Large Biological Samples Research Articles

Innovative sample preparation using alcohol dehydration and high refractive index medium enables acquisition of two-channel super-resolution 3D STED image of an entire oocyte.

Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and large biological structures have posed a challenge. To overcome this, the key step is to optimise sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique two-channel 3D STED SR image of an entire mouse oocyte. By optimising sample preparation, it is possible to overcome current limitations of SR methods and obtain high-resolution images of large biological structures, such as oocytes, in order to study fundamental biological processes. Lay Abstract: Super-resolution (SR) microscopy is a cutting-edge tool that allows scientists to view incredibly fine details in biological samples. However, it struggles with larger, thicker specimens, as they need to be optically clear and uniform for the best imaging results. In this study, we refined the sample preparation process to make it more suitable for SR microscopy. Our method includes carefully dehydrating biological samples with alcohol and then transferring them into a mounting medium that enhances optical clarity. This improved protocol enables high-resolution imaging of thick biological structures, which was previously challenging. By optimizing this preparation method, we hope to expand the use of SR microscopy for studying large biological samples, helping scientists better understand complex biological structures.

Flexible solid immersion meniscus lens (SIMlens) approach for enhancing biological imaging of cleared samples.

Tissue-clearing techniques have revolutionized the field of biological imaging by rendering biological specimens transparent and enabling inside optical detection. Light-sheet fluorescence microscopy (LSFM) is a powerful tool for three-dimensional imaging of large biological samples. Combining tissue-clearing techniques with LSFM has advanced the efficient 3D visualization of these samples. A crucial challenge with LSFM is the requirement for the objective to operate within the clearing reagent, which can cause aberrations. To address this issue, we introduce a novel, to our knowledge, approach for the flexible design of the solid immersion refractive meniscus lens (SIMlens), facilitating the use of air objectives with cleared samples. Compared to the previous SIMlens, this method not only eliminates aberrations but also offers customized options for enhancing the numerical aperture and working distance of the objective lens, achieving at least a 10% improvement. We have demonstrated the feasibility of this new method using mouse brain samples.

Optimize Electron Beam Energy toward In Situ Imaging of Thick Frozen Bio-Samples with Nanometer Resolution Using MeV-STEM.

To optimize electron energy for in situ imaging of large biological samples up to 10 μm in thickness with nanoscale resolutions, we implemented an analytical model based on elastic and inelastic characteristic angles. This model has been benchmarked by Monte Carlo simulations and can be used to predict the transverse beam size broadening as a function of electron energy while the probe beam traverses through the sample. As a result, the optimal choice of the electron beam energy can be realized. In addition, the impact of the dose-limited resolution was analysed. While the sample thickness is less than 10 μm, there exists an optimal electron beam energy below 10 MeV regarding a specific sample thickness. However, for samples thicker than 10 μm, the optimal beam energy is 10 MeV or higher depending on the sample thickness, and the ultimate resolution could become worse with the increase in the sample thickness. Moreover, a MeV-STEM column based on a two-stage lens system can be applied to reduce the beam size from one micron at aperture to one nanometre at the sample with the energy tuning range from 3 to 10 MeV. In conjunction with the state-of-the-art ultralow emittance electron source that we recently implemented, the maximum size of an electron beam when it traverses through an up to 10 μm thick bio-sample can be kept less than 10 nm. This is a critical step toward the in situ imaging of large, thick biological samples with nanometer resolution.

Development of innovative system of universal stent graft for endovascular treatment of aneurysm and aortic displacement in various locations

This study presents the technology of developing a universal stent graft for endovascular treatment of aneurysms and aortic dissection of various localizations, without considering the vessel diameter. A self-expanding nitinol stent was used as the frame of the main trunk of the stent graft. During the study, several variants of the aortic linear graft were manufactured and tested. The optimal stiffness and diameter of the nitinol wire were selected based on the results. When creating a bifurcation module, special attention was paid to simplifying the positioning and intravascular assembly of the structure. Implantable modules have been developed for the prosthetics of the main branches of the aorta. Dacron, optimal in terms of fiber structure, was chosen as the material of the woven shell of the graft. Linear extensibility, compactness of the pile, and tensile strength during fenestration were evaluated. To determine the heparin-controlled surgical porosity, experimental samples of stent grafts were tested on a stand simulating arterial blood flow. The wall material of the developed device had a heparin-controlled surgical porosity of 50150 mL/min/cm2 at 120 mm Hg with the possibility of maintaining a controlled endolic. The graft wall created a pressure gradient of no more than 3 mm Hg, and the flow velocity indicators were quite sufficient for adequate perfusion of vital organs. After the inactivation of heparin, blood permeability became zero. The implantation technique of the developed product was implemented on a silicone aortic phantom simulating aneurysm expansion with and without dissection. The phantom contour was filled with a solution simulating the rheological properties of native blood. Pulsating blood flow was simulated using a perfusion pump. Under X-ray control, a stent graft was installed on five large biological samples (sheep). Implantation was performed in the aortic arch with prosthetics of the brachiocephalic trunk and the suprarenal aorta with prosthetics of the visceral branches. With the experiment, we hope that the result will allow us to minimally invasively help patients suffering from aneurysms of any localization.

Preparation of large biological samples for high-resolution, hierarchical, synchrotron phase-contrast tomography with multimodal imaging compatibility.

Imaging across different scales is essential for understanding healthy organ morphology and pathophysiological changes. The macro- and microscale three-dimensional morphology of large samples, including intact human organs, is possible with X-ray microtomography (using laboratory or synchrotron sources). Preparation of large samples for high-resolution imaging, however, is challenging due to limitations such as sample shrinkage, insufficient contrast, movement of the sample and bubble formation during mounting or scanning. Here, we describe the preparation, stabilization, dehydration and mounting of large soft-tissue samples for X-ray microtomography. We detail the protocol applied to whole human organs and hierarchical phase-contrast tomography at the European Synchrotron Radiation Facility, yet it is applicable to a range of biological samples, including complete organisms. The protocol enhances the contrast when using X-ray imaging, while preventing sample motion during the scan, even with different sample orientations. Bubbles trapped during mounting and those formed during scanning (in the case of synchrotron X-ray imaging) are mitigated by multiple degassing steps. The sample preparation is also compatible with magnetic resonance imaging, computed tomography and histological observation. The sample preparation and mounting require 24-36 d for a large organ such as a whole human brain or heart. The preparation time varies depending on the composition, size and fragility of the tissue. Use of the protocol enables scanning of intact organs with a diameter of 150 mm with a local voxel size of 1 μm. The protocol requires users with expertise in handling human or animal organs, laboratory operation and X-ray imaging.

Experiments and simulations demonstrating the rapid ultrasonic rewarming of frozen tissue cryovials

The development of methods to safely rewarm large cryopreserved biological samples remains a barrier to the widespread adoption of cryopreservation. Here, experiments and simulations were performed to demonstrate that ultrasound can increase rewarming rates relative to thermal conduction alone. An ultrasonic rewarming setup based on a custom 444 kHz tubular piezoelectric transducer was designed, characterized, and tested with 2 ml cryovials filled with frozen ground beef. Rewarming rates were characterized in the -20 °C to 5 °C range. Thermal conduction-based rewarming was compared to thermal conduction plus ultrasonic rewarming, demonstrating a tenfold increase in rewarming rate when ultrasound was applied. The maximum recorded rewarming rate with ultrasound was 57° C/min, approximately 2.5 times faster than with thermal conduction alone. Coupled acoustic and thermal simulations were developed and showed good agreement with the heating rates demonstrated experimentally and were also used to demonstrate spatial heating distributions with small (<3° C) temperature differentials throughout the sample when the sample was below 0° C. The experiments and simulations demonstrate the potential for ultrasonic cryovial rewarming with a possible application to large volume rewarming, as faster rewarming rates may improve the viability of cryopreserved tissues and reduce the time needed for cells to regain normal function.

In situ X-ray-assisted electron microscopy staining for large biological samples.

Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks such as whole brains into the realm of the feasible. However, homogeneous, high-quality electron microscopy staining of large biological samples is still a major challenge. To date, assessing the staining quality in electron microscopy requires running a sample through the entire staining protocol end-to-end, which can take weeks or even months for large samples, rendering protocol optimization for such samples to be inefficient. Here, we present an in situ time-lapsed X-ray-assisted staining procedure that opens the 'black box' of electron microscopy staining and allows observation of individual staining steps in real time. Using this novel method, we measured the accumulation of heavy metals in large tissue samples immersed in different staining solutions. We show that the measured accumulation of osmium in fixed tissue obeys empirically a quadratic dependence between the incubation time and sample size. We found that potassium ferrocyanide, a classic reducing agent for osmium tetroxide, clears the tissue after osmium staining and that the tissue expands in osmium tetroxide solution, but shrinks in potassium ferrocyanide reduced osmium solution. X-ray-assisted staining gave access to the in situ staining kinetics and allowed us to develop a diffusion-reaction-advection model that accurately simulates the measured accumulation of osmium in tissue. These are first steps towards <i>in silico</i> staining experiments and simulation-guided optimization of staining protocols for large samples. Hence, X-ray-assisted staining will be a useful tool for the development of reliable staining procedures for large samples such as entire brains of mice, monkeys, or humans.

The effect of exercise on cytokine concentration in equine autologous conditioned serum.

Autologous conditioned serum (ACS) is a commonly administered intra-articular treatment for the management of osteoarthritis in athletic horses. To investigate the influence of exercise on the concentration of cytokines in a non-commercial method of ACS production. Non-randomised cross over design. Whole blood was obtained from eight healthy Standardbred horses immediately prior to, 1h and 24 h following a single bout of exhaustive exercise. Blood was processed using a non-commercial method of ACS production. Fluorescent microsphere immunoassay (FMIA) analysis was performed to quantify Interleukin 1 receptor antagonist (IL-1Ra), Interleukin-10 (IL-10), Interleukin 1β (IL-1β) and tumour necrosis factor α (TNF-α) concentrations at each time point. Mixed effect repeated measures analysis of variance (ANOVA) was used to compare the pre-exercise and post-exercise cytokine concentrations. Significance was set at P < 0.05. A reduced concentration of IL-1Ra (median 584.4, IQR 81.9-5098 pg/ml, p= 0.004) and an increased concentration of TNF-α (11.92, 9.28-39.75 pg/ml, P= .05) at 1h post-exercise were observed when compared with baseline values (IL-Ra 7349, 1272-10760 pg/ml; TNF TNF-α 11.16, 8.36-32.74 pg/ml). No difference in cytokine concentrations of IL-10 or IL-1β were found between any of the time points. The large biological variability and small sample size represents limitations of this study. These results suggest that a single bout of intense exercise can reduce the concentration of the anti-inflammatory cytokine IL-1Ra and increase the concentration of the pro-inflammatory cytokine TNF-α, reducing the 'anti-inflammatory' cytokine composition of ACS. Our findings suggest that collection of blood for ACS production should be performed no sooner than 24 h following a single episode of intense exercise.

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease.

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 µm) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 µm) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Towards a Comprehensive Optical Connectome at Single Synapse Resolution via Expansion Microscopy.

Mapping and determining the molecular identity of individual synapses is a crucial step towards the comprehensive reconstruction of neuronal circuits. Throughout the history of neuroscience, microscopy has been a key technology for mapping brain circuits. However, subdiffraction size and high density of synapses in brain tissue make this process extremely challenging. Electron microscopy (EM), with its nanoscale resolution, offers one approach to this challenge yet comes with many practical limitations, and to date has only been used in very small samples such as C. elegans, tadpole larvae, fruit fly brain, or very small pieces of mammalian brain tissue. Moreover, EM datasets require tedious data tracing. Light microscopy in combination with tissue expansion via physical magnification—known as expansion microscopy (ExM)—offers an alternative approach to this problem. ExM enables nanoscale imaging of large biological samples, which in combination with multicolor neuronal and synaptic labeling offers the unprecedented capability to trace and map entire neuronal circuits in fully automated mode. Recent advances in new methods for synaptic staining as well as new types of optical molecular probes with superior stability, specificity, and brightness provide new modalities for studying brain circuits. Here we review advanced methods and molecular probes for fluorescence staining of the synapses in the brain that are compatible with currently available expansion microscopy techniques. In particular, we will describe genetically encoded probes for synaptic labeling in mice, zebrafish, Drosophila fruit flies, and C. elegans, which enable the visualization of post-synaptic scaffolds and receptors, presynaptic terminals and vesicles, and even a snapshot of the synaptic activity itself. We will address current methods for applying these probes in ExM experiments, as well as appropriate vectors for the delivery of these molecular constructs. In addition, we offer experimental considerations and limitations for using each of these tools as well as our perspective on emerging tools.

Use of High-Refractive Index Hydrogels and Tissue Clearing for Large Biological Sample Imaging.

Recent advances in tissue clearing and light sheet fluorescence microscopy have improved insights into and understanding of tissue morphology and disease pathology by imaging large samples without the requirement of histological sectioning. However, sample handling and conservation of sample integrity during lengthy staining and acquisition protocols remains a challenge. This study overcomes these challenges with acrylamide hydrogels synthesised to match the refractive index of solutions typically utilised in aqueous tissue clearing protocols. These hydrogels have a high-water content (82.0 ± 3.7% by weight). The gels are stable over time and FITC-IgG readily permeated into and effluxed out of them. Whilst the gels deformed and/or swelled over time in some commonly used solutions, this was overcome by using a previously described custom refractive index matched solution. To validate their use, CUBIC cleared mouse tissues and whole embryos were embedded in hydrogels, stained using fluorescent small molecule dyes, labels and antibodies and successfully imaged using light sheet fluorescence microscopy. In conclusion, the high water content, high refractive index hydrogels described in this study have broad applicability to research that delves into pathophysiological processes by stabilising and protecting large and fragile samples.

Adaptive coherence volume in full-field optical coherence tomography

Optical sectioning is instrumental for the observation of extended biological samples. It allows the observation of only a slice of the sample while rejecting contributions from out of focus depths. The acquisition of the whole volume then requires an axial displacement of the sample or the focus. To satisfy Nyquist sampling, this axial displacement has to be equal to half the axial resolution. As lateral and axial resolutions are coupled by the numerical aperture of the microscope objective in most imaging techniques, high-resolution imaging of a volume is a time-consuming task, especially caused by the slow axial scanning. Here, we propose to adapt the axial resolution, or axial extent of the coherence volume, by filtering the spectrum of the illumination of an interferometric imaging technique. We applied our approach on full-field optical coherence tomography and show a tuning of this axial extent from 1.5 to 15 μm, allowing to adapt both the acquisition time and the amount of data. We finally demonstrate that the method is especially suited to image large biological samples such as millimetric engineered tissues.

An alternative approach to tracing the volumic proliferation development of an entire tumor spheroid in 3D through a mini-Opto tomography platform

Microscopy, which is listed among the major in-situ imaging applications, allows to derive information from a biological sample on the existing architectural structures of cells and tissues and their changes over time. Large biological samples such as tumor spheroids cannot be imaged within one field of view, regional imaging in different areas and subsequent stitching are required to attain the full picture. Microscopy is not typically used to produce full-size visualization of tumor spheroids measuring a few millimeters in size. In this study, we propose a 3D volume imaging technique for tracing the growth of an entire tumor spheroid measuring up to 10 mm using a miniaturized optical (mini-Opto) tomography platform. We performed a primary analysis of the 3D imaging for the MIA PaCa-2 pancreatic tumoroid employing its 2D images produced with the mini-Opto tomography from different angles ranging from -25 ° to +25 ° at six different three-day-apart time points of consecutive image acquisition. These 2D images were reconstructed by using a 3D image reconstruction algorithm that we developed based on the algebraic reconstruction technique (ART). We were able to reconstruct the 3D images of the tumoroid to achieve 800 × 800-pixel 50-layer images at resolutions of 5–25 μm. We also created its 3D visuals to understand more clearly how its volume changed and how it looked over weeks. The volume of the tumor was calculated to be 6.761 mm3 at the first imaging time point and 46.899 mm3 15 days after the first (at the sixth time point), which is 6.94 times larger in volume. The mini-Opto tomography can be considered more advantageous than commercial microscopy because it is portable, more cost-effective, and easier to use, and enables full-size visualization of biological samples measuring a few millimeters in size.

An adaptive microscope for the imaging of biological surfaces

Scanning fluorescence microscopes are now able to image large biological samples at high spatial and temporal resolution. This comes at the expense of an increased light dose which is detrimental to fluorophore stability and cell physiology. To highly reduce the light dose, we designed an adaptive scanning fluorescence microscope with a scanning scheme optimized for the unsupervised imaging of cell sheets, which underly the shape of many embryos and organs. The surface of the tissue is first delineated from the acquisition of a very small subset (~0.1%) of sample space, using a robust estimation strategy. Two alternative scanning strategies are then proposed to image the tissue with an improved photon budget, without loss in resolution. The first strategy consists in scanning only a thin shell around the estimated surface of interest, allowing high reduction of light dose when the tissue is curved. The second strategy applies when structures of interest lie at the cell periphery (e.g. adherens junctions). An iterative approach is then used to propagate scanning along cell contours. We demonstrate the benefit of our approach imaging live epithelia from Drosophila melanogaster. On the examples shown, both approaches yield more than a 20-fold reduction in light dose -and up to more than 80-fold- compared to a full scan of the volume. These smart-scanning strategies can be easily implemented on most scanning fluorescent imaging modality. The dramatic reduction in light exposure of the sample should allow prolonged imaging of the live processes under investigation.

Multifluorescence High‐Resolution Episcopic Microscopy for 3D Imaging of Adult Murine Organs

3D microscopy of large biological samples (&gt;0.5 cm3) is transforming biological research. Many existing techniques require trade‐offs between image resolution, sample size, and method complexity. A simple robust instrument with the potential to conduct large‐volume 3D imaging currently exists in the form of the optical high‐resolution episcopic microscopy (HREM). However, the development of the instrument to date is limited to single‐fluorescent wavelength imaging with nonspecific eosin staining. Herein, developments to realize the potential of the HREM to become multifluorescent high‐resolution episcopic microscopy (MF‐HREM) are presented. MF‐HREM is a serial‐sectioning and block‐facing wide‐field fluorescence imaging technique, which does not require tissue clearing or optical sectioning. Multiple developments are detailed in sample preparation and image postprocessing to enable multiple specific stains in large samples and show how these enable segmentation and quantification of the data. The application of MF‐HREM is demonstrated in a variety of biological contexts: 3D imaging of whole tumor vascular networks and tumor cell invasion in xenograft tumors up to 7.5 mm3 at resolutions of 2.75 μm, quantification of glomeruli volume in the adult mouse kidney, and quantification of vascular networks and white‐matter track orientation in adult mouse brain.

Electrochemiluminescence imaging of respiratory activity of cellular spheroids using sequential potential steps

The respiratory activity of cultured cells can be electrochemically monitored using scanning electrochemical microscopy (SECM) with high spatial resolution. However, in SECM, the electrode takes a long time to scan, limiting simultaneous measurements with large biological samples such as cell spheroids. Therefore, for rapid electrochemical imaging, a novel strategy is needed. Herein, we report electrochemiluminescence (ECL) imaging of spheroid respiratory activity for the first time using sequential potential steps. L-012, a luminol analog, was used as an ECL luminophore, and H2O2, a sensitizer for ECL of L-012, was generated by the electrochemical reduction of dissolved O2. The ECL imaging visualized spheroid respiratory activity—evidenced by ECL suppression—corresponding to O2 distribution around the spheroids. This method enabled the time-lapse imaging of respiratory activity in multiple spheroids with good spatial resolution comparable to that of SECM. Our work provides a promising high-throughput imaging strategy for elucidating spheroid cellular dynamics.

High-throughput optical sectioning via line-scanning imaging with digital structured modulation.

Optical sectioning with high-throughput, a high signal-to-noise ratio (SNR), and submicrometer resolution is crucial, but challenging, to three-dimensional visualization of large biological tissue samples. Here we propose line-scanning imaging with digital structured modulation for optical sectioning. Our method generates images with a significantly improved SNR, compared to wide-field structured illumination microscopy (WF-SIM), without residual modulation artifacts. We image a 14.5mm×11.5mm horizontal view of mouse brain tissue at a pixel resolution of 0.32µm×0.32µm in 101 s, which, compared to WF-SIM, represents a significant improvement on imaging throughput. These results provide development opportunities for high-throughput, high-resolution large-area optical imaging methods.

Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH.

Advances in light-sheet and confocal microscopy now allow imaging of cleared large biological tissue samples and enable the 3D appreciation of cell and protein localization in their native organ environment. However, the sample preparations for such imaging are often onerous, and their capability for antigen detection is limited. Here, we describe FLASH (fast light-microscopic analysis of antibody-stained whole organs), a simple, rapid, fully customizable technique for molecular phenotyping of intact tissue volumes. FLASH utilizes non-degradative epitope recovery and membrane solubilization to enable the detection of a multitude of membranous, cytoplasmic and nuclear antigens in whole mouse organs and embryos, human biopsies, organoids and Drosophila. Retrieval and immunolabeling of epithelial markers, an obstacle for previous clearing techniques, can be achieved with FLASH. Upon volumetric imaging, FLASH-processed samples preserve their architecture and integrity and can be paraffin-embedded for subsequent histopathological analysis. The technique can be performed by scientists trained in light microscopy and yields results in <1 week.

C-ECi: a CUBIC-ECi combined clearing method for three-dimensional follicular content analysis in the fish ovary†.

Deciphering mechanisms of oocyte development in the fish ovary still remain challenging, and a comprehensive overview of this process at the level of the organ is still needed. The recent development of optical tissue clearing methods has tremendously boosted the three-dimensional (3D) imaging of large size biological samples that are naturally opaque. However, no attempt of clearing on fish ovary that accumulates extremely high concentration of lipids within oocytes has been reported to date. To face with this ovarian-specific challenge, we combined two existing clearing methods, the nontoxic solvent-based ethyl cinnamate (ECi) method for efficient clearing and the Clear Unobstructed Brain Imaging Cocktails and Computational (CUBIC) method to enhance lipid removal and reduce nonspecific staining. The methyl green fluorescent dye was used to stain nuclei and delineate the follicular structures that include oocytes. Using this procedure (named CUBIC-ECi [C-ECi]), ovaries of both medaka and trout could be imaged in 3D and follicles analyzed. To our knowledge, this is the first procedure elaborated for clearing and imaging fish ovary in 3D. The C-ECi method thus provides an interesting tool for getting precise quantitative data on follicular content in fish ovary and promises to be useful for further developmental and morphological studies.