Light-sheet Microscopy Research Articles

Rapid reconstruction of neural circuits using tissue expansion and light sheet microscopy.

Brain function is mediated by the physiological coordination of a vast, intricately connected network of molecular and cellular components. The physiological properties of neural network components can be quantified with high throughput. The ability to assess many animals per study has been critical in relating physiological properties to behavior. By contrast, the synaptic structure of neural circuits is presently quantifiable only with low throughput. This low throughput hampers efforts to understand how variations in network structure relate to variations in behavior. For neuroanatomical reconstruction, there is a methodological gulf between electron microscopic (EM) methods, which yield dense connectomes at considerable expense and low throughput, and light microscopic (LM) methods, which provide molecular and cell-type specificity at high throughput but without synaptic resolution. To bridge this gulf, we developed a high-throughput analysis pipeline and imaging protocol using tissue expansion and light sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many animals with single-synapse resolution and molecular contrast. Using Drosophila to validate this approach, we demonstrate that it yields synaptic counts similar to those obtained by EM, enables synaptic connectivity to be compared across sex and experience, and can be used to correlate structural connectivity, functional connectivity, and behavior. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.

Resolution doubling in light-sheet microscopy via oblique plane structured illumination.

Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photobleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle three-dimensional (3D) imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, an LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150 nm, combined with lower phototoxicity compared to traditional SIM systems and volumetric acquisition speed exceeding 1 Hz.

Doubling the resolution of light-sheet fluorescence microscopy.

Doubling the resolution of light-sheet fluorescence microscopy.

Practical considerations for quantitative light sheet fluorescence microscopy.

Fluorescence microscopy has evolved from a purely observational tool to a platform for quantitative, hypothesis-driven research. As such, the demand for faster and less phototoxic imaging modalities has spurred a rapid growth in light sheet fluorescence microscopy (LSFM). By restricting the excitation to a thin plane, LSFM reduces the overall light dose to a specimen while simultaneously improving image contrast. However, the defining characteristics of light sheet microscopes subsequently warrant unique considerations in their use for quantitative experiments. In this Perspective, we outline many of the pitfalls in LSFM that can compromise analysis and confound interpretation. Moreover, we offer guidance in addressing these caveats when possible. In doing so, we hope to provide a useful resource for life scientists seeking to adopt LSFM to quantitatively address complex biological hypotheses.

Axial resolution and imaging contrast enhancement in inverted light-sheet microscopy by natural illumination modulation.

Inverted light-sheet microscopy (ILSM) is widely employed for fast large-volume imaging of biological tissue. However, the scattering especially in an uncleared sample, and the divergent propagation of the illumination beam lead to a trade-off between axial resolution and imaging depth. Herein, we propose naturally modulated ILSM (NM-ILSM) as a technique to improve axial resolution while simultaneously maintaining the wide field-of-view (FOV), and enhancing imaging contrast via background suppression. Theoretical derivations, simulations, and experimental imaging demonstrate 15% axial resolution increases, and fivefold greater image contrast compared with conventional ILSM. Therefore, NM-ILSM allows convenient imaging quality improvement for uncleared tissue and could extend the biological application scope of ILSM.

Extended depth-of-field light-sheet microscopy improves imaging of large volumes at high numerical aperture.

Light-sheet microscopes must compromise among field of view, optical sectioning, resolution, and detection efficiency. High-numerical-aperture (NA) detection objective lenses provide higher resolution, but their narrow depth of field inefficiently captures the fluorescence signal generated throughout the thickness of the illumination light sheet when imaging large volumes. Here, we present ExD-SPIM (extended depth-of-field selective-plane illumination microscopy), an improved light-sheet microscopy strategy that solves this limitation by extending the depth of field (DOF) of high-NA detection objectives to match the thickness of the illumination light sheet. This extension of the DOF uses a phase mask to axially stretch the point-spread function of the objective lens while largely preserving lateral resolution. This matching of the detection DOF to the illumination-sheet thickness increases the total fluorescence collection, reduces the background, and improves the overall signal-to-noise ratio (SNR), as shown by numerical simulations, imaging of bead phantoms, and imaging living animals. In comparison to conventional light sheet imaging with low-NA detection that yields equivalent DOF, the results show that ExD-SPIM increases the SNR by more than threefold and dramatically reduces the rate of photobleaching. Compared to conventional high-NA detection, ExD-SPIM improves the signal sensitivity and volumetric coverage of whole-brain activity imaging, increasing the number of detected neurons by over a third.

Single-molecule tracking of Nodal and Lefty in live zebrafish embryos supports hindered diffusion model

The hindered diffusion model postulates that the movement of a signaling molecule through an embryo is affected by tissue geometry and binding-mediated hindrance, but these effects have not been directly demonstrated in vivo. Here, we visualize extracellular movement and binding of individual molecules of the activator-inhibitor signaling pair Nodal and Lefty in live developing zebrafish embryos using reflected light-sheet microscopy. We observe that diffusion coefficients of molecules are high in extracellular cavities, whereas mobility is reduced and bound fractions are high within cell-cell interfaces. Counterintuitively, molecules nevertheless accumulate in cavities, which we attribute to the geometry of the extracellular space by agent-based simulations. We further find that Nodal has a larger bound fraction than Lefty and shows a binding time of tens of seconds. Together, our measurements and simulations provide direct support for the hindered diffusion model and yield insights into the nanometer-to-micrometer-scale mechanisms that lead to macroscopic signal dispersal.

Adaptive optics in single objective inclined light sheet microscopy enables three-dimensional localization microscopy in adult Drosophila brains.

Single-molecule localization microscopy (SMLM) enables the high-resolution visualization of organelle structures and the precise localization of individual proteins. However, the expected resolution is not achieved in tissue as the imaging conditions deteriorate. Sample-induced aberrations distort the point spread function (PSF), and high background fluorescence decreases the localization precision. Here, we synergistically combine sensorless adaptive optics (AO), in-situ 3D-PSF calibration, and a single-objective lens inclined light sheet microscope (SOLEIL), termed (AO-SOLEIL), to mitigate deep tissue-induced deteriorations. We apply AO-SOLEIL on several dSTORM samples including brains of adult Drosophila. We observed a 2x improvement in the estimated axial localization precision with respect to widefield without aberration correction while we used synergistic solution. AO-SOLEIL enhances the overall imaging resolution and further facilitates the visualization of sub-cellular structures in tissue.

Rationalized deep learning super-resolution microscopy for sustained live imaging of rapid subcellular processes.

The goal when imaging bioprocesses with optical microscopy is to acquire the most spatiotemporal information with the least invasiveness. Deep neural networks have substantially improved optical microscopy, including image super-resolution and restoration, but still have substantial potential for artifacts. In this study, we developed rationalized deep learning (rDL) for structured illumination microscopy and lattice light sheet microscopy (LLSM) by incorporating prior knowledge of illumination patterns and, thereby, rationally guiding the network to denoise raw images. Here we demonstrate that rDL structured illumination microscopy eliminates spectral bias-induced resolution degradation and reduces model uncertainty by five-fold, improving the super-resolution information by more than ten-fold over other computational approaches. Moreover, rDL applied to LLSM enables self-supervised training by using the spatial or temporal continuity of noisy data itself, yielding results similar to those of supervised methods. We demonstrate the utility of rDL by imaging the rapid kinetics of motile cilia, nucleolar protein condensation during light-sensitive mitosis and long-term interactions between membranous and membrane-less organelles.

Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy.

Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization1. Aging or presbycusis is a primary cause of sensorineural hearing loss and is characterized by damage to hair cells, spiral ganglion neurons (SGNs), and the stria vascularis. These structures reside within the cochlea, which has a complex, spiral-shaped anatomy of membranous tissues suspended in fluid and surrounded by bone. These properties make it technically difficult to investigate and quantify histopathological changes. To address this need, we developed a light-sheet microscope (TSLIM) that can image and digitize the whole cochlea to facilitate the study of structure-function relationships in the inner ear. Well-aligned serial sections of the whole cochlea result in a stack of images for three-dimensional (3D) volume rendering and segmentation of individual structures for 3D visualization and quantitative analysis (i.e., length, width, surface, volume, and number). Cochleae require minimal processing steps (fixation, decalcification, dehydration, staining, and optical clearing), all of which are compatible with subsequent high-resolution imaging by scanning and transmission electron microscopy. Since all the tissues are present in the stacks, each structure can be assessed individually or relative to other structures. In addition, since imaging uses fluorescent probes, immunohistochemistry and ligand binding can be used to identify specific structures and their 3D volume or distribution within the cochlea. Here we used TSLIM to examine cochleae from aged mice to quantify the loss of hair cells and spiral ganglion neurons. In addition, advanced analyses (e.g., cluster analysis) were used to visualize local reductions of spiral ganglion neurons in Rosenthal's canal along its 3D volume. These approaches demonstrate TSLIM microscopy's ability to quantify structure-function relationships within and between cochleae.

Airy beam assisted NIR-II light-sheet microscopy

Near-infrared II (1000–1700 nm) three-dimensional (3D) microscopy addresses a significant improvement on the penetration depth for bioimaging, but is challenging in high z-direction spatial resolution, leading to imaging distortion in deep tissue bioimaging. Here, we developed Airy beam-assisted NIR-II light-sheet Microscope (NIR-II Airy LSM) to improve z-direction spatial resolution with large field of view (FOV) on thick biological specimens via self-healing properties of the propagation-invariant field. The axial resolution was measured to be consistently 3.5 µm across the whole FOV (∼ 600 µm), about 4 times higher than traditional Gaussian-beam based microscopy (∼ 15 µm). Besides, the NIR-II Airy LSM demonstrates 2–3 times higher signal background ratio (SBR) than NIR-I imaging, at the penetration depth of approximately 2.5 mm. The increased spatial resolution and SBR allows us to observe pathological conditions and progression of glomeruli and tubules of radiation induced kidney injury and discriminated integral damage degree of intestinal mucosal cells from intestinal tissue accurately via 3D volumetric multiplexed imaging. The improved NIR-II light-sheet microscopy exhibit complete 3D imaging capabilities of whole bio tissues without sacrificing axial resolution and avoid imaging distortion under deep tissue.

Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy

Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization1. Aging or presbycusis is a primary cause of sensorineural hearing loss and is characterized by damage to hair cells, spiral ganglion neurons (SGNs), and the stria vascularis. These structures reside within the cochlea, which has a complex, spiral-shaped anatomy of membranous tissues suspended in fluid and surrounded by bone. These properties make it technically difficult to investigate and quantify histopathological changes. To address this need, we developed a light-sheet microscope (TSLIM) that can image and digitize the whole cochlea to facilitate the study of structure-function relationships in the inner ear. Well-aligned serial sections of the whole cochlea result in a stack of images for three-dimensional (3D) volume rendering and segmentation of individual structures for 3D visualization and quantitative analysis (i.e., length, width, surface, volume, and number). Cochleae require minimal processing steps (fixation, decalcification, dehydration, staining, and optical clearing), all of which are compatible with subsequent high-resolution imaging by scanning and transmission electron microscopy. Since all the tissues are present in the stacks, each structure can be assessed individually or relative to other structures. In addition, since imaging uses fluorescent probes, immunohistochemistry and ligand binding can be used to identify specific structures and their 3D volume or distribution within the cochlea. Here we used TSLIM to examine cochleae from aged mice to quantify the loss of hair cells and spiral ganglion neurons. In addition, advanced analyses (e.g., cluster analysis) were used to visualize local reductions of spiral ganglion neurons in Rosenthal's canal along its 3D volume. These approaches demonstrate TSLIM microscopy's ability to quantify structure-function relationships within and between cochleae.

Integration of mouse ovary morphogenesis with developmental dynamics of the oviduct, ovarian ligaments, and rete ovarii.

Morphogenetic events during the development of the fetal ovary are crucial to the establishment of female fertility. However, the effects of structural rearrangements of the ovary and surrounding reproductive tissues on ovary morphogenesis remain largely uncharacterized. Using tissue clearing and lightsheet microscopy, we found that ovary folding correlated with regionalization into cortex and medulla. Relocation of the oviduct to the ventral aspect of the ovary led to ovary encapsulation, and mutual attachment of the ovary and oviduct to the cranial suspensory ligament likely triggered ovary folding. During this process, the rete ovarii (RO) elaborated into a convoluted tubular structure extending from the ovary into the ovarian capsule. Using genetic mouse models in which the oviduct and RO are perturbed, we found the oviduct is required for ovary encapsulation. This study reveals novel relationships among the ovary and surrounding tissues and paves the way for functional investigation of the relationship between architecture and differentiation of the mammalian ovary.

Three-dimensional visualization of cerebral blood vessels and neural changes in thick ischemic rat brain slices using tissue clearing

Blood vessels are three-dimensional (3D) in structure and precisely connected. Conventional histological methods are unsuitable for their analysis because of the destruction of functionally important topological 3D vascular structures. Tissue optical clearing techniques enable extensive volume imaging and data analysis without destroying tissue. This study therefore applied a tissue clearing technique to acquire high-resolution 3D images of rat brain vasculature using light-sheet and confocal microscopies. Rats underwent middle cerebral artery occlusion for 45 min followed by 24 h reperfusion with lectin injected directly into the heart for vascular staining. For acquiring 3D images of rat brain vasculature, 3-mm-thick brain slices were reconstructed using tissue clearing and light-sheet microscopy. Subsequently, after 3D rendering, the fitting of blood vessels to a filament model was used for analysis. The results revealed a significant reduction in vessel diameter and density in the ischemic region compared to those in contralesional non-ischemic regions. Immunostaining of 0.5-mm-thick brain slices revealed considerable neuronal loss and increased astrocyte fluorescence intensity in the ipsilateral region. Thus, these methods can provide more accurate data by broadening the scope of the analyzed regions of interest for examining the 3D cerebrovascular system and neuronal changes occurring in various brain disorders.

Modeling Biochemical Gradients In Vitro to Control Cell Compartmentalization in a Microengineered 3D Model of the Intestinal Epithelium.

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they sensed by the epithelium is still not fully understood. Here a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and precisely controlled biomolecular gradients of the ISC niche (v) is presented. Biochemical gradients are formed through hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of spatiotemporally controlled gradients, light-sheet fluorescence microscopy and in-silico modeling are employed. The ISC niche biochemical gradients coming from the stroma and applied along the villus axis lead to the in vivo-like compartmentalization of the proliferative and differentiated cells, while changing the composition and concentration of the biochemical factors affects the cellular organization along the villus axis. This novel 3D in vitro intestinal model derived from organoids recapitulates both the villus-like architecture and the gradients of ISC biochemical factors, thus opening the possibility to study in vitro the nature of such gradients and the resulting cellular response.

Circulatory disturbance of the cochlear spiral modiolar artery in a type 2 diabetic mouse model.

ObjectiveThis study aimed to identify significant differences in cochlea microvessel size between a diabetic mouse model (db/db) and normal mice using three‐dimensional (3D) quantitative analysis.MethodsSix control heterozygote db/+ as well as 18 male B6/BKS(D)‐Leprdb/J (db/db) mice aged 14 (n = 9) and 28 (n = 9) weeks were examined. After clearing the cochlea, we reconstructed the 3D volumes of the spiral modiolar artery (SMA) in the cochlea using light‐sheet microscopy and analyzed vessel wall thickness, cross‐sectional area, short and long diameter, and vessel height.ResultsThe average SMA‐wall thickness in the db/db‐mouse group (3.418 ± 0.328 μm) was greater than that in the control group (2.388 ± 0.411 μm). The average cross‐sectional outer area, short diameter, and long diameter of the SMA in db/db mice were significantly larger than those in control mice (all p < 0.001). The cross‐sectional areas increased with age (control: 221.782 ± 121.230 μm, 14 weeks; 294.378 ± 151.008 μm, and 28 weeks; 312.925 ± 147.943 μm).ConclusionThe db/db mice had thicker and larger proximal‐SMA vessel walls and diameters than control mice, respectively, thus potentially inducing increased blood viscosity or vascular insufficiency and aggravating hearing loss in type 2 diabetes mellitus.Level of EvidenceIIb

A proliferative to invasive switch is mediated by srGAP1 downregulation through the activation of TGF-β2 signaling.

SUMMARYMany breast cancer (BC) patients suffer from complications of metastatic disease. To form metastases, cancer cells must become migratory and coordinate both invasive and proliferative programs at distant organs. Here, we identify srGAP1 as a regulator of a proliferative-to-invasive switch in BC cells. High-resolution light-sheet microscopy demonstrates that BC cells can form actin-rich protrusions during extravasation. srGA-P1low cells display a motile and invasive phenotype that facilitates their extravasation from blood vessels, as shown in zebrafish and mouse models, while attenuating tumor growth. Interestingly, a population of srGAP1low cells remain as solitary disseminated tumor cells in the lungs of mice bearing BC tumors. Overall, srGAP1low cells have increased Smad2 activation and TGF-β2 secretion, resulting in increased invasion and p27 levels to sustain quiescence. These findings identify srGAP1 as a mediator of a proliferative to invasive phenotypic switch in BC cells in vivo through a TGF-β2-mediated signaling axis.

Axial scanning of dual focus to improve light sheet microscopy.

Axially swept light sheet microscopy (ASLM) is an emerging technique that enables isotropic, subcellular resolution imaging with high optical sectioning capability over a large field-of-view (FOV). Due to its versatility across a broad range of immersion media, it has been utilized to image specimens that may range from live cells to intact chemically cleared organs. However, because of its design, the performance of ASLM-based microscopes is impeded by a low detection signal and the maximum achievable frame-rate for full FOV imaging. Here we present a new optical concept that pushes the limits of ASLM further by scanning two staggered light sheets and simultaneously synchronizing the rolling shutter of a scientific camera. For a particular peak-illumination-intensity, this idea can make ASLMs image twice as fast without compromising the detection signal. Alternately, for a particular frame rate our method doubles the detection signal without requiring to double the peak-illumination-power, thereby offering a gentler illumination scheme compared to tradition single-focus ASLM. We demonstrate the performance of our instrument by imaging fluorescent beads and a PEGASOS cleared-tissue mouse brain.

Acid sphingomyelinase deactivation post-ischemia promotes brain angiogenesis and remodeling by small extracellular vesicles

Antidepressants have been reported to enhance stroke recovery independent of the presence of depressive symptoms. They have recently been proposed to exert their mood-stabilizing actions by inhibition of acid sphingomyelinase (ASM), which catalyzes the hydrolysis of sphingomyelin to ceramide. Their restorative action post-ischemia/reperfusion (I/R) still had to be defined. Mice subjected to middle cerebral artery occlusion or cerebral microvascular endothelial cells exposed to oxygen–glucose deprivation were treated with vehicle or with the chemically and pharmacologically distinct antidepressants amitriptyline, fluoxetine or desipramine. Brain ASM activity significantly increased post-I/R, in line with elevated ceramide levels in microvessels. ASM inhibition by amitriptyline reduced ceramide levels, and increased microvascular length and branching point density in wildtype, but not sphingomyelinase phosphodiesterase-1 ([Smpd1]−/−) (i.e., ASM-deficient) mice, as assessed by 3D light sheet microscopy. In cell culture, amitriptyline, fluoxetine, and desipramine increased endothelial tube formation, migration, VEGFR2 abundance and VEGF release. This effect was abolished by Smpd1 knockdown. Mechanistically, the promotion of angiogenesis by ASM inhibitors was mediated by small extracellular vesicles (sEVs) released from endothelial cells, which exhibited enhanced uptake in target cells. Proteomic analysis of sEVs revealed that ASM deactivation differentially regulated proteins implicated in protein export, focal adhesion, and extracellular matrix interaction. In vivo, the increased angiogenesis was accompanied by a profound brain remodeling response with increased blood–brain barrier integrity, reduced leukocyte infiltrates and increased neuronal survival. Antidepressive drugs potently boost angiogenesis in an ASM-dependent way. The release of sEVs by ASM inhibitors disclosed an elegant target, via which brain remodeling post-I/R can be amplified.

Optogenetic manipulation of cell migration with high spatiotemporal resolution using lattice lightsheet microscopy

Lattice lightsheet microscopy (LLSM) featuring three-dimensional recording is improved to manipulate cellular behavior with subcellular resolution through optogenetic activation (optoLLSM). A position-controllable Bessel beam as a stimulation source is integrated into the LLSM to achieve spatiotemporal photoactivation by changing the spatial light modulator (SLM) patterns. Unlike the point-scanning in a confocal microscope, the lattice beams are capable of wide-field optical sectioning for optogenetic activation along the Bessel beam path.We show that the energy power required for optogenetic activations is lower than 1 nW (or 24 mWcm-2) for time-lapses of CRY2olig clustering proteins, and membrane ruffling can be induced at different locations within a cell with subcellular resolution through light-triggered recruitment of phosphoinositide 3-kinase. Moreover, with the epidermal growth factor receptor (EGFR) fused with CRY2olig, we are able to demonstrate guided cell migration using optogenetic stimulation for up to 6 h, where 463 imaging volumes are collected, without noticeable cellular damages.