Light-sheet Fluorescence Imaging Research Articles

A 3D atlas of the human developing pancreas to explore progenitor proliferation and differentiation

Aims/hypothesisRodent pancreas development has been described in great detail. On the other hand, there are still gaps in our understanding of the developmental trajectories of pancreatic cells during human ontogenesis. Here, our aim was to map the spatial and chronological dynamics of human pancreatic cell differentiation and proliferation by using 3D imaging of cleared human embryonic and fetal pancreases.MethodsWe combined tissue clearing with light-sheet fluorescence imaging in human embryonic and fetal pancreases during the first trimester of pregnancy. In addition, we validated an explant culture system enabling in vitro proliferation of pancreatic progenitors to determine the mitogenic effect of candidate molecules.ResultsWe detected the first insulin-positive cells as early as five post-conceptional weeks, two weeks earlier than previously observed. We observed few insulin-positive clusters at five post-conceptional weeks (mean ± SD 9.25±5.65) with a sharp increase to 11 post-conceptional weeks (4307±152.34). We identified a central niche as the location of onset of the earliest insulin cell production and detected extra-pancreatic loci within the adjacent developing gut. Conversely, proliferating pancreatic progenitors were located in the periphery of the epithelium, suggesting the existence of two separated pancreatic niches for differentiation and proliferation. Additionally, we observed that the proliferation ratio of progenitors ranged between 20% and 30%, while for insulin-positive cells it was 1%. We next unveiled a mitogenic effect of the platelet-derived growth factor AA isoform (PDGFAA) in progenitors acting through the pancreatic mesenchyme by increasing threefold the number of proliferating progenitors.Conclusions/interpretationThis work presents a first 3D atlas of the human developing pancreas, charting both endocrine and proliferating cells across early development.Graphical

Photonic neural probe enabled microendoscopes for light-sheet light-field computational fluorescence brain imaging.

Light-sheet fluorescence microscopy is widely used for high-speed, high-contrast, volumetric imaging. Application of this technique to in vivo brain imaging in non-transparent organisms has been limited by the geometric constraints of conventional light-sheet microscopes, which require orthogonal fluorescence excitation and collection objectives. We have recently demonstrated implantable photonic neural probes that emit addressable light sheets at depth in brain tissue, miniaturizing the excitation optics. Here, we propose a microendoscope consisting of a light-sheet neural probe packaged together with miniaturized fluorescence collection optics based on an image fiber bundle for lensless, light-field, computational fluorescence imaging. Foundry-fabricated, silicon-based, light-sheet neural probes can be packaged together with commercially available image fiber bundles to form microendoscopes for light-sheet light-field fluorescence imaging at depth in brain tissue. Prototype microendoscopes were developed using light-sheet neural probes with five addressable sheets and image fiber bundles. Fluorescence imaging with the microendoscopes was tested with fluorescent beads suspended in agarose and fixed mouse brain tissue. Volumetric light-sheet light-field fluorescence imaging was demonstrated using the microendoscopes. Increased imaging depth and enhanced reconstruction accuracy were observed relative to epi-illumination light-field imaging using only a fiber bundle. Our work offers a solution toward volumetric fluorescence imaging of brain tissue with a compact size and high contrast. The proof-of-concept demonstrations herein illustrate the operating principles and methods of the imaging approach, providing a foundation for future investigations of photonic neural probe enabled microendoscopes for deep-brain fluorescence imaging in vivo.

In Situ Visualization of Membrane Fouling Evolution during Ultrafiltration Using Label-Free Hyperspectral Light Sheet Fluorescence Imaging.

Profound understanding of fouling behaviors and underlying mechanisms is fundamentally important for fouling control in membrane-based environmental applications. Therefore, it entails novel noninvasive analytical approaches for in situ characterizing the formation and development of membrane fouling processes. This work presents a characterization approach based on hyperspectral light sheet fluorescence microscopy (HSPEC-LSFM), which is capable of discriminating various foulants and providing their 2-dimensional/3-dimensional spatial distributions on/in membranes in a label-free manner. A fast, highly sensitive and noninvasive imaging platform was established by developing a HSPEC-LSFM system and further extending it to incorporate a laboratory-scale pressure-driven membrane filtration system. Hyperspectral data sets with a spectral resolution of ∼1.1 nm and spatial resolution of ∼3 μm as well as the temporal resolution of ∼8 s/plane were obtained, and the fouling formation and development process of foulants onto membrane surfaces, within the pores and on the pore walls were clearly observed during the ultrafiltration of protein and humic substances solutions. Pore blocking/constriction at short times while cake growth/concentration polarization at longer times was found to have coupled effects for the flux decline in these filtration tests, and yet the contribution of each effect as well as the transition of the governing mechanisms was found distinct. These results demonstrate in situ label-free characterization of membrane fouling evolution with the recognition of foulant species during filtration and provide new insights into membrane fouling. This work offers a powerful tool to investigate dynamic processes for a wide range of membrane-based explorations.

Hyperstabilization of T cell microvilli contacts by chimeric antigen receptors.

T cells typically recognize their ligands using a defined cell biology-the scanning of their membrane microvilli (MV) to palpate their environment-while that same membrane scaffolds T cell receptors (TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise and a tractable means to study the interplay between receptor affinity, MV dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high-resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize MV scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing receptor affinity, antigen density, and/or multiplicity of receptor binding sites normalized microvillar dynamics and synapse resolution, and effector functions improved with reduced affinity and/or antigen density, highlighting the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement.

Abstract P2001: Vessel Normalization By Targeting Endothelial Cell Plasticity To Improve Cardiac Repair After Myocardial Infarction

Ischemic heart disease is a leading cause of death in the world. Formation of new blood vessels is fundamental to cardiac reperfusion and repair after myocardial infarction (MI), and therapeutic promotion of angiogenesis, therefore, has emerged as a promising strategy for treating ischemic heart diseases. However, the therapeutic efficacy is small and transient in most of pro-angiogenic therapy. Here, we characterize unexpectedly prominent vessel aberrancy, albeit a robust angiogenesis, in MI-associated cardiac tissue by 3-dementional light sheet fluorescence imaging. Our single-cell transcriptome analysis of mouse MI tissues with Cdh5-Cre ; Rosa-LSL-tdTomato -mediated endothelial lineage tracing identifies a robust EC plasticity to acquire mesenchymal gene signature in MI-associated ECs, peaked at 1 week and partially retained at 4 and 8 weeks after MI. Moreover, cardiac ECs undergo transcriptional activation to drive mesenchymal phenotypes including over-proliferation, migration and increased permeability, which lead to vessel abnormality after exposure to in vitro ischemic microenvironment. Furthermore, our single-cell and bulk RNA-seq analysis uncovers a PDGF/NF-κB/HIF-1α axis that induces Snail expression and mesenchymal phenotypes in ECs under hypoxia, culminating in aberrant vascularization. Notably, EC-specific knockout of PDGFR-β abrogates mesenchymal gene expression in ECs, attenuates vascular abnormalities in the infarcted tissue, and improves cardiac repair after MI. Interestingly, our single-nuclei analysis uncovers that genetic ablation of PDGFR-β in ECs temporally reprograms cardiomyocyte metabolism and enhances myocyte survival and proliferation. Pharmacological PDGFR inhibition or nanoparticle-based targeted PDGFR-β siRNA delivery normalizes vasculature and improves cardiac function recovery after MI. These findings illustrate a mechanism controlling aberrant neovascularization after ischemia, and suggest that targeting PDGF/Snail-mediated endothelial plasticity may offer exciting opportunities for normalizing vasculature and treating ischemic heart disease.

Open-top light-sheet imaging of CLEAR emulsion for high-throughput loss-free analysis of massive fluorescent droplets

Digital droplet PCR (ddPCR) is classified as the third-generation PCR technology that enables absolute quantitative detection of nucleic acid molecules and has become an increasingly powerful tool for clinic diagnosis. We previously established a CLEAR-dPCR technique based on the combination of CLEAR droplets generated by micro-centrifuge-based microtubule arrays (MiCA) and insitu 3D readout by light-sheet fluorescence imaging. This CLEAR-dPCR technique attains very high readout speed and dynamic range. Meanwhile, it is free from sample loss and contamination, showing its advantages over commercial d-PCR technologies. However, a conventional orthogonal light-sheet imaging setup in CLEAR d-PCR cannot image multiple centrifuge tubes, thereby limiting its widespread application to large-scale, high-speed dd-PCR assays. Herein, we propose an in-parallel 3D dd-PCR readout technique based on an open-top light-sheet microscopy setup. This approach can continuously scan multiple centrifuge tubes which contain CLEAR emulsions with highly diverse concentrations, and thus further boost the scale and throughput of our 3D dd-PCR technique.

Charting the meningeal lymphatic network.

A genuine network of lymphatic vessels can be found in the dural layer of the meninges that ensheathe the brain and spinal cord of mammalians. In this issue, Jacob et al. (2022. J. Exp. Med.https://doi.org/10.1084/jem.20220035) employ light sheet fluorescence imaging of intact mouse heads to provide a more comprehensive chart of the meningeal lymphatic vasculature and draw a parallel between lymphatic drainage of cerebrospinal fluid in mice and humans.

In situ visualization of combined membrane fouling behaviors using multi-color light sheet fluorescence imaging: A study with BSA and dextran mixture

The understanding of fouling behaviors and mechanism is critical for membrane fouling that is a bottleneck for the sustainable application of membrane technology. Therefore, it is essential to develop new analytical tools that can in situ characterize membrane fouling processes during filtration. In this work, we demonstrate a capability of high-resolution large-scale in situ visualization that can rapidly differentiate foulants and their temporal-spatial-distributions on/in/over the membrane matrix during filtration process based on multi-color light sheet fluorescence imaging platform. The rapid-evolving fouling process of different foulants were directly monitored and analyzed during the ultrafiltration of bovine serum albumin (BSA), dextran and their mixture solutions. Inner adsorption was found to play a more critical role for dextran-induced fouling on polymeric membranes compared with BSA-induced fouling. During the ultrafiltration of BSA/dextran mixture solutions, BSA was directly observed as the major contributor to the decrease of permeate flux, which could be employed as a complementary evidence for the interfacial free energy calculations during membrane fouling and vice versa. This work provides new insights into combined membrane fouling process and offers a highly-sensitive, non-invasive characterization tool for a wide range of explorations in membrane-based processes and interfacial processes. In situ large-scale characterization of combined membrane fouling behaviors Strong relations between flux decline rate and foulant distributions in/on membrane Direct observation of key foulant in combined fouling process Facilitation of exploring interfacial processes (dynamic and thermodynamic)

Microscopic Imaging Methods for Organ-on-a-Chip Platforms.

Microscopic imaging is essential and the most popular method for in situ monitoring and evaluating the outcome of various organ-on-a-chip (OOC) platforms, including the number and morphology of mammalian cells, gene expression, protein secretions, etc. This review presents an overview of how various imaging methods can be used to image organ-on-a-chip platforms, including transillumination imaging (including brightfield, phase-contrast, and holographic optofluidic imaging), fluorescence imaging (including confocal fluorescence and light-sheet fluorescence imaging), and smartphone-based imaging (including microscope attachment-based, quantitative phase, and lens-free imaging). While various microscopic imaging methods have been demonstrated for conventional microfluidic devices, a relatively small number of microscopic imaging methods have been demonstrated for OOC platforms. Some methods have rarely been used to image OOCs. Specific requirements for imaging OOCs will be discussed in comparison to the conventional microfluidic devices and future directions will be introduced in this review.

Internal and external hydrodynamics of Taylor cone under constant and alternating voltage actuation

Electrohydrodynamic atomization (EHDA) is carried out in the Taylor cone mode for generating unimodal particle distribution, which can be achieved by either constant voltage actuation (CVA) or alternating voltage actuation (AVA). The present study reports an experimental investigation of the flow field both inside and outside the Taylor cone using light sheet fluorescence imaging and time-resolved particle image velocimetry measurements. Liquid ethanol is used as the working fluid and the amplitude of both constant and alternating electric potential difference is set at the same value, i.e., VC=VA=5.2 kV with an actuation frequency of 200 Hz in the case of alternating EHDA. The hydrodynamic behavior both inside and outside the Taylor cone is presented for the first time. The flow field measurements demonstrate meridional circulation from the nozzle exit toward the apex of the Taylor cone along the generatrix followed by flow from the apex of the Taylor cone along the central axis. A symmetric toroidal vortex is observed inside the Taylor cone in the case of CVA and an asymmetric toroidal vortex is observed for AVA. The flow field shows streamline-like flow in the ambient medium from the nozzle toward the ground electrode along the interface of the Taylor cone jet for CVA. In contrast, two vortical structures are observed around the apex of the Taylor cone for AVA. The velocity profile near the liquid–air interface of the Taylor cone indicates no direct correlation between the flow field inside the Taylor cone with the flow of the ambient medium. This difference may be attributed to the corona wind generated due to asymmetric electrode configuration. The unsteady flow field generated by alternating EHDA has great potential for enhanced heat transfer using spray cooling.

Deep-learning super-resolution light-sheet add-on microscopy (Deep-SLAM) for easy isotropic volumetric imaging of large biological specimens.

Isotropic 3D histological imaging of large biological specimens is highly desired but remains highly challenging to current fluorescence microscopy technique. Here we present a new method, termed deep-learning super-resolution light-sheet add-on microscopy (Deep-SLAM), to enable fast, isotropic light-sheet fluorescence imaging on a conventional wide-field microscope. After integrating a minimized add-on device that transforms an inverted microscope into a 3D light-sheet microscope, we further integrate a deep neural network (DNN) procedure to quickly restore the ambiguous z-reconstructed planes that suffer from still insufficient axial resolution of light-sheet illumination, thereby achieving isotropic 3D imaging of thick biological specimens at single-cell resolution. We apply this easy and cost-effective Deep-SLAM approach to the anatomical imaging of single neurons in a meso-scale mouse brain, demonstrating its potential for readily converting commonly-used commercialized 2D microscopes to high-throughput 3D imaging, which is previously exclusive for high-end microscopy implementations.

Light-sheet fluorescence imaging charts the gastrula origin of vascular endothelial cells in early zebrafish embryos

It remains challenging to construct a complete cell lineage map of the origin of vascular endothelial cells in any vertebrate embryo. Here, we report the application of in toto light-sheet fluorescence imaging of embryos to trace the origin of vascular endothelial cells (ECs) at single-cell resolution in zebrafish. We first adapted a previously reported method to embryo mounting and light-sheet imaging, created an alignment, fusion, and extraction all-in-one software (AFEIO) for processing big data, and performed quantitative analysis of cell lineage relationships using commercially available Imaris software. Our data revealed that vascular ECs originated from broad regions of the gastrula along the dorsal–ventral and anterior–posterior axes, of which the dorsal–anterior cells contributed to cerebral ECs, the dorsal–lateral cells to anterior trunk ECs, and the ventral–lateral cells to posterior trunk and tail ECs. Therefore, this work, to our knowledge, charts the first comprehensive map of the gastrula origin of vascular ECs in zebrafish, and has potential applications for studying the origin of any embryonic organs in zebrafish and other model organisms.

Instantaneous non-diffracting light-sheet generation by controlling spatial coherence

We demonstrate single-shot non-diffracting light-sheet generation by controlling the spatial coherence of light. A one-dimensional coherent beam, created by either increasing the spatial coherence of an LED or decreasing the spatial coherence of a laser, makes it unnecessary to scan non-diffracting beams to generate light-sheets. We theoretically and experimentally demonstrate the equivalence between our method and a scanned light-sheet, and investigate the characteristics of the light-sheet in detail. Our method is easily implementable and universally applicable for high-resolution multicolor light-sheet fluorescence imaging.

Efficient and cost-effective 3D cellular imaging by sub-voxel-resolving light-sheet add-on microscopy.

Light-sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high-speed and with low photo-toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub-voxel-resolving (SVR) light-sheet add-on microscopy (SLAM), is presented to enable fast, resolution-enhanced light-sheet fluorescence imaging from a conventional wide-field microscope. This method contains two components: a miniature add-on device to regular wide-field microscopes, which contains a horizontal laser light-sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off-axis scanning strategy and a SVR algorithm that utilizes sub-voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost-effective solution to convert the vast number of in-service microscopes for fast 3D live imaging with voxel-super-resolved capability.

Efficient and Cost-Effective 3D Cellular Imaging by Sub-Voxel-Resolving Light-Sheet Add-On Microscopy

Light-sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high-speed and with low photo-toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub-voxel-resolving light-sheet add-on microscopy (SLAM), is presented to enable fast, resolution-enhanced light-sheet fluorescence imaging from a conventional wide-field microscope. This method contains two components: a miniature add-on device to regular wide-field microscopes, which contains a horizontal laser light-sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off-axis scanning strategy and a sub-voxel-resolving (SVR) algorithm that utilizes sub-voxel spatial shifts to reconstruct the image volume that results in a two-fold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost-effective solution to convert the vast number of in-service microscopes for fast 3D live volume imaging with voxel-super-resolved capability.

Holistic Monte-Carlo optical modelling of biological imaging

The invention and advancement of biological microscopy depends critically on an ability to accurately simulate imaging of complex biological structures embedded within complex scattering media. Unfortunately no technique exists for rigorous simulation of the complete imaging process, including the source, instrument, sample and detector. Monte-Carlo modelling is the gold standard for the modelling of light propagation in tissue, but is somewhat laborious to implement and does not incorporate the rejection of scattered light by the microscope. On the other hand microscopes may be rigorously and rapidly modelled using commercial ray-tracing software, but excluding the interaction with the biological sample. We report a hybrid Monte-Carlo optical ray-tracing technique for modelling of complete imaging systems of arbitrary complexity. We make the software available to enable user-friendly and rigorous virtual prototyping of biological microscopy of arbitrary complexity involving light scattering, fluorescence, polarised light propagation, diffraction and coherence. Examples are presented for the modelling and optimisation of representative imaging of neural cells using light-sheet and micro-endoscopic fluorescence microscopy and imaging of retinal vasculature using confocal and non-confocal scanning-laser ophthalmoscopes.

Fabrication of YAG/Cr:YAG transparent composite ceramics and characterization by light sheet fluorescence imaging

Transparent composite ceramics based on YAG/Cr:YAG bilayer samples were manufactured by the solid-state reaction route and sequential slip-casting technique. They were characterized by light sheet fluorescence imaging (LSFI). This work shows that LSFI allows determining the spatial distribution of chromium with different valence states (Cr4+ and Cr3+) with a resolution close to 15 μm and high sensitivity in regards to the concentration. LSFI thus appears well adapted to the characterization of transparent ceramic materials with luminescent dopant gradient used as solid-state lasers optical components.

On-chip light-sheet fluorescence imaging flow cytometry at a high flow speed of 1 m/s.

We present on-chip fluorescence imaging flow cytometry by light-sheet excitation on a mirror-embedded microfluidic chip. The method allows us to obtain microscopy-grade fluorescence images of cells flowing at a high speed of 1 m/s, which is comparable to the flow speed of conventional non-imaging flow cytometers. To implement the light-sheet excitation of flowing cells in a microchannel, we designed and fabricated a mirror-embedded PDMS-based microfluidic chip. To show its broad utility, we used the method to classify large populations of microalgal cells (Euglena gracilis) and human cancer cells (human adenocarcinoma cells). Our method holds promise for large-scale single-cell analysis.

Note: Eliminating stripe artifacts in light-sheet fluorescence imaging

We report two techniques to mitigate stripe artifacts in light-sheet fluorescence imaging. The first uses an image processing algorithm called the multidirectional stripe remover method to filter stripes from an existing image. The second uses an elliptical holographic diffuser with strong scattering anisotropy to prevent stripe formation during image acquisition. These techniques facilitate accurate interpretation of image data, especially in denser samples. They are also facile and cost-effective.

Dynamic light sheet generation and fluorescence imaging behind turbid media

BackgroundLight sheet microscopy became a popular tool allowing fast imaging with reduced out of focus light. However, when light penetrates turbid media such as biological tissues, multiple scattering scrambles the illumination into a speckle pattern and severely challenges conventional fluorescence imaging with focused light or with a light sheet. In this article, we present generation of light sheet type illumination patterns despite scattering.MethodsWe optimize the wave-front of the incoming light to transform the speckle pattern behind the scattering layer into a light sheet within the region of interest. We utilize a fast spatial light modulator for phase modulation and a genetic optimization algorithm. The light pattern behind the scattering layer is detected via a clear detection path and acts as a feedback signal for the algorithm.ResultsWe enabled homogenous light sheet illumination behind turbid media and enhanced the signal of fluorescent beads selectively at the desired focal plane up to eight times on average. The technique is capable to compensate the dynamic changes of the speckle pattern as well, as shown on samples consisting of living drosophila pupae.ConclusionOur technique shows that not only single foci, but also a homogenous light sheet illumination can directly be created and maintained behind static and dynamic scattering media. To make the technique suitable for common biological settings, where the detection path is turbid as well, a fluorescent probe can be used to provide the feedback signal.