Light Sheet Microscopy Optimized for Depth Penetration to Study Embryogenesis

Three-Dimensional Cross-Sectional Light-Sheet Microscopy Imaging of the Inflamed Mouse Gut

Selective Plane Illumination Microscopy (SPIM) is attractive for its ability to acquire 3D images with high 3D spatial resolution, good optical sectioning capability and high imaging speed. However, tradeoffs have to be made when a large field of view (FOV) is required, results in lower axial resolution or worse optical sectioning capability. Here, we present a novel method for 3D imaging by SPIM that is capable to maintain its high 3D spatial resolution and good optical sectioning capability within a large FOV. Instead of trying to generate a large and uniformly thick excitation light sheet, the method tiles a relative small light sheet quickly to multiple positions within the image plane by defocusing the excitation beam used to create the light sheet, and takes one additional image at each position, so that a large FOV can be imaged by repeating this process and stitching all images together. By implementing this method, light sheets with thin thickness and good excitation light confinement can be used for SPIM imaging with slightly compromised imaging speed. The method was investigated through both numerical simulation and experiments, and the imaging performance was demonstrated by imaging fluorescent particles embedded in agarose gel and live C. elegans embryos.

Re-scan confocal microscopy (RCM) is a new super-resolution technique based on a standard confocal microscope extended with a re-scan unit in the detection path that projects the emitted light onto a sensitive camera. In this paper the fundamental properties of RCM, lateral resolution, axial resolution and signal-to-noise ratio, are characterized and compared with properties of standard confocal microscopy. The results show that the lateral resolution of RCM is ~170 nm compared to ~240 nm of confocal microscopy for 488 nm excitation and 1.49 NA. As the theory predicts, this improved lateral resolution is independent of the pinhole diameter. In standard confocal microscopy, the same lateral resolution can only be achieved with an almost closed pinhole and, consequently, with a major loss of signal. We show that the sectioning capabilities of the standard confocal microscope are preserved in RCM and that the axial resolution of RCM is slightly better (~15%) than the standard confocal microscope. Furthermore, the signal-to-noise ratio in RCM is a factor of 2 higher than in standard confocal microscopy, also due to the use of highly sensitive modern cameras. In case the pinhole of a confocal microscope is adjusted in such way that the lateral resolution is comparable to that of RCM, the signal-to-noise ratio in RCM is 4 times higher than standard confocal microscopy. Therefore, RCM offers a good alternative to standard confocal microscopy for higher lateral resolution with the main advantage of strongly improved sensitivity.

Light sheet fluorescence microscopy (LSFM) utilizes a light sheet to optically section samples, giving it the advantages of high signal-to-noise ratio, low phototoxicity and rapid three-dimensional imaging, which is an ideal tool for long-time microscopic observation of living samples. However, due to the inherent characteristics of Gaussian light sheet, there is a contradiction between the field of view and depth resolution of LSFM. In life science research, high depth resolution is necessary to resolve fine structures of biological systems such as neurons. However, in order to maintain a large field of view, the thickness of Gaussian light sheet is limited. Here, we propose a depth resolution enhancement method for LSFM. The incorporation of light field imaging into LSFM allows the acquirement of light field information within the Gaussian light sheet. By using the light field back projection (LFBP) reconstruction algorithm and depth estimation algorithm, it is possible to obtain the depth information in a light sheet, so as to improve the depth resolution. We built a light field light sheet fluorescence microscope (LFLSFM), and demonstrated it by fluorescent microsphere experiments. The proposed method can achieve a depth resolution of 0.5um when the thickness of the light sheet is kept at 6um to ensure a large field of view. Therefore, the method has potentialities in visualizing neural network of tissues and organs such as mouse brain.

Light sheet fluorescence microscopy is an excellent tool for imaging larger and thicker model organisms such as zebrafish larvae. Samples can be imaged with high spatial and temporal resolution over large fields of view without photodamage. But light sheet microscopy still suffers from optical aberrations due to the thickness of the samples and the high resolution. We have previously demonstrated imaging of the zebrafish central nervous system and correction of aberrations using sensorless Adaptive Optics. Sensorless AO is slow because many images are required to achieve a correction. In this work, we demonstrate measuring the wavefront using a Shack-Hartmann wavefront sensor and a confocal spot as the guide star. This approach does not require special sample preparation or an additional laser. Here we demonstrate our approach by imaging fluorescent beads and inducing wavefront errors with the deformable mirror.

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis.

Light-Sheet Fluorescence Microscopy (LSFM) has proven to be an excellent tool for imaging the nervous system zebrafish and other model organisms. However, these LSFMs typically have low resolution and suffer from striping artifacts. Here, we present a light sheet system using a novel single-objective light-sheet geometry to allow for multi-direction light-sheet illumination with structured light. This geometry is designed to mitigate striping artifacts and achieve lateral resolutions of better than 200 nm over a 277 × 277 × 100 µm3 volume. We demonstrate the system by imaging the central nervous system of dissected Drosophila embryos as well as cerebellum organoids.

For the investigation of three‐dimensional (3D) tumor spheroids and migrating cells embedded in a hydrogel using light sheet fluorescence microscopy (LSFM), a novel approach has been developed. LSFM has become a powerful tool for visualizing 3D structures of fluorescently labeled biological samples, providing high‐resolution imaging with minimal phototoxicity. Unlike conventional widefield or confocal microscopy methods, LSFM selectively illuminates a sample using a thin light sheet, thereby minimizing out‐of‐focus signals and facilitating quick volumetric data collection. However, sample mounting for LSFM still requires improvement, as conventional strategies, such as capillary‐based methods, present challenges especially for larger or delicate specimens, and may include mechanical stress. In this work, a novel sample holder compatible with the Zeiss Lightsheet 7 microscope is presented, specifically designed for the analysis of spheroids embedded in collagen gels. The novel holder enables dual‐sided illumination, improving effective image quality and facilitating detailed analysis of cell migration within the collagen matrix. Images acquired with this setup are analyzed using Arivis Vision4D to assess parameters such as cell number, cell density, and cell distance. The results demonstrated significant improvements not only in studying cancer cell migration and early metastatic behavior but also in evaluating chemotherapy within 3D extracellular matrix models.

We study the feasibility of volume imaging from a few angular views/scans in a light sheet fluorescence microscopy. Two-dimensional (2D) images (angular views) were acquired at an angular separation of 10° and volume images were constructed with merely 18, 9, and 6 views. We study the structural changes in a 5-day old Zebrafish embryo labeled with Phalloidin TRITC that binds to F-Actin of embryo cell. To collect the data, the specimen is rotated (for varying sampling angles Δθ) with respect to a fixed vertical axis passing through the volume-of-interest (yolk sac). In the proposed realization of selective plane illumination microscopy (SPIM) technique, the translation is completely avoided. Analysis shows rich structural information with marginal reduction in contrast. Comparison with the state-of-the-art SPIM shows appreciable volume reconstruction (from an order less 2D scans) that may be good enough for rapid monitoring of macroscopic specimens. Microsc. Res. Tech. 79:455-458, 2016. © 2016 Wiley Periodicals, Inc.

Stiffness-related eye diseases such as keratoconus require comprehensive visualization of the complex morphological matrix changes. The aim of this study was to use three-dimensional (3D) light sheet fluorescence microscopy (LSFM) to analyze unlabeled corneal tissue samples, qualitatively visualizing changes in corneal stiffness. Isolated porcine corneal tissue samples were treated with either NaCl or 0.1% glutaraldehyde (GTA) prior to clearing with benzyl alcohol/benzyl benzoate (BABB) and subsequently scanned with LSFM. After analysis of the LSFM data sets, the samples were embedded in paraffin to validate the results by conventional planar microscopy. In the unlabeled corneal tissue samples the 2D/3D morphology of the entire tissue volume was identified by specific autofluorescence signals. An enhancement of collagen crosslinking was induced by applying GTA to the corneal tissue. Subsequent LSFM scans showed specific morphological changes due to altered autofluorescence signals of the corneal stroma, which were confirmed by conventional histology. Therefore, LSFM analysis of corneal tissue samples allowed label-free 3D autofluorescence assessment of the corneal morphology in its anatomical context. It provides the technical basis for the examination of the pathologically altered cornea and facilitates ophthalmologic examinations of corneal diseases based on the altered tissue stiffness.

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.

As the general population ages, more people are affected by eye diseases, such as retinopathies. It is therefore critical to improve imaging of eye disease mouse models. Here, we demonstrate that 1) rapid, quantitative 3D and 4D (time lapse) imaging of cellular and subcellular processes in the mouse eye is feasible, with and without tissue clearing, using light-sheet fluorescent microscopy (LSFM); 2) flat-mounting retinas for confocal microscopy significantly distorts tissue morphology, confirmed by quantitative correlative LSFM-Confocal imaging of vessels; 3) LSFM readily reveals new features of even well-studied eye disease mouse models, such as the oxygen-induced retinopathy (OIR) model, including a previously unappreciated 'knotted' morphology to pathological vascular tufts, abnormal cell motility and altered filopodia dynamics when live-imaged. We conclude that quantitative 3D/4D LSFM imaging and analysis has the potential to advance our understanding of the eye, in particular pathological, neurovascular, degenerative processes.

Assessment of preclinical models of vascular disease is paramount in the successful translation of novel treatments. The results of these models have traditionally relied on two-dimensional (2D) histological methodologies. Light sheet fluorescence microscopy (LSFM) is an imaging platform that allows for three-dimensional (3D) visualization of whole organs and tissues. In this study, we describe an improved methodological approach utilizing LSFM for imaging of preclinical vascular injury models while minimizing analysis bias. The rat carotid artery segmental pressure-controlled balloon injury and mouse carotid artery ligation injury were performed. Arteries were harvested and processed for LSFM imaging and 3D analysis, as well as for 2D area histological analysis. Artery processing for LSFM imaging did not induce vessel shrinkage or expansion and was reversible by rehydrating the artery, allowing for subsequent sectioning and histological staining a posteriori. By generating a volumetric visualization along the length of the arteries, LSFM imaging provided different analysis modalities including volumetric, area, and radial parameters. Thus, LSFM-imaged arteries provided more precise measurements compared to classic histological analysis. Furthermore, LSFM provided additional information as compared to 2D analysis in demonstrating remodelling of the arterial media in regions of hyperplasia and periadventitial neovascularization around the ligated mouse artery. LSFM provides a novel and robust 3D imaging platform for visualizing and quantifying arterial injury in preclinical models. When compared with classic histology, LSFM outperformed traditional methods in precision and quantitative capabilities. LSFM allows for more comprehensive quantitation as compared to traditional histological methodologies, while minimizing user bias associated with area analysis of alternating, 2D histological artery cross-sections.

Light sheet fluorescence microscopy is an imaging method in which the sample is illuminated from the side by a wide and relatively thin light sheet. This method minimizes the out-of-focus excitation, provides a good balance between spatial resolution and temporal resolution, and mitigates photobleaching and photodamage. Unfortunately, the axial resolution of light sheet fluorescence microscopy is much lower than lateral resolution, which limits the achievement of isotropic imaging. We propose to combine the subtraction method with light sheet fluorescence microscopy to improve the axial resolution. The experimental results demonstrate that the proposed method can produce higher axial resolution than that of conventional light sheet fluorescence microscopy. In addition, a way to reduce negative values in subtraction method is also discussed.

In higher plants, germline differentiation occurs during a relatively short period within developing flowers. Understanding of the mechanisms that govern germline differentiation lags behind other plant developmental processes. This is largely because the germline is restricted to relatively few cells buried deep within floral tissues, which makes them difficult to study. To overcome this limitation, we have developed a methodology for live imaging of the germ cell lineage within floral organs of Arabidopsis using light sheet fluorescence microscopy. We have established reporter lines, cultivation conditions, and imaging protocols for high-resolution microscopy of developing flowers continuously for up to several days. We used multiview imagining to reconstruct a three-dimensional model of a flower at subcellular resolution. We demonstrate the power of this approach by capturing male and female meiosis, asymmetric pollen division, movement of meiotic chromosomes, and unusual restitution mitosis in tapetum cells. This method will enable new avenues of research into plant sexual reproduction.