Biology subjects

Gratton, E.

Publications and source records attributed to Gratton, E..

14 recordsLinked to original sources

bioRxiv2025

Highly multiplexed spectral FLIM via physics informed data analysis

Spectral fluorescence lifetime imaging (S-FLIM) allows for the simultaneous deconvolution of signal from multiple fluorophore species by leveraging both spectral and lifetime information. However, existing analyses still face multiple difficulties in decoding information collected from typical S-FLIM experiments. These include: using information from pre-calibrated spectra in environments that may differ from the cellular context in which S-FLIM experiments are performed; limitations in the ability to deconvolute species due to overlapping spectra; high photon budget requirements, typically about a hundred photons per pixel per species. Yet information on the spectra themselves are already encoded in the data and do not require pre-calibration. What is more, efficient photon-by-photon analyses are possible reducing both the required photon budget and making it possible to use larger budgets in order to discriminate small differences in spectra to resolve spatially co-localized fluorophore species. To achieve this, we propose a Bayesian S-FLIM framework capable of simultaneously learning spectra and lifetimes photon-by-photon ultimately using limited photon counts and being highly data efficient. We demonstrate the proposed framework using a range of synthetic and experimental data and show that it can deconvolve up to 9 species with heavily overlapped spectra.

biophysics↗

bioRxiv2023

Organelle phenotyping and multi-dimensional microscopy identify C1q as a novel regulator of microglial function.

Microglia, the immune cells of the central nervous system (CNS), are incredibly dynamic and heterogenous cells. While single cell RNA sequencing has become the conventional methodology for evaluating microglial state, transcriptomics do not provide insight into functional changes. Here, we propose a novel organelle phenotyping approach where we treat live human induced pluripotent stem cell-derived microglia (iMGL) with organelle dyes (mitochondria, lipids, lysosomes) and acquire data by live-cell spectral microscopy. Dimensionality reduction techniques and unbiased cluster identification allow for recognition of microglial subpopulations based in organelle function. We validate this methodology using lipopolysaccharide (LPS) and IL-10 treatment to polarize iMGL to an inflammatory" and "anti-inflammatory state, respectively, and then apply it to identify a novel regulator of iMGL function, complement protein C1q. C1q is traditionally known as the initiator of the complement cascade, but here we use organelle phenotyping to identify a role for C1q in regulating iMGL fatty acid storage and mitochondria membrane potential. Follow up evaluation of microglia with more traditional read outs of activation state confirm that C1q drives an increase in microglia pro-inflammatory cytokine production and migration, while suppressing microglial proliferation. These data together validate the use of a novel organelle phenotyping approach and enable better mechanism investigation of molecular regulators of microglial state, such as C1q.

neuroscience↗

bioRxiv2022

Building Fluorescence Lifetime Maps Photon-by-photon by Leveraging Spatial Correlations

Fluorescence lifetime imaging microscopy (FLIM) has become a standard tool in the quantitative analysis of sub-cellular environments. However, quantitative FLIM analyses face several challenges. First, spatial correlations between pixels are often ignored as signal from individual pixels is analyzed independently thereby limiting spatial resolution. Second, existing methods deduce photon ratios instead of absolute lifetime maps. Next, the number of lifetime components contributing to the signal is unknown, while excited state lifetimes with <1 ns difference are difficult to discriminate. Finally, existing analyses require high photon budgets, and often cannot rigorously propagate experimental uncertainty into values over lifetime maps and number of components involved. To overcome all of these challenges simultaneously and self-consistently at once, we propose the first doubly nonparametric framework. That is, we learn the number of fluorescent species (through beta-Bernoulli process priors) and absolute lifetime maps of these species (through Gaussian process priors) by leveraging information from pulses not leading to observed photon. We benchmark our algorithm using a broad range of synthetic and experimental data and demonstrate its robustness across a number of scenarios including cases where we recover lifetime differences between components as small as 0.3 ns with merely 1000 photons.

biophysics↗

bioRxiv2022

Fluorescence Lifetime: Beating the IRF and interpulse window

Fluorescence lifetime imaging (FLIM) has been essential in capturing spatial distributions 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, that 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 (IRF) 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 Bayesian nonparametrics (BNP). We benchmark our algorithm, termed BNP-lifetime analysis of BNP-LA, using a range of synthetic and experimental data. Moreover, we show that the BNP-LA method can distinguish and deduce lifetimes using photon counts as small as 500.

biophysics↗

bioRxiv2022

Live cell biosensors based on the fluorescence lifetime of environment-sensing dyes

Most biosensors used with fluorescence lifetime imaging (FLIM) have been based on fluorescence resonance energy transfer (FRET). Here we examined the capabilities of FLIM biosensors based on environment-sensing dyes. We screened merocyanine dyes to find an optimal combination of environment-induced FLIM changes, photostability and brightness at wavelengths suitable for live cell imaging. A biosensor reporting conformational changes of endogenous Cdc42 protein was tested in vitro and in live cells. In addition to the known advantages of FLIM (quantitation independent of photobleaching, intracellular biosensor distribution, and excitation intensity), the spectral properties of the merocyanine-FLIM biosensor (mcFLIM) provided enhanced sensitivity at low activation levels (<10%) where FRET is typically least sensitive. We leveraged these properties and the phasor representation of FLIM to determine the specific concentration of activated Cdc42 across the cell.

cell biology↗

bioRxiv2022

Epigenetic dysregulation from chromosomal transit in micronuclei

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers [1-4], yet whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei [5, 6], and subsequent micronuclear envelope rupture [7] profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice as well as cancer and non-transformed cells. Some of the changes to histone PTMs occur due to micronuclear envelope rupture whereas others are inherited from mitotic abnormalities prior to micronucleus formation. Using orthogonal techniques, we show that micronuclei exhibit extensive differences in chromatin accessibility with a strong positional bias between promoters and distal or intergenic regions. Finally, we show that inducing CIN engenders widespread epigenetic dysregulation and that chromosomes which transit in micronuclei experience durable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, in addition to genomic copy number alterations, CIN can serve as a vehicle for epigenetic reprogramming and heterogeneity in cancer.

cancer biology↗

bioRxiv2021

Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis

Multiplexed mRNA profiling in the spatial context provides important new information enabling basic research and clinical applications. Unfortunately, most existing spatial transcriptomics methods are limited due to either low multiplexing or assay complexity. Here, we introduce a new 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 target decoding. This technology is the first application combining the biophotonic techniques, Spectral and Fluorescence Lifetime Imaging and Microscopy (FLIM), to the field of spatial transcriptomics. By integrating the time dimension with conventional spectrum-based measurements, MOSAICA enables direct and highly-multiplexed in situ spatial biomarker profiling in a single round of staining and imaging while providing error correction removal of background autofluorescence. We demonstrate mRNA encoding using combinatorial spectral and lifetime labeling and target decoding and quantification using a phasor-based image segmentation and machine learning clustering technique. We then showcase MOSAICAs multiplexing scalability in detecting 10-plex targets in fixed colorectal cancer cells using combinatorial labeling of only five fluorophores with facile error-correction and removal of autofluorescent moieties. MOSAICAs analysis is strongly correlated with sequencing data (Pearsons r = 0.9) and was further benchmarked using RNAscopeand LGC Stellaris. We further apply MOSAICA for multiplexed analysis of clinical melanoma Formalin-Fixed Paraffin-Embedded (FFPE) tissues that have a high degree of tissue scattering and intrinsic autofluorescence, demonstrating the robustness of the approach. We then demonstrate simultaneous co-detection of protein and mRNA in colorectal cancer cells. MOSAICA represents a simple, versatile, and scalable tool for targeted spatial transcriptomics analysis that can find broad utility in constructing human cell atlases, elucidating biological and disease processes in the spatial context, and serving as companion diagnostics for stratified patient care.

bioengineering↗

bioRxiv2021

In vivo macromolecular crowding is differentially modulated by Aquaporin 0 in zebrafish lens: insights from a nano-environment sensor and spectral imaging.

Macromolecular crowding is crucial for cellular homeostasis. In vivo studies of macromolecular crowding and ultimately water-dynamics are needed to understand their role in cellular fates. The macromolecular crowding in the lens is essential for understanding normal optics of the lens, and moreover for understanding and prevention of cataract and presbyopia. Here we combine the use of the water nano-environmentally sensitive sensor (6-acetyl-2-dimethylaminonaphthalene, ACDAN) with in vivo studies of Aquaporin zero zebrafish mutants to understand the lens macromolecular crowding. Spectral phasor analysis of ACDAN fluorescence reveal the extent of water dipolar relaxation and demonstrate that the mutations in the duplicated zebrafish Aquaporin 0s, Aqp0a and Aqp0b, alter the water state and macromolecular crowding in the living zebrafish lens. Our results provide in vivo evidence that Aqp0a promotes fluid influx in the deeper lens cortex, whereas Aqp0b facilitates fluid efflux. This work opens new perspectives for in vivo studies on macromolecular crowding. TeaserIn this study we uncover the roles of Aquaporin 0 in macromolecular crowding required for lens development and vision.

biophysics↗

bioRxiv2020

Ultrafast phasor-based hyperspectral snapshot microscopy for biomedical imaging

Hyperspectral imaging is highly sought after in many fields including mineralogy and geology, environment and agriculture, astronomy and, importantly, biomedical imaging and biological fluorescence. We developed ultrafast phasor-based hyperspectral snapshot microscopy based on sine/cosine interference filters for biomedical imaging not feasible with conventional hyperspectral detection methods. Current approaches rely on slow spatial or spectral scanning limiting their application in living biological tissues, while faster snapshot methods such as image mapping spectrometry and multispectral interferometry are limited in spatial and/or spectral resolution, are computationally demanding, and imaging devices are very expensive to manufacture. Leveraging light sheet microscopy, phasor-based hyperspectral snapshot microscopy improved imaging speed 10-100 fold which, combined with minimal light exposure and high detection efficiency, enabled hyperspectral metabolic imaging of live, three-dimensional mouse tissues not feasible with other methods. As a fit-free method that does not require any a priori information often unavailable in complex and evolving biological systems, the rule of linear combinations of the phasor could spectrally resolve subtle differences between cell types in the developing zebrafish retina and spectrally separate and track multiple organelles in 3D cultured cells over time. The sine/cosine snapshot method is adaptable to any microscope or imaging device thus making hyperspectral imaging and fit-free analysis based on linear combinations broadly available to researchers and the public.

biophysics↗

bioRxiv2020

A Modular Microarray Imaging System for Highly Specific COVID-19 Antibody Testing

To detect the presence of antibodies in blood against SARS-CoV-2 in a highly sensitive and specific manner, here we describe a robust, inexpensive ($200), 3D-printable portable imaging platform (TinyArray imager) that can be deployed immediately in areas with minimal infrastructure to read coronavirus antigen microarrays (CoVAMs) that contain a panel of antigens from SARS-CoV-2, SARS-1, MERS, and other respiratory viruses. Application includes basic laboratories and makeshift field clinics where a few drops of blood from a finger prick could be rapidly tested in parallel for the presence of antibodies to SARS-CoV-2 with a test turnaround time of only 2-4 h. To evaluate our imaging device, we probed and imaged coronavirus microarrays with COVID-19-positive and negative sera and achieved a performance on par with a commercial microarray reader 100x more expensive than our imaging device. This work will enable large scale serosurveillance, which can play an important role in the months and years to come to implement efficient containment and mitigation measures, as well as help develop therapeutics and vaccines to treat and prevent the spread of COVID-19.

microbiology↗

bioRxiv2019

Defining epidermal basal cell states during skin homeostasis and wound healing using single-cell transcriptomics

Our knowledge of transcriptional heterogeneities in epithelial stem/progenitor cell compartments is limited. Epidermal basal cells sustain cutaneous tissue maintenance and drive wound healing. Previous studies have probed basal cell heterogeneity in stem/progenitor potential, but a non-biased dissection of basal cell dynamics during differentiation is lacking. Using single-cell RNA-sequencing coupled with RNAScope and fluorescence lifetime imaging, we identify three non-proliferative and one proliferative basal cell transcriptional states in homeostatic skin that differ in metabolic preference and become spatially partitioned during wound re-epithelialization. Pseudotemporal trajectory and RNA velocity analyses produce a quasi-linear differentiation hierarchy where basal cells progress from Col17a1high/Trp63high state to early response state, proliferate at the juncture of these two states, or become growth arrested before differentiating into spinous cells. Wound healing induces plasticity manifested by dynamic basal-spinous interconversions at multiple basal states. Our study provides a systematic view of epidermal cellular dynamics supporting a revised \"hierarchical-lineage\" model of homeostasis.

developmental biology↗

bioRxiv2019

Nanoscale distribution of nuclear sites analyzed by superresolution STED-ICCS

Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, superresolution microscopy has gained considerable interest as it can be used to probe the spatial organization of functional sites in intact single cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a pre-segmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet.\n\nHere we combine dual color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that STED-ICCS provides not only a value of colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the Proliferating cell nuclear antigen (PCNA) protein have a high level of proximity and are correlated at a nanometer distance which is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of nanoscale distribution of functional sites in the nucleus.\n\nStatement of significanceSeveral methods are available to quantify the proximity of two labeled molecules from dual color images. Among them, image cross-correlation spectroscopy (ICCS) is attractive as it does not require a pre-segmentation of the image into objects and can be used to detect dynamic interactions. Here, we combine for the first time ICCS with superresolution stimulated emission depletion (STED) microscopy (STED-ICCS) to quantify the spatial distribution of functional sites in the nucleus. Our results show that STED-ICCS, in addition to quantifying the colocalized fraction, detects characteristic nanometer distances associated to correlated nuclear sites. This work shows that STED-ICCS can be a powerful tool to quantify the nanoscale distribution of functional sites in the nucleus.

biophysics↗

bioRxiv2019

Syncrip/hnRNPQ is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation

Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganisation at new synapses, often requiring post-transcriptional regulation a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organises actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is not yet known. Here, we show that the local translation of msp300 is promoted during activity-dependent plasticity by the conserved RNA binding protein Syncrip/hnRNP Q, which binds to msp300 transcripts and is essential for plasticity. Single molecule imaging shows that Syncrip is associated in vivo with msp300 mRNA in ribosome-rich particles. Elevated neural activity alters the dynamics of Syncrip RNP granules at the synapse, suggesting a change in particle composition or binding that facilitates translation. These results introduce Syncrip as an important early-acting activity-dependent translational regulator of a plasticity gene that is strongly associated with human ataxias.\n\nSyncrip regulates synaptic plasticity via msp300Titlow et al. find that Syncrip (hnRNPQ RNA binding protein) acts directly on msp300 to modulate activity-dependent synaptic plasticity. In vivo biophysical experiments reveal activity-dependent changes in RNP complex sizes compatible with an increase in translation at the synapse.

neuroscience↗

bioRxiv2018

Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response.

To investigate how chromatin architecture is spatiotemporally organised 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 (ICS) of histone FLIM-FRET microscopy data acquired in live cells co-expressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that when coupled with laser micro-irradiation induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ATM and RNF8 regulate rapid chromatin decompaction at DSBs and formation of a compact chromatin ring surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.\n\nSIGNIFICANCE STATEMENTChromatin dynamics play a central role in the DNA damage response (DDR). A long-standing obstacle in the DDR field was the lack of technology capable of visualising chromatin dynamics at double strand break (DSB) sites. Here we describe novel biophysical methods that quantify spatiotemporal chromatin compaction dynamics in living cells. Using these novel tools, we identify how chromatin architecture is reorganised at a DSB locus to enable repair factor access and demarcate the lesion from the surrounding nuclear environment. Further, we identify novel regulatory roles for key DDR enzymes in this process. Finally, we demonstrate method utility with physical, pharmacological and genetic manipulation of the chromatin environment, identifying method potential for use in future studies of chromatin biology.

biophysics↗