Light Sheet Fluorescence Microscopy Research Articles

SPIM-Flow: An Integrated Light Sheet and Microfluidics Platform for Hydrodynamic Studies of Hydra.

Selective plane illumination microscopy (SPIM), or light sheet microscopy, is a powerful imaging approach. However, access to and interfacing microscopes with microfluidics have remained challenging. Complex interfacing with microfluidics has limited the SPIM's utility for studying the hydrodynamics of freely moving multicellular organisms. We developed SPIM-Flow, an inexpensive light sheet platform that enables easy integration with microfluidics. We used SPIM-Flow to investigate the hydrodynamics of a freely moving Hydra polyp via particle tracking in millimeter-sized chambers. Initial experiments across multiple animals, feeding on a chip (Artemia franciscana nauplii used as food), and baseline behaviors (tentacle swaying, elongation, and bending) indicated the organisms' health inside the system. Fluidics were used to investigate Hydra's response to flow. The results suggested that the animals responded to an established flow by bending and swaying their tentacles in the flow direction. Finally, using SPIM-Flow in a proof-of-concept experiment, the shear stress required to detach an animal from a surface was demonstrated. Our results demonstrated SPIM-Flow's utility for investigating the hydrodynamics of freely moving animals.

Oblique plane microscope for mesoscopic imaging of freely moving organisms with cellular resolution.

Several important questions in biology require non-invasive and three-dimensional imaging techniques with an appropriate spatiotemporal resolution that permits live organisms to move in an unconstrained fashion over an extended field-of-view. While selective-plane illumination microscopy (SPIM) has emerged as a powerful method to observe live biological specimens at high spatio-temporal resolution, typical implementations often necessitate constraining sample mounting or lack the required volumetric speed. Here, we report on an open-top, dual-objective oblique plane microscope (OPM) capable of observing millimeter-sized, freely moving animals at cellular resolution. We demonstrate the capabilities of our mesoscopic OPM (MesOPM) by imaging the behavioral dynamics of the sea anemone Nematostella vectensis over 1.56 × 1.56 × 0.25 mm at 1.5 × 2.8 × 5.3 µm resolution and 0.5 Hz volume rate.

Linearly modulated multi-focal diffractive lens for multi-sheet excitation of flow driven samples in a light-sheet fluorescence microscope

Light sheet fluorescence microscope with single light sheet illumination enables rapid 3D imaging of living cells. In this paper we show the design, fabrication and characterization of a diffractive optical element producing several light sheets along a 45° inclined tube. The element, which is based on a multi-focal diffractive lens and a linear grating, generates five thin light sheets with equal intensities when combined with a refractive cylindrical lens. The generated uniform light sheets can be applied for the scanning of samples in tubes enabling flow-driven 3-dimensional imaging.

Imaging of calcium gradient oscillations in plant root hairs by light sheet fluorescence microscopy

Root hairs are a delicate single-cell system whose growth is regulated by a fine mechanism characterised by the presence of a tip-high Ca2+ gradient that shows regular oscillations in growing root hairs. We show a method based on the use of Light sheet fluorescence microscopy (LSFM) which allows the quasi-physiological analysis of Arabidopsis thaliana plant roots hairs with excellent spatial and temporal resolution over a wide field of view. We show how the healthy growing root hairs are linked to precise oscillations and how a disruption of this mechanism can be associated to specific genes.

Selective plane illumination optical endomicroscopy with polymer imaging fibers

Imaging fibers are used to perform real-time fluorescence endomicroscopy, in vivo, in situ, with the goal of increasing diagnostic information for a plethora of organ systems and diseases. Widefield fiber endomicroscopy systems are simple, cost-effective, and come with fast image acquisition times. However, alternative approaches such as scanning systems produce higher contrast images with intrinsic optical sectioning, improving the visibility of histological features, albeit at the expense of simplicity, cost, and acquisition rate. We developed a selective plane illumination microscopy endoscopic fiber platform, consisting of an ultrafast laser fabricated end-cap, integrated with a polymer coherent fiber bundle, and an epifluorescence microscope. Polymer fibers are known to fluoresce when pumped with blue light, enhancing the background and noise in images. Our end-cap design circumvents this challenge. We demonstrate a reduction of out-of-focus features, along with improved contrast of in-focus features, in images of a tissue phantom. Moreover, we demonstrate the utility of our platform for endomicroscopy using a whole, ex vivo human lung model.

Multiple airy beams light-sheet fluorescence microscopy

Light-sheet fluorescence microscopy (LSFM) is a kind of volumetric imaging methodology suited for long term living specimens at high temporal-spatial resolution. A single Airy beam (SAB) light-sheet can extend the field of view of Light-sheet fluorescence microscopy benefiting from its non-diffracting nature, but at the cost of out-of-focus background and low imaging contrast caused by side lobes illumination. Here, we propose a method to generate a sort of multiple Airy beams (MAB), which are linearly superimposed of multiple single Airy beams with different scale factors. Compared to the SAB light-sheet, the energy of the multiple Airy beams light-sheet is more concentrated on the focal plane of the detection objective, which can improve the imaging contrast and decrease the photodamage effect. Furthermore, we combined the complementary beam subtraction (CBS) strategy to increase the axial resolution, termed as multiple Airy beams-complementary beam subtraction method, which enables the axial resolution of 1.2 μm while keeping the field of view of 450 μm × 450 μm. The effectiveness of the method is demonstrated by imaging of fluorescent beads and aspergillus conidiophores.

Three-dimensional Imaging Reveals Immune-driven Tumor-associated High Endothelial Venules as a Key Correlate of Tumor Rejection Following Depletion of Regulatory T Cells.

High endothelial venules (HEV) are specialized post capillary venules that recruit naïve T cells and B cells into secondary lymphoid organs (SLO) such as lymph nodes (LN). Expansion of HEV networks in SLOs occurs following immune activation to support development of an effective immune response. In this study, we used a carcinogen-induced model of fibrosarcoma to examine HEV remodeling after depletion of regulatory T cells (Treg). We used light sheet fluorescence microscopy imaging to visualize entire HEV networks, subsequently applying computational tools to enable topological mapping and extraction of numerical descriptors of the networks. While these analyses revealed profound cancer- and immune-driven alterations to HEV networks within LNs, these changes did not identify successful responses to treatment. The presence of HEV networks within tumors did however clearly distinguish responders from nonresponders. Finally, we show that a successful treatment response is dependent on coupling tumor-associated HEV (TA-HEV) development to T-cell activation implying that T-cell activation acts as the trigger for development of TA-HEVs which subsequently serve to amplify the immune response by facilitating extravasation of T cells into the tumor mass.

3D Optical Molecular Imaging of the Rodent Pancreas by OPT and LSFM.

The rodent pancreas is the prevalent model system for preclinical diabetes research. However, due to the compound endocrine-exocrine organization of the gland, with the endocrine islets of Langerhans scattered by the thousands throughout the much greater exocrine parenchyma, stereological assessments of endocrine cell mass, commonly insulin-producing ß-cells, are exceedingly challenging. In recent years, optical mesoscopic imaging techniques such as optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) have seen dramatic developments, enabling 3D visualization of fluorescently labeled cells in mm- to cm-sized tissues with μm resolution. Here we present a protocol for 3D visualization and "absolute" quantitative assessments of, for example, islet mass throughout the volume of rodent pancreata with maintained spatial context.

Laterally swept light-sheet microscopy enhanced by pixel reassignment for photon-efficient volumetric imaging

In light-sheet fluorescence microscopy, the axial resolution and field of view are mutually constrained. Axially swept light-sheet microscopy (ASLM) can decouple the trade-off, but the confocal detection scheme using a rolling shutter also rejects fluorescence signals from the specimen in the field of interest, which sacrifices the photon efficiency. Here, we report a laterally swept light-sheet microscopy (LSLM) scheme in which the focused beam is first scanned along the axial direction and subsequently laterally swept with the rolling shutter. We show that LSLM can obtain a higher photon efficiency when similar axial resolution and field of view can be achieved. Moreover, based on the principle of image scanning microscopy, applying the pixel reassignment to the LSLM images, hereby named iLSLM, improves the optical sectioning. Both simulation and experimental results demonstrate the higher photon efficiency with similar axial resolution and optical sectioning. Our proposed scheme is suitable for volumetric imaging of specimens that are susceptible to photobleaching or phototoxicity.

Single Plane Illumination Microscopy for Microfluidic Device Imaging

Three-dimensional imaging of live processes at a cellular level is a challenging task. It requires high-speed acquisition capabilities, low phototoxicity, and low mechanical disturbances. Three-dimensional imaging in microfluidic devices poses additional challenges as a deep penetration of the light source is required, along with a stationary setting, so the flows are not perturbed. Different types of fluorescence microscopy techniques have been used to address these limitations; particularly, confocal microscopy and light sheet fluorescence microscopy (LSFM). This manuscript proposes a novel architecture of a type of LSFM, single-plane illumination microscopy (SPIM). This custom-made microscope includes two mirror galvanometers to scan the sample vertically and reduce shadowing artifacts while avoiding unnecessary movement. In addition, two electro-tunable lenses fine-tune the focus position and reduce the scattering caused by the microfluidic devices. The microscope has been fully set up and characterized, achieving a resolution of 1.50 μm in the x-y plane and 7.93 μm in the z-direction. The proposed architecture has risen to the challenges posed when imaging microfluidic devices and live processes, as it can successfully acquire 3D volumetric images together with time-lapse recordings, and it is thus a suitable microscopic technique for live tracking miniaturized tissue and disease models.

Polarization effects on the fluorescence emission of zebrafish neurons using light-sheet microscopy.

Light-sheet fluorescence microscopy (LSFM) makes use of a thin plane of light to optically section and image transparent tissues or organisms in vivo, which has the advantages of fast imaging speed and low phototoxicity. In this paper, we have employed light-sheet microscopy to investigate the polarization effects on fluorescence emission of zebrafish neurons via modifying the electric oscillation orientation of the excitation light. The intensity of the fluorescence emission from the excited zebrafish larvae follows a cosine square function with respect to the polarization state of the excitation light and reveals a 40% higher fluorescence emission when the polarization orientation is orthogonal to the illumination and detection axes. Through registration and subtraction of fluorescence images under different polarization states, we have demonstrated that most of the enhanced fluorescence signals are from the neuronal cells rather than the extracellular substance. This provides us a way to distinguish the cell boundaries and observe the organism structures with improved contrast and resolution.

Evaluation and manipulation of tissue and cellular distribution of cardiac progenitor cell-derived extracellular vesicles.

Cardiac progenitor cell-derived extracellular vesicles (CPC-EVs) have been successfully applied via different delivery routes for treating post-myocardial infarction injury in several preclinical models. Hence, understanding the in vivo fate of CPC-EVs after systemic or local, i.e. myocardial, delivery is of utmost importance for the further therapeutic application of CPC-EVs in cardiac repair. Here, we studied the tissue- and cell distribution and retention of CPC-EVs after intramyocardial and intravenous injection in mice by employing different EV labeling and imaging techniques. In contrast to progenitor cells, CPC-EVs demonstrated no immediate flush-out from the heart upon intramyocardial injection and displayed limited distribution to other organs over time, as determined by near-infrared imaging in living animals. By employing CUBIC tissue clearing and light-sheet fluorescent microscopy, we observed CPC-EV migration in the interstitial space of the myocardium shortly after EV injection. Moreover, we demonstrated co-localization with cTnI and CD31-positive cells, suggesting their interaction with various cell types present in the heart. On the contrary, after intravenous injection, most EVs accumulated in the liver. To potentiate such a potential systemic cardiac delivery route, targeting the cardiac endothelium could provide openings for directed CPC-EV therapy. We therefore evaluated whether decorating EVs with targeting peptides (TPs) RGD-4C or CRPPR connected to Lamp2b could enhance EV delivery to endothelial cells. Expression of both TPs enhanced CPC-EV uptake under in vitro continuous flow, but did not affect uptake under static cell culture conditions. Together, these data demonstrate that the route of administration influences CPC-EV biodistribution pattern and suggest that specific TPs could be used to target CPC-EVs to the cardiac endothelium. These insights might lead to a better application of CPC-EV therapeutics in the heart.

Studying the trafficking of labeled trodusquemine and its application as nerve marker for light-sheet and expansion microscopy.

Trodusquemine is an aminosterol with a variety of biological and pharmacological functions, such as acting as an antimicrobial, stimulating body weight loss and interfering with the toxicity of proteins involved in the development of Alzheimer's and Parkinson's diseases. The mechanisms of interaction of aminosterols with cells are, however, still largely uncharacterized. Here, by using fluorescently labeled trodusquemine (TRO-A594 and TRO-ATTO565), we show that trodusquemine binds initially to the plasma membrane of living cells, that the binding affinity is dependent on cholesterol, and that trodusquemine is then internalized and mainly targeted to lysosomes after internalization. We also found that TRO-A594 is able to strongly and selectively bind to myelinated fibers in fixed mouse brain slices, and that it is a marker compatible with tissue clearing and light-sheet fluorescence microscopy or expansion microscopy. In conclusion, this work contributes to further characterize the biology of aminosterols and provides a new tool for nerve labeling suitable for the most advanced microscopy techniques.

Detection and Morphological Analysis of Micro-Ruptured Cortical Arteries in Subdural Hematoma: Three-Dimensional Visualization Using the Tissue-Clearing Clear, Unobstructed, Brain/Body Imaging Cocktails and Computational Analysis Method.

One of the causes of bleeding in subdural hematoma is cortical artery rupture, which is difficult to detect at autopsy. Therefore, reports of autopsy cases with this condition are limited and hence, the pathogenesis of subdural hematoma remains unclear. Herein, for the detection and morphological analysis of cortical artery ruptures as the bleeding sources of subdural hematoma, we used the tissue-clearing CUBIC (clear, unobstructed, brain/body imaging cocktails and computational analysis) method with light-sheet fluorescence microscopy and reconstructed the two-dimensional and three-dimensional images. Using the CUBIC method, we could clearly visualize and detect cortical artery ruptures that were missed by conventional methods. Indeed, the CUBIC method enables three-dimensional morphological analysis of cortical arteries including the ruptured area, and the creation of cross-sectional two-dimensional images in any direction, which are similar to histopathological images. This highlights the effectiveness of the CUBIC method for subdural hematoma analysis.

Identification of Nodal-dependent enhancer of amphioxus Chordin sufficient to drive gene expression into the chordate dorsal organizer.

The core molecular mechanisms of dorsal organizer formation during gastrulation are highly conserved within the chordate lineage. One of the key characteristics is that Nodal signaling is required for the organizer-specific gene expression. This feature appears to be ancestral, as evidenced by the presence in the most basally divergent chordate amphioxus. To provide a better understanding of the evolution of organizer-specific gene regulation in chordates, we analyzed the cis-regulatory sequence of amphioxus Chordin in the context of the vertebrate embryo. First, we generated stable zebrafish transgenic lines, and by using light-sheet fluorescent microscopy, characterized in detail the expression pattern of GFP driven by the cis-regulatory sequences of amphioxus Chordin. Next, we performeda 5'deletion analysis and identified an enhancer sufficient to drivethe expression of the reporter gene into a chordate dorsal organizer. Finally, we found that the identified enhancer element strongly depends on Nodal signaling, which is consistent with the well-established role of this pathway in the regulation of the expression of dorsal organizer-specific genes across chordates. The enhancer identified in our study may represent a suitable simple system to study the interplay of the evolutionarily conserved regulatory mechanisms operating during early chordate development.

Hemocytes in Drosophila melanogaster embryos move via heterogeneous anomalous diffusion

Drosophila melanogaster hemocytes are highly motile cells that are crucial for successful embryogenesis and have important roles in the organism’s immunological response. Here we measure the motion of hemocytes using selective plane illumination microscopy. Every hemocyte cell in one half of an embryo is tracked during embryogenesis and analysed using a deep learning neural network. We show that the anomalous transport of the cells is well described by fractional Brownian motion that is heterogeneous in both time and space. LanB1 and SCAR mutants disrupt the collective cellular motion and reduce its persistence due to the modification of laminin and actin-based motility respectively. The anomalous motility of the hemocytes oscillated in time with alternating periods of varying persistent motion. Touching hemocytes appear to experience synchronised contact inhibition of locomotion. A quantitative statistical framework is presented for hemocyte motility which provides biological insights.

A new protocol for whole-brain biodistribution analysis of AAVs by tissue clearing, light-sheet microscopy and semi-automated spatial quantification.

Recombinant adeno-associated virus (rAAV) has become one of the most promising gene delivery systems for both in vitro and in vivo applications. However, a key challenge is the lack of suitable imaging technologies to evaluate delivery, biodistribution and tropism of rAAVs and efficiently monitor disease amelioration promoted by AAV-based therapies at a whole-organ level with single-cell resolution. Therefore, we aimed to establish a new pipeline for the biodistribution analysis of natural and new variants of AAVs at a whole-brain level by tissue clearing and light-sheet fluorescence microscopy (LSFM). To test this platform, neonatal C57BL/6 mice were intravenously injected with rAAV9 encoding EGFP and, after sacrifice, brains were processed by standard immunohistochemistry and a recently released aqueous-based clearing procedure. This clearing technique required no dedicated equipment and rendered highly cleared brains, while simultaneously preserving endogenous fluorescence. Moreover, three-dimensional imaging by LSFM allowed the quantitative analysis of EGFP at a whole-brain level, as well as the reconstruction of Purkinje cells for the retrieval of valuable morphological information inaccessible by standard immunohistochemistry. In conclusion, the pipeline herein described takes the AAVs to a new level when coupled to LSFM, proving its worth as a bioimaging tool in tropism and gene therapy studies.

Three-dimensional characterization of developing and adult ocular vasculature in mice using in toto clearing

The ocular vasculature is critically involved in many blinding diseases and is also a popular research model for the exploration of developmental and pathological angiogenesis. The development of ocular vessels is a complex, finely orchestrated sequence of events, involving spatial and temporal coordination of hyaloid, choroidal and retinal networks. Comprehensive studies of the tridimensional dynamics of microvascular remodeling are limited by the fact that preserving the spatial disposition of ocular vascular networks is cumbersome using classical histological procedures. Here, we demonstrate that light-sheet fluorescence microscopy (LFSM) of cleared mouse eyes followed by extensive virtual dissection offers a solution to this problem. To the best of our knowledge, this is the first 3D quantification of the evolution of the hyaloid vasculature and of post-occlusive venous remodeling together with the characterization of spatial distribution of various cell populations in ocular compartments, including the vitreous. These techniques will prove interesting to obtain other insights in scientific questions addressing organ-wide cell interactions.

Cochlear implant imaging in the mouse and guinea pig using light-sheet microscopy

Postmortem examination of the cochlea with a cochlear implant in the scala tympani presents several challenges. It is technologically difficult to section a cochlea with an implant due to the presence of its wires and metal components that are adjacent to the membranous and bony tissues of the cochlea. These metal components damage traditional steel blades of a microtome in celloidin, paraffin or frozen embedded tissues. However, plastic embedded implanted cochleas have been successfully sectioned using specialized methods (Irving et al., 2013). An alternative non-destructive method is to optically section a chemically cleared cochlea using light-sheet microscopy, which we will describe in this publication. However, since this method uses a light-sheet to section the cochlea the opaque and reflective metal components of the implant results in some artifacts in the 2D optical sections. The best image quality using light-sheet fluorescent microscopy is when the implant is removed prior to imaging.

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.