Biology subjects

Fraser, S. E.

Publications and source records attributed to Fraser, S. E..

At least 19 recordsLinked to original sources

bioRxiv2026

Bright Probes, Blurred Metabolism: Navigating Fluorescent Protein Cross-Excitation in NADH FLIM

Fluorescence lifetime imaging microscopy (FLIM) of endogenous NAD(P)H enables the label-free assessment of cellular metabolic state. Although metabolic imaging is increasingly combined with fluorescent protein (FP) reporters to enhance biological specificity, the potential cross-talk between the intrinsic and extrinsic labels remain ill-defined. Here, we systematically evaluate cross-talk from FPs in metabolic FLIM using phasor analysis of two-photon fluorescence microscopy. The results clearly show that many widely used fluorescent proteins are excited under the conditions used for NADH imaging; they emit blue-shifted, short-lifetime fluorescence that can interfere with imaging NADH metabolic signatures. This overlap persists across excitation wavelengths and FP classes, posing a significant challenge for multiplexed metabolic imaging. This cautionary tale argues against unvalidated multiplexing strategies in metabolic FLIM studies. Our study aims to identify acceptable imaging partners, offer a pipeline for assaying potential cross-talk, and provide practical guidance for experimental design. SignificanceFluorescence lifetime imaging of NADH autofluorescence is a powerful, label-free approach to map cellular metabolism in living tissues. A growing number of studies combine NADH imaging with fluorescent protein (FP) reporters to simultaneously identify specific cell types or subcellular compartments. This study reveals that many FPs, spanning the visible spectrum, are unexpectedly excited under NADH conditions. Commonly used green, yellow, and red variants produce short-lifetime, blue-shifted fluorescence that directly overlaps with metabolic NADH signals. This cross-excitation can be falsely interpreted as a shift in cellular metabolic state, posing a significant risk for multiplexed metabolic imaging studies. Our studies establish a pipeline to assess and manage this risk. We identify StayGoldE138D and mNeonGreen as the most compatible FPs for co-imaging with NADH, and provide a practical framework to guide experimental design and control strategies for multiplexed metabolic FLIM.

biophysics↗

bioRxiv2026

Quantitative comparison of fluorescent reporters by FCS excitation scan

Fluorescent reporters such as fluorescent proteins or chemigenetic indicators are indispensable tools for studying biological processes using light microscopy. Choosing an appropriate fluorescent tag is a crucial step in experimental design not only for imaging but also for quantitative measurements such as fluorescence fluctuation spectroscopy. Two key parameters should be considered: Fluorescent brightness and photo-bleaching. Change to fluorescence intensity due to photobleaching is relatively easy to assess in different biological environments, while brightness is more elusive. Here, we develop and employ a fluorescence correlation spectroscopy (FCS) based excitation scan assay that determines fluorescent protein performance and validate it in tissue culture and zebrafish embryos. We employ our FCS pipeline to compare a set of 10 established fluorescent proteins as well as HALO and SNAP tags for both cellular imaging and measurements of diffusion dynamics with FCS. We show that mNeonGreen outperforms mEGFP in tissue culture and zebrafish embryos. We also compare StayGold variants against other green fluorescent proteins and chemigenetic reporters in tissue culture. Overall, we present a broadly applicable approach for determining fluorescent reporter brightness in the living system of interest.

biophysics↗

bioRxiv2025

Negative-Valence Neurons in the Larval Zebrafish Pallium

An organisms survival depends on the rapid classification of sensory events as either harmful or beneficial. In mammals, this computation is partially performed by neurons in the amygdala that respond to signals with negative or positive valence. The larval zebrafish pallium is thought to contain homologs of the mammalian amygdala, isocortex, and hippocampus; however, the signals encoded by pallial neurons remain largely uncharacterized. Using two-photon light-sheet microscopy to image 7-9-day-old zebrafish that express pan-neuronal GCaMP6s, we recorded calcium dynamics throughout the brain while presenting a panel of strongly aversive stimuli including infrared heat, electric shock, and a whole-field looming shadow, together with milder threats including vibration, loud sound, light transitions, and a partial looming stimulus. A compact cluster of neurons in the rostrolateral dorsal pallium (Rl) responded vigorously to strongly noxious and fully looming stimuli, but not to the milder cues. In contrast, neurons in the ventromedial pallium and habenula responded to all stimuli tested. Rl neurons are characterized by high Tiam2a expression, suggesting they can be genetically accessed. Thus, our results identify a locus of negative valence neurons in the teleost pallium.

neuroscience↗

bioRxiv2025

Prolactin receptor localization and dynamics: Insights from quantitative imaging and mathematical modeling

Signal transduction through the prolactin receptor (PRLR) is crucial in pancreatic {beta}-cell pro-liferation, impacting pancreatic homeostasis. PRLR-induced JAK/STAT signaling is dynamic, involving changes in spatial organization of signaling molecules. Thus, the spatial organization of PRLR could have strong implications on signaling output. Internalization has been shown and modeled in other signaling pathways but has not been considered in a mathematical model of PRLR signaling. Here, we use live-cell fluorescence imaging, reconstitution approaches, and fluorescence correlation spectroscopy (FCS) to inform a mathematical model of PRLR signaling. Internal PRLR localization is observed in primary pancreatic tissue and in an engineered PRLR expression system. Our imaging data indicate the presence of intracellular and plasma membrane-bound receptor populations. We use FCS to resolve the membrane-bound PRLR population. Based on our data, we include internalization dynamics within an ordinary differential equation (ODE) model of PRLR signaling. We employ the model to explore how the spatial heterogeneity of PRLR affects downstream signaling. We show that the model is more sensitive to PRLR trafficking rates and ability to promote signaling than to its initial spatial distribution. Our data underscore the versatility of a modeling-imaging framework to quantitatively understand signal transduction in and beyond {beta}-cells. Significance StatementO_LIProlactin receptor (PRLR) signal transduction impacts the growth and survival of insulin-secreting cells, making this pathway a target for building our understanding of pancreatic homeostasis and exploring potential diabetes therapeutics. C_LIO_LILive fluorescence imaging techniques applied within an engineered PRLR expression platform indicate PRLR localization patterns consistent with primary pancreatic tissue and the presence of two spatially distinct PRLR populations. These observations inform a predictive mathematical model of PRLR signaling. C_LIO_LIIntegrating experimental data tailored to computational approaches shapes our understanding of complex, multiscale systems such as signal transduction. A generalizable modeling-imaging framework enables the study of molecular dynamics beyond {beta}-cells. C_LI

cell biology↗

bioRxiv2025

Transforming Hyperspectral Data into Insight: The DREAM Approach for Pathology

Quantitative pathology remains limited due to the need for chemical staining and subjective interpretation of tissue features. Autofluorescence imaging offers a label-free alternative: however, high-dimensional excitation-emission datasets pose challenges for visualization and reproducible analysis. Here, we present Dimensionality Reduction for Enhanced Autofluorescence Microscopy (DREAM), a method that condenses multi-excitation emission spectra into a compact, information-rich format using phasor-based tools. Applied to unstained esophageal tissue samples, DREAM enables high-contrast visualizations that distinguish key histological structures without the need for exogenous labeling. Quantitative assessments across multiple datasets show DREAM improves colorfulness, sharpness, and consistency over single-laser acquisitions, supporting its potential to advance objective, label-free diagnostics through enhanced spectral visualization.

biophysics↗

bioRxiv2024

Neuronally differentiated endothelial cell subtype regulates organ blood flow and immune balance

Vascular endothelial cells (ECs) perform key pleiotropic functions to maintain body homeostasis via the regulation of organ blood flow, vascular permeability, tissue growth and inflammation, and angiogenesis. Recent transcriptomic studies uncovered many EC subtypes across organs; however their specific functions are incompletely understood. Here we identified and characterized a novel, minority subtype of scattered ECs with a well-defined arteriovenous zonal localization exclusively in small resistance (strain) arterioles, and with the highest density in the brain>retina>kidney. Due to their expression of both endothelial and neuron-like functional and gene transcriptomic signatures, they were termed neuro-endothelial cells (NECs). High resolution single-cell transcriptome analysis of mouse brain and kidney ECs identified neuronal nitric oxide synthase (Nos1) and cytokine-like 1 (Cytl1) as top NEC biomarkers. Intravital multiphoton imaging of optogenetic mouse models with NEC gain/loss-of-function revealed NEC and Nos1-dependent vasodilation/vasoconstriction of intact brain and kidney arterioles and elevation/reduction in blood flow. Silencing NEC Nos1 and Cytl1 gene expression in vivo caused marked segmental arteriolar vasoconstrictions, reductions in vascular density and organ blood flow, increased vascular permeability and immune cell homing. Cytl1 administration triggered vasodilation and increased blood flow acutely, and increased capillary density and clonal EC remodeling chronically. NECs play major vasodilatory, angiogenic and anti-inflammatory functions that may be therapeutically targeted for vascular and inflammatory diseases.

physiology↗

bioRxiv2024

A Versatile Light Field Microscopy Platform for Multi-purpose Dynamic Volumetric Bio-imaging

Light field microscopy (LFM) has emerged in recent years as a unique solution for fast, scan-free volumetric imaging of dynamic biological samples. This is achieved by using a microlens array in the detection path to record both the lateral and angular information of the light fields coming from the sample, capturing a 3-dimensional (3D) volume in a single 2-dimensional (2D) snapshot. In post-acquisition, the 3D sample volume is computationally reconstructed from the recorded 2D images, thus enabling unprecedented 3D capture speed, not limited by the typical constraint of physically scanning the focal plane over the sample volume. Up to date, most published LFM imaging setups have been specialized single-purpose platforms, optimized for a narrow performance window in field of view and resolution, thus hampering widespread adoption of LFM for biomedical research. Here, we present a versatile LFM platform for fast 3D imaging across multiple scales, enabling applications from cell to system-level biology on the same imaging setup. Our multiscale LFM is built as an add-on module to a conventional commercially available wide field microscope, and the various imaging applications, with different ranges of field of view and resolution, are achieved by simply switching between the standard microscope objectives available on the wide field microscope. Importantly, we provide an open-source end-to-end software package for calculating the system performance parameters, processing the experimentally measured point spread function, and light field 3D image reconstruction. We demonstrate the performance of our multiscale LFM platform through imaging the whole-brain activity map of seizures in larval zebrafish, calcium dynamics in ex vivo mouse pancreatic islets, and subcellular protein dynamics in cultured cells.

bioengineering↗

bioRxiv2024

Neural Basis of Number Sense in Larval Zebrafish

Number sense, the ability to discriminate discrete quantity, is widespread across animals, yet how this capacity develops remains unknown. Using two-photon light-sheet imaging, we recorded whole-brain activity at single-cell resolution in larval zebrafish (Danio rerio) exposed to controlled visual numerosity stimuli. We discovered that number-selective neurons emerge in a striking developmental sequence: cells tuned to numerosity 1 are already present at 3 days post-fertilization (dpf), and neurons selective for 2, 3, and higher quantities appear in increasing abundance later, at 5 and 7 dpf, accompanied by a relative reduction in 1-tuned cells. The proportion of number-selective cells, summed over all tested numerosities, relative to all identified neurons in the brain, was found to decrease over time. We further showed that a machine-learning decoder based on the activity of the number-selective neurons can predict the number stimulus seen by the animals with accuracies at better than twice chance level. These results reveal how neuronal circuits develop structured numerical codes and provide a framework for studying the emergence of cognitive primitives at cellular resolution.

neuroscience↗

bioRxiv2024

Far-red chemigenetic biosensors for multi-dimensional and super-resolved kinase activity imaging

Fluorescent biosensors revolutionized biomedical science by enabling the direct measurement of signaling activities in living cells, yet the current technology is limited in resolution and dimensionality. Here, we introduce highly sensitive chemigenetic kinase activity biosensors that combine the genetically encodable self-labeling protein tag HaloTag7 with bright far-red-emitting synthetic fluorophores. This technology enables five-color biosensor multiplexing, 4D activity imaging, and functional super-resolution imaging via stimulated emission depletion (STED) microscopy.

bioengineering↗

bioRxiv2023

Hand2 represses non-cardiac cell fates through chromatin remodeling at cis-regulatory elements

Developmental studies have revealed the importance of the transcription factor Hand2 in cardiac development. Hand2 promotes cardiac progenitor differentiation and epithelial maturation, while repressing other tissue types. The mechanisms underlying the promotion of cardiac fates are far better understood than those underlying the repression of alternative fates. Here, we assess Hand2-dependent changes in gene expression and chromatin remodeling in cardiac progenitors of zebrafish embryos. Cell-type specific transcriptome analysis shows a dual function for Hand2 in activation of cardiac differentiation genes and repression of pronephric pathways. We identify functional cis-regulatory elements whose chromatin accessibility are increased in hand2 mutant cells. These regulatory elements associate with non-cardiac gene expression, and drive reporter gene expression in tissues associated with Hand2-repressed genes. We find that functional Hand2 is sufficient to reduce non-cardiac reporter expression in cardiac lineages. Taken together, our data support a model of Hand2-dependent coordination of transcriptional programs, not only through transcriptional activation of cardiac and epithelial maturation genes, but also through repressive chromatin remodeling at the DNA regulatory elements of non-cardiac genes.

developmental biology↗

bioRxiv2023

Episodic live imaging of cone photoreceptor maturation in GNAT2-EGFP retinal organoids

Fluorescent reporter pluripotent stem cell (PSC) derived retinal organoids are powerful tools to investigate cell type-specific development and disease phenotypes. When combined with live imaging, they enable direct and repeated observation of cell behaviors within a developing retinal tissue. Here, we generated a human cone photoreceptor reporter line by CRISPR/Cas9 genome editing of WTC11-mTagRFPT-LMNB1 human induced pluripotent stem cells (iPSCs) by inserting enhanced green fluorescent protein (EGFP) coding sequences and a 2A self-cleaving peptide at the N-terminus of Guanine Nucleotide-Binding Protein Subunit Alpha Transducin 2 (GNAT2). In retinal organoids generated from these iPSCs, the GNAT2-EGFP allele robustly and exclusively labeled both immature and mature cones starting at culture day 34. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of morphological maturation of individual cones for >18 weeks and revealed inner segment accumulation of mitochondria and growth at 12.2 cubic microns per day from day 126 to day 153. Immobilized GNAT2-EGFP cone reporter organoids provide a valuable tool for investigating human cone development and disease.

developmental biology↗

bioRxiv2022

Patterning and growth are coordinated early in the cell cycle

Precise control of cell division is essential for proper patterning and growth during the development of multicellular organisms. Coordination of formative (asymmetric) divisions that generate new tissue patterns with proliferative (symmetric) divisions that promote growth is poorly understood. Here, we employed quantitative 4D light sheet and confocal microscopy to probe in vivo the dynamics of two transcription factors, SHORTROOT (SHR) and SCARECROW (SCR), which are required for asymmetric division in the stem cell niche of Arabidopsis roots [1,2]. Long-term (up to 48 hours), frequent (every 15 minutes) imaging of the two regulators in tandem in single cells, in conjunction with a SHR induction system, enabled us to challenge an existing bistable model[3] of the SHR/SCR gene regulatory network. By directly controlling SHR and SCR expression dynamics, we were able to identify key features that are essential for rescue of asymmetric division in shr mutants. We show that instead of high stable levels of nuclear SHR and SCR, only low transient levels of expression are required. Nuclear SHR kinetics do not follow predictions of the bistable model, and the regulatory relationship between SHR and SCR can be modeled by monostable alternatives. Furthermore, expression of these two regulators early in the cell cycle determines the orientation of the division plane, resulting in either formative or proliferative cell division. Our findings provide evidence for an uncharacterized mechanism by which developmental regulators directly coordinate patterning and growth.

developmental biology↗

bioRxiv2022

The kinase occupancy of T-cell coreceptors reconsidered

The sensitivity of the {beta} T-cell receptor (TCR) is enhanced by the coreceptors CD4 and CD8{beta}, which are expressed primarily by cells of the helper and cytotoxic T-cell lineages, respectively. The coreceptors bind to major histocompatibility complex (MHC) molecules and associate intracellularly with the Src-family kinase Lck, which catalyzes TCR phosphorylation during receptor triggering. Although coreceptor-kinase occupancy was initially believed to be high, a recent study suggested that most coreceptors exist in an Lck-free state, and that this low occupancy helps to effect TCR antigen discrimination. Here, using the same method, we found instead that the CD4-Lck interaction was stoichiometric (~100%) and that the CD8{beta}-Lck interaction was also substantial (~60%). We confirmed our findings in live cells using fluorescence cross-correlation spectroscopy (FCCS) to measure coreceptor-Lck co-diffusion in situ. After introducing structurally guided mutations into the intracellular domain of CD4, we used FCCS to show that stoichiometric Lck coupling required an amphipathic -helix present in CD4 but not CD8. In double-positive cells expressing equal numbers of both coreceptors, but limiting amounts of kinase, CD4 out-competed CD8{beta} for Lck. In T cells, TCR signaling induced CD4-Lck oligomerization but did not affect the high levels of CD4-Lck occupancy. These findings help settle the question of kinase occupancy and suggest that the binding advantages that CD4 has over CD8 could be important when Lck levels are limiting. Significance statementCD4 and CD8{beta} are archetypal coreceptor proteins that potently enhance T-cell antigen sensitivity but how they function is still debated. A fundamental question that remains incompletely resolved is: what fractions of the coreceptors bind the signal-initiating kinase, Lck? Using in vitro assays and non-invasive fluorescence fluctuation spectroscopy in live cells, we show that most coreceptors are occupied by Lck at the surface of live cells. The structural basis for important differences in the kinase occupancy of CD4 and CD8{beta} is also identified. These results provide important context for refining current models of both TCR antigen recognition and cell fate decisions made during thymopoiesis.

immunology↗

bioRxiv2022

Behavioural analysis of loss of function zebrafish supports baz1b as master regulator of domestication

Domestication is associated with both morphological and behavioural phenotypic changes that differentiate domesticated species from their wild counterparts. Some of the traits are those purposely targeted by the selection process, whilst others co-occur as a result of selection. The combination of traits is referred to as the domestication syndrome and their shared characteristics has given rise to the neural crest domestication syndrome (NCDS) hypothesis. According to this hypothesis, the phenotypic changes are a consequence of a selection towards animals with mild underdevelopment of the neural crest. A similar mechanism is suggested to affect some species of "self-domesticated" animals, including humans. Recent research supports a role for BAZ1B, one of the haplo-insufficient genes in Williams syndrome (WS), in the evolution of craniofacial features of modern humans. Interestingly, WS recapitulates some of the traits observed in the domestication syndrome, including hypersociability and reduced facial bones. However, the evidence linking BAZ1B to behavioural phenotypes associated with domestication is presumptive. Here, we use zebrafish as a model to test the hypothesis that baz1b loss-of-function leads to both morphological and behavioural phenotypes associated with the domestication syndrome by influencing the development of the neural crest. Our research provides further evidence supporting the NCDS hypothesis and bazlbs role in this process.

animal behavior and cognition↗

bioRxiv2022

FRaeppli, a multispectral imaging toolbox for cell tracing and dense tissue analysis in zebrafish

Visualizing cell shapes, interactions and lineages of differentiating cells is instrumental for understanding organ development and repair. Across species, strategies for stochastic multicolour labelling have greatly facilitated tracking cells in in vivo and mapping neuronal connectivity. Nevertheless, integrating multi-fluorophore information into the context of developing tissues in zebrafish is challenging given their cytoplasmic localization and spectral incompatibility with commonly used fluorescent markers. Here, we developed FRaeppli (Fish-Raeppli) expressing bright membrane-or nuclear-targeted fluorescent proteins for efficient cell shape analysis and tracking. High spatiotemporal activation flexibility is provided by the Gal4/UAS system together with Cre/lox and/or PhiC31integrase. The distinct spectra of the FRaeppli fluorescent proteins allow simultaneous imaging with GFP and infrared subcellular reporters or tissue landmarks. By tailoring hyperspectral protocols for time-efficient acquisition, we demonstrate FRaepplis suitability for live imaging of complex internal organs, like the liver. Combining FRaeppli with polarity markers revealed previously unknown canalicular topologies between differentiating hepatocytes, reminiscent of the mammalian liver, suggesting shared developmental mechanisms. The multispectral FRaeppli toolbox thus enables the comprehensive analysis of intricate cellular morphologies, topologies and tissue lineages at single-cell resolution in zebrafish.

developmental biology↗

bioRxiv2021

Kinetic and data-driven modeling of pancreatic β-cell central carbon metabolism and insulin secretion

Pancreatic {beta}-cells respond to increased extracellular glucose levels by initiating a metabolic shift. That change in metabolism is part of the process of glucose-stimulated insulin secretion and is of particular interest in the context of diabetes. However, we do not fully understand how the coordinated changes in metabolic pathways and metabolite products influence insulin secretion. In this work, we apply systems biology approaches to develop a detailed kinetic model of the intracellular central carbon metabolic pathways in pancreatic {beta}-cells upon stimulation with high levels of glucose. The model is calibrated to published metabolomics datasets for the INS1 823/13 cell line, accurately capturing the measured metabolite fold-changes. We first employed the calibrated mechanistic model to estimate the stimulated cells fluxome. We then used the predicted network fluxes in a data-driven approach to build a partial least squares regression model. By developing the combined kinetic and data-driven modeling framework, we gain insights into the link between {beta}-cell metabolism and glucose-stimulated insulin secretion. The combined modeling framework was used to predict the effects of common anti-diabetic pharmacological interventions on metabolite levels, flux through the metabolic network, and insulin secretion. Our simulations reveal targets that can be modulated to enhance insulin secretion. The model is a promising tool to contextualize and extend the usefulness of metabolomics data and to predict dynamics and metabolite levels that are difficult to measure in vitro. In addition, the modeling framework can be applied to identify, explain, and assess novel and clinically-relevant interventions that may be particularly valuable in diabetes treatment. Author SummaryDiabetes is among the most common chronic illnesses, occurring when the {beta}-cells in the pancreas are unable to produce enough insulin to properly manage the bodys blood sugar levels. {beta}-cells metabolize nutrients to produce energy needed for insulin secretion in response to high glucose, and there is a potential to harness {beta}-cell metabolism for treating diabetes. However, {beta}-cell metabolism is not fully characterized. We have developed a computational modeling framework to better understand the relationship between cellular metabolism and insulin production in the pancreatic {beta}-cell. With this modeling framework, we are able to simulate metabolic perturbations, such as the knockdown of the activity of a metabolic enzyme, and predict the effect on the metabolic network and on insulin production. This work can therefore be applied to investigate, in a time- and cost-efficient manner, {beta}-cell metabolism and predict effective therapies that target the cells metabolic network.

systems biology↗

bioRxiv2020

Neurons in the dorso-central division of zebrafish pallium respond to change in visual numerosity

Non-symbolic number cognition based on an approximate sense of magnitude has been documented in zebrafish. Here we investigated for the first time its neural bases. Zebrafish were habituated to a set of three or nine small dots associated with food reward. During habituation trials, the dots changed in their individual size, position and density maintaining their numerousness and overall surface area. In the dishabituation test, zebrafish faced a change (i) in number (from three to nine or vice versa with the same overall surface), (ii) in shape (with the same overall surface and number), or (iii) in size (with the same shape and number); in a control group (iv) zebrafish faced the same familiar stimuli as during the habituation. Using qPCR to measure modulation of the expression of the immediate early genes c-fos and egr-1 and in-situ hybridization to count egr1-positive cells we found a specific and selective activation of the caudal part of the dorso-central (Dc) division of the zebrafish pallium upon change in numerosity. As pallial regions are implicated in number cognition in mammals and birds, these findings support the existence of an evolutionarily conserved mechanism for approximate magnitude and provide an avenue for exploring the underlying molecular correlates.

neuroscience↗

bioRxiv2019

A versatile twin-microscope system for light-sheet imaging

Light-sheet microscopy offers faster imaging and reduced phototoxicity in comparison to conventional point-scanning microscopy, making it a preferred technique for imaging biological dynamics for durations of hours or days. Such extended imaging sessions pose a challenge, as it reduces the number of specimens that can be imaged in a given day. Here we present an instrument, the flex-SPIM, that combines two independently controlled light-sheet microscope-twins, built so that they can share an ultrafast near-infrared laser and a bank of continuous-wave visible lasers, increasing throughput and decreasing cost. To permit a wide variety of specimens to be imaged, each microscope-twin provides flexible imaging parameters, including (i) operation in one-photon and/or two-photon excitation modes, (ii) delivery of one to three light-sheets via a trio of orthogonal excitation arms, (iii) sub-micron to micron imaging resolution, (iv) multicolor compatibility, and (v) upright and/or inverted detection geometry. We offer a detailed description of the flex-SPIM design to aid instrument builders who wish to construct and use similar systems. We demonstrate the instruments versatility for biological investigation by performing fast imaging of the beating heart in an intact zebrafish embryo, deep imaging of thick patient-derived tumor organoids, and gentle whole-brain imaging of neural activity in behaving larval zebrafish.

biophysics↗