
Biophys J. 2023; 122(3, Suppl 1): 132a, 642-Pos.Metabolic plasticity, i.e., the capability of cells to modify their metabolic state, is a hallmark of cancer and an important factor in the formation of metastases as well as a major contributor to chemoresistance and tumor recurrence. Furthermore, the tumor microenvironment, namely the collection of environmental properties (pH, nutrient availability, fatty acids) and cell types that surround the main tumor mass, is regarded to as the main driver of the metabolic state of tumor cells. Unfortunately, no technique can non-invasively assess the metabolic state of single cells in living samples, limiting our understanding of the heterogeneity and dynamics of metabolic state transitions. Here, we apply a technique, named ESPRESSO (Environmental Sensors Profiling Relayed by Subcellular Structures and Organelles) that combines organelle-specific environment-sensitive probes (ESPs) with hyperspectral imaging and quantitative bioimage analysis. We use a mixture of lipid droplet-, mitochondria- and lysosome-specific ESPs to quantify of morphological (e.g., number, size, organization) ... [truncated at 150 words]
Zhu S, Digman MA, Thompson J, Campos-Chillon F.
IVF bovine oocyte classification and selection.
67th Annual Meeting of the Biophysical Society. San Diego, California. February 18-22.
Biophys J. 2023; 122(3, Suppl 1): 415a, 2017-Pos.Metabolic plasticity, i.e., the capability of cells to modify their metabolic state, is a hallmark of cancer and an important factor in the formation of metastases as well as a major contributor to chemoresistance and tumor recurrence. Furthermore, the tumor microenvironment, namely the collection of environmental properties (pH, nutrient availability, fatty acids) and cell types that surround the main tumor mass, is regarded to as the main driver of the metabolic state of tumor cells. Unfortunately, no technique can non-invasively assess the metabolic state of single cells in living samples, limiting our understanding of the heterogeneity and dynamics of metabolic state transitions. Here, we apply a technique, named ESPRESSO (Environmental Sensors Profiling Relayed by Subcellular Structures and Organelles) that combines organelle-specific environment-sensitive probes (ESPs) with hyperspectral imaging and quantitative bioimage analysis. We use a mixture of lipid droplet-, mitochondria- and lysosome-specific ESPs to quantify of morphological (e.g., number, size, organization) ... [truncated at 150 words]
Huang Y, Zhuang M, Digman MA.
Metabolic profiling of transferred mitochondria using FLIM intensity based image segmentation (FIBIS).
67th Annual Meeting of the Biophysical Society. San Diego, California. February 18-22.
Biophys J. 2023; 122(3, Suppl 1): 129a, 626-Pos.Bulk metabolic assays on cell populations have shown that mitochondria transfer trigger breast cancer cells turn towards oxidative phosphorylation (OXPHOS) in favor of proliferation, migration, and cell growth. Yet, the current methods lack single cell resolution of interaction between endogenous and exogenous mitochondria to help understand the consequence metabolic alterations and other cell fate decisions. The phasor approach to fluorescence lifetime imaging microscopy (FLIM) has been widely used to measure the free to bound fraction of reduced form of NADH to quantify metabolic changes in live cells but not at the single mitochondrial scale. Here, we developed the FIBIS algorithm as a robust approach to recognize mitochondria from NADH intensity and further analyze mitochondrial metabolic states to investigate mitochondria transfer. We demonstrate that the NADH autofluorescence perfectly localizes with Mito7-mRuby labeled mitochondria and is free from artifactual emission spectral overlaps. Our data indicates that there was a 40% and a ... [truncated at 150 words]
Torrado B, Vallmitjana A, Dvornikov A, Gratton E.
Simultaneous FLIM, harmonic generation and hyperspectral imaging using a 4-channel detector.
67th Annual Meeting of the Biophysical Society. San Diego, California. February 18-22.
Biophys J. 2023; 122(3, Suppl 1): 129a, 1358-Pos.We describe a method that utilizes a PMT detector with an array of 4 independent channels allowing simultaneous acquisition of high-resolution Fluorescence Lifetime Imaging Microscopy (FLIM), Phasor-based Hyperspectral Imaging (HI), and Harmonic Generation images (HG). For FLIM acquisition our system is coupled with multichannel high digital frequency domain card which allows high photon counts per unit time and channel. HI is done based on sin/cos transmission optical filters placed in the emission path of a microscope to resolve the emission wavelength of every point in an image. The sin/cos filters used cover a broad spectral range and do not require collimated light, which makes this method very suitable for imaging in scattering media. The transmission geometry of our microscope provides a high efficiency in photon harvesting from scattering media than conventional detection methods used in commercial microscopes, making it ideal for HG imaging. The combined data acquired is analyzed using ... [truncated at 150 words]
Scipioni L, Tedeschi G, Atwood S, Digman MA, Gratton E.
Spatiotemporal single-cell phenotyping in living 3D skin organoids.
67th Annual Meeting of the Biophysical Society. San Diego, California. February 18-22.
Biophys J. 2023; 122(3, Suppl 1): 129a, 628-Pos.Continuous monitoring of cell states in living tissues remains elusive despite the explosion of single cell technologies. Here, we use organelle-specific environment-sensitive probes (ESPs) combined with hyperspectral imaging and a dedicated quantitative analysis pipeline to spatially and temporally track keratinocyte cell states in living 3D skin organoids. Our technique, named ESPRESSO (Environmental Sensors Profiling Relayed by Subcellular Structures and Organelles) combines hyperspectral imaging and phasor unmixing, enabling imaging of up to 6 ESPs (targeting chromatin, mitochondria, lysosomes, tubulin, Golgi apparatus and lipid droplets) at the same time with a single laser excitation. The quantification of morphological (e.g., number, size, organization) and functional (e.g., membrane potential, pH, polarity) characteristics of the organelles and subcellular structures allows us to identify the cell state at the single-cell level and is applicable to living cells in 2D and 3D. Cell state and cell-cell interactions are tightly regulated in space and time to determine tissue ... [truncated at 150 words]
Fang T, Huang YK, Wei J, Mena JEM, Lakey PSJ, Kleinman MT, Digman MA, Shiraiwa M.
Superoxide Release by Macrophages through NADPH Oxidase Activation Dominating Chemistry by Isoprene Secondary Organic Aerosols and Quinones to Cause Oxidative Damage on Membranes.
Environ Sci Technol. 2022; 56(23): 17029-17038. PMCID: PMC9730850Oxidative stress mediated by reactive oxygen species (ROS) is a key process for adverse aerosol health effects. Secondary organic aerosols (SOA) account for a major fraction of fine particulate matter, and their inhalation and deposition into the respiratory tract causes the formation of ROS by chemical and cellular processes, but their relative contributions are hardly quantified and their link to oxidative stress remains uncertain. Here, we quantified cellular and chemical superoxide generation by 9,10-phenanthrenequinone (PQN) and isoprene SOA using a chemiluminescence assay combined with electron paramagnetic resonance spectroscopy as well as kinetic modeling. We also applied cellular imaging techniques to study the cellular mechanism of superoxide release and oxidative damage on cell membranes. We show that PQN and isoprene SOA activate NADPH oxidase in macrophages to release massive amounts of superoxide, overwhelming the superoxide formation by aqueous chemical reactions in the epithelial lining fluid. The activation dose for PQN is ... [truncated at 150 words]
Halbrook CJ, Thurston G, Boyer S, Anaraki C, Jiménez JA, McCarthy A, Steele NG, Kerk SA, Hong HS, Lin L, Law FV, Felton C, Scipioni L, Sajjakulnukit P, Andren A, Beutel AK, Singh R, Nelson BS, van Bergh FD, Krall AS, Mullen PJ, Zhang L, Batra S, Morton JP, Stanger BZ, Christofk HR, Digman MA, Beard DA, Viale A, Zhang J, Crawford HC, di Magliano MP, Jorgensen C, Lyssiotis CA.
Differential integrated stress response and asparagine production drive symbiosis and therapy resistance of pancreatic adenocarcinoma cells.
Nat Cancer. 2022; 3(11): 1386-1403. PMCID: PMC9701142The pancreatic tumor microenvironment drives deregulated nutrient availability. Accordingly, pancreatic cancer cells require metabolic adaptations to survive and proliferate. Pancreatic cancer subtypes have been characterized by transcriptional and functional differences, with subtypes reported to exist within the same tumor. However, it remains unclear if this diversity extends to metabolic programming. Here, using metabolomic profiling and functional interrogation of metabolic dependencies, we identify two distinct metabolic subclasses among neoplastic populations within individual human and mouse tumors. Furthermore, these populations are poised for metabolic cross-talk, and in examining this, we find an unexpected role for asparagine supporting proliferation during limited respiration. Constitutive GCN2 activation permits ATF4 signaling in one subtype, driving excess asparagine production. Asparagine release provides resistance during impaired respiration, enabling symbiosis. Functionally, availability of exogenous asparagine during limited respiration indirectly supports maintenance of aspartate pools, a rate-limiting biosynthetic precursor. Conversely, depletion of extracellular asparagine with PEG-asparaginase sensitizes tumors to mitochondrial ... [truncated at 150 words]
Wang XX, Xie C, Libby AE, Ranjit S, Levi J, Myakala K, Bhasin K, Jones BA, Orlicky DJ, Takahashi S, Dvornikov A, Kleiner DE, Hewitt SM, Adorini L, Kopp JB, Krausz KW, Rosenberg A, McManaman JL, Robertson CE, Ir D, Frank DN, Luo Y, Gonzalez FJ, Gratton E, Levi M.
The role of FXR and TGR5 in reversing and preventing progression of Western diet-induced hepatic steatosis, inflammation, and fibrosis in mice.
J Biol Chem. 2022; 298(11), 102530. PMCID: PMC9638804Nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease in the US, partly due to the increasing incidence of metabolic syndrome, obesity, and type 2 diabetes. The roles of bile acids and their receptors, such as the nuclear receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, on the development of NASH are not fully clear. C57BL/6J male mice fed a Western diet (WD) develop characteristics of NASH, allowing determination of the effects of FXR and TGR5 agonists on this disease. Here we show that the FXR-TGR5 dual agonist INT-767 prevents progression of WD-induced hepatic steatosis, inflammation, and fibrosis, as determined by histological and biochemical assays and novel label-free microscopy imaging techniques, including third harmonic generation, second harmonic generation, and fluorescence lifetime imaging microscopy. Furthermore, we show INT-767 decreases liver fatty acid synthesis and fatty acid and cholesterol uptake, as well as liver inflammation. INT-767 markedly changed ... [truncated at 150 words]
Rahim MK, Zhao J, Patel HV, Lagouros HA, Kota R, Fernandez I, Gratton E, Haun JB.
Phasor analysis of fluorescence lifetime enables quantitative multiplexed molecular imaging of three probes.
Anal Chem. 2022; 94(41): 14185-14194.The excited-state lifetime is an intrinsic property of fluorescent molecules that can be leveraged for multiplexed imaging. An advantage of fluorescence lifetime-based multiplexing is that signals from multiple probes can be gathered simultaneously, whereas traditional spectral fluorescence imaging typically requires multiple images at different excitation and emission wavelengths. Additionally, lifetime and spectra could both be utilized to expand the multiplexing capacity of fluorescence. However, resolving exogenous molecular probes based exclusively on the fluorescence lifetime has been limited by technical challenges in analyzing lifetime data. The phasor approach to lifetime analysis offers a simple, graphical solution that has increasingly been used to assess endogenous cellular autofluorescence to quantify metabolic factors. In this study, we employed the phasor analysis of FLIM to quantitatively resolve three exogenous, antibody-targeted fluorescent probes with similar spectral properties based on lifetime information alone. First, we demonstrated that three biomarkers that were spatially restricted to the cell membrane, ... [truncated at 150 words]
Guaglianone G, Torrado B, Lin YF, Watkins MC, Wysocki VH, Gratton E, Nowick JS.
Elucidating the oligomerization and cellular interactions of a trimer derived from Aβ through fluorescence and mass spectrometric studies.
ACS Chem Neurosci. 2022; 13(16): 2473-2482. PMCID: PMC9389591Aβ oligomers play a central role in the neurodegeneration observed with Alzheimer's disease. Our laboratory has developed covalently stabilized trimers derived from residues 17-36 of Aβ as model systems for studying Aβ oligomers. In the current study, we apply the emerging techniques of fluorescence lifetime imaging microscopy (FLIM) and native mass spectrometry (native MS) to better understand the assembly and interactions of the oligomer model system 2AT-L in aqueous solutions and with cells. 2AT-L and fluorescently labeled 2AT-L analogues assemble in the membrane-like environment of SDS-PAGE, showing diffuse bands of oligomers in equilibrium. Native ion mobility-mass spectrometry (native IM-MS) of 2AT-L allows for the identification of discrete oligomers in solution and shows similar patterns of oligomer formation between 2AT-L and fluorescently labeled analogues. Fluorescence microscopy with SH-SY5Y cells reveals that fluorescently labeled 2AT-L analogues colocalize within lysosomes. FLIM studies with phasor analysis further elucidate the assembly of 2AT-L within cells ... [truncated at 150 words]
Chun SK, Fortin BM, Fellows RC, Habowski AN, Verlande A, Song WA, Mahieu AL, Lefebvre AEYT, Sterrenberg JN, Velez LM, Digman MA, Edwards RA, Pannunzio NR, Seldin MM, Waterman ML, Masri S.
Disruption of the circadian clock drives Apc loss of heterozygosity to accelerate colorectal cancer.
Sci Adv. 2022; 8(32), eabo2389. PMCID: PMC9365282An alarming rise in young onset colorectal cancer (CRC) has been reported; however, the underlying molecular mechanism remains undefined. Suspected risk factors of young onset CRC include environmental aspects, such as lifestyle and dietary factors, which are known to affect the circadian clock. We find that both genetic disruption and environmental disruption of the circadian clock accelerate Apc-driven CRC pathogenesis in vivo. Using an intestinal organoid model, we demonstrate that clock disruption promotes transformation by driving Apc loss of heterozygosity, which hyperactivates Wnt signaling. This up-regulates c-Myc, a known Wnt target, which drives heightened glycolytic metabolism. Using patient-derived organoids, we show that circadian rhythms are lost in human tumors. Last, we identify that variance between core clock and Wnt pathway genes significantly predicts the survival of patients with CRC. Overall, our findings demonstrate a previously unidentified mechanistic link between clock disruption and CRC, which has important implications for young onset ... [truncated at 150 words]
Sallaberry I, Luszczak A, Philipp N, Navarro GSC, Gabriel MV, Gratton E, Gamarnik AV, Estrada LC.
Author Correction: In vivo pair correlation microscopy reveals dengue virus capsid protein nucleocytoplasmic bidirectional movement in mammalian infected cells.
Sci Rep. 2022; 12(1): 11343. PMCID: PMC9256625No abstract available
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; 19(7): 893-898.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; 14(25): 8901-8905.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]
Morris MA, Vallmitjana A, Grein F, Schneider T, Arts M, Jones CR, Nguyen BT, Hashemian MH, Malek M, Gratton E, Nowick JS.
Visualizing the mode of action and supramolecular assembly of teixobactin analogues in Bacillus subtilis.
Chem Sci. 2022; 13(26): 7747-7754. PMCID: PMC9258396Teixobactin has been the source of intensive study and interest as a promising antibiotic, because of its excellent activity against drug-resistant Gram-positive pathogens and its novel but not yet fully understood mechanism of action that precludes drug resistance. Recent studies have demonstrated that the mode of action of teixobactin is more complicated than initially thought, with supramolecular assembly of the antibiotic appearing to play a critical role in the binding process. Further studies of the interactions of teixobactin with bacteria and its molecular targets offer the promise of providing deeper insights into its novel mechanism of action and guiding the design of additional drug candidates and analogues. The current study reports the preparation and study of teixobactin analogues bearing a variety of fluorophores. Structured illumination microscopy of the fluorescent teixobactin analogues with B. subtilis enables super-resolution visualization of the interaction of teixobactin with bacterial cell walls and permits the observation ... [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; 9(3): 1015-1025. PMCID: PMC9278809Fluorescence 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]
