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Examples of immunostaining and preserving GFP fluorescence with tissue clearing on the developing mouse heart (A and B) Immunohistochemistry combined with BABB on ED 9.5 mouse embryo, smooth muscle actin antibody (SMA) in red labeling the myocardium, CD31 (PECAM-1) in green staining the endocardium (B), and DAPI nuclear staining (not very distinct at this low magnification) in blue. (C and D) Preservation of natural GFP fluorescence with CUBIC tissue clearing on ED 10.5 (C) and ED 14.5 (D) in mouse Connexin 40 -GFP (Cx40-GFP) embryo hearts with superimposed autofluorescence in red. Ventricular trabeculae and atria are positive for Cx40 at these stages. Autofluorescent blood is present in the ventricles (C). All images were captured with confocal microscopy. Scale bar represents 100 mm in all figures.
Source publication
Tissue imaging in 3D using visible light is limited and various clearing techniques were developed to increase imaging depth, but none provides universal solution for all tissues at all developmental stages. In this review, we focus on different tissue clearing methods for 3D imaging of heart and vasculature, based on chemical composition (solvent-...
Contexts in source publication
Context 1
... few studies have directly analyzed compatibility of tissue clearing and fluorescence preservation in the adult or embryonic heart tissue. In our previous study ( Kolesova et al., 2016) we used CLARITY, SCALE, CU-BIC, and DBE clearing methods and compared their GFP fluorescence preservation ability in embryonic hearts (illustrated in Figures 2C, 2D and 3B with CUBIC tissue clearing on Cx40:GFP trabeculae and coronary vasculature in embryonic hearts). We found that CUBIC cleared the tissue to a deeper level compared with SCALE and therefore was more suitable for analysis of intact hearts. ...
Context 2
... few studies have directly analyzed compatibility of tissue clearing and fluorescence preservation in the adult or embryonic heart tissue. In our previous study ( Kolesova et al., 2016) we used CLARITY, SCALE, CU-BIC, and DBE clearing methods and compared their GFP fluorescence preservation ability in embryonic hearts (illustrated in Figures 2C, 2D and 3B with CUBIC tissue clearing on Cx40:GFP trabeculae and coronary vasculature in embryonic hearts). We found that CUBIC cleared the tissue to a deeper level compared with SCALE and therefore was more suitable for analysis of intact hearts. ...
Citations
... Large and detailed 3D information is important to understand complex cellular interactions during organogenesis or to fully appreciate the topography of systems spanning the entire body like neuronal projections or vasculature. As most embryos are non-transparent, tissue clearing is usually required to get workable 3D information from whole mount embryo images [19][20][21][22][23][24]. Although chicken embryos are initially transparent, the tissues rapidly gain opacity making the imaging of cells inside the embryo difficult after the second day of development. ...
Background
Fine characterization of gene expression patterns is crucial to understand many aspects of embryonic development. The chicken embryo is a well-established and valuable animal model for developmental biology. The period spanning from the third to sixth embryonic days (E3 to E6) is critical for many organ developments. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) enables multiplex RNA detection in thick samples including embryos of various animal models. However, its use is limited by tissue opacity.
Results
We optimized HCR RNA-FISH protocol to efficiently label RNAs in whole mount chicken embryos from E3.5 to E5.5 and adapted it to ethyl cinnamate (ECi) tissue clearing. We show that light sheet imaging of HCR RNA-FISH after ECi clearing allows RNA expression analysis within embryonic tissues with good sensitivity and spatial resolution. Finally, whole mount immunofluorescence can be performed after HCR RNA-FISH enabling as exemplified to assay complex spatial relationships between axons and their environment or to monitor GFP electroporated neurons.
Conclusions
We could extend the use of HCR RNA-FISH to older chick embryos by optimizing HCR RNA-FISH and combining it with tissue clearing and 3D imaging. The integration of immunostaining makes possible to combine gene expression with classical cell markers, to correlate expressions with morphological differentiation and to depict gene expressions in gain or loss of function contexts. Altogether, this combined procedure further extends the potential of HCR RNA-FISH technique for chicken embryology.
... Combining tissue clearing with semi-automated tracing has provided great insight into how angiogenesis occurs in the skin and other tissues (11)(12)(13). This approach has been recently applied to tumors (14,15). ...
Tumors that arise in the epidermis must develop a vascular supply to grow beyond a millimeter in depth. Despite this dependence on angiogenesis for tumor growth, melanoma tumors are minimally responsive to anti-angiogenesis agents. Existing methods to measure changes in angiogenesis rely on two dimensional approaches that have limited ability to capture changes in the three-dimensional architecture of the tumor vascular network. Here we integrate antibody infusion, optical tissue clearing, multiphoton imaging, and three-dimensional semi-automated tracing to quantify changes in the vascular architecture of skin and tumors. Initial studies used this new vessel visualization approach to demonstrate that RhoJ knockout mice have marked differences in vessel arborization in the skin despite having similar numbers of endothelial cells. Small molecules that inhibit CDC42 GTPases, which include RhoJ, RhoQ, and CDC42, inhibit tumor growth and vessel branching within tumors to a similar degree as Braf inhibitors. In contrast to Braf inhibitors, which only affect vessel arborization in tumors, CDC42 inhibitors alter vessel arborization in normal skin, colon, and human skin organoids. Taken together, these studies identify a new class of pharmacologic agents that inhibit vessel branching in both normal skin and tumors which could have utility in treating skin cancer and skin disorders characterized by pathologic angiogenesis.
... Tissue clearing techniques use chemicals to remove the light scattering/attenuation components of the cells while largely retaining their cytoarchitectures, thereby generating an "optically transparent" tissue in its nearly intact status [19,20]. Three major types of clearing strategies, classified as hydrophobic, hydrophilic, and hydrogel-based approaches, have been widely used for processing diverse organ tissues, such as the brain, kidney, heart, lung, pancreas, and even bone [21][22][23][24][25][26][27]. Grüneboom et al. ...
Rationale: The composition and spatial structure of the lymphoma tumor microenvironment (TME) provide key pathological insights for tumor survival and growth, invasion and metastasis, and resistance to immunotherapy. However, the 3D lymphoma TME has not been well studied owing to the limitations of current imaging techniques. In this work, we take full advantage of a series of new techniques to enable the first 3D TME study in intact lymphoma tissue.
Methods: Diverse cell subtypes in lymphoma tissues were tagged using a multiplex immunofluorescence labeling technique. To optically clarify the entire tissue, immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO+), clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) and stabilization to harsh conditions via intramolecular epoxide linkages to prevent degradation (SHIELD) were comprehensively compared with the ultimate dimensional imaging of solvent-cleared organs (uDISCO) approach selected for clearing lymphoma tissues. A Bessel-beam light-sheet fluorescence microscope (B-LSFM) was developed to three-dimensionally image the clarified tissues at high speed and high resolution. A customized MATLAB program was used to quantify the number and colocalization of the cell subtypes based on the acquired multichannel 3D images. By combining these cutting-edge methods, we successfully carried out high-efficiency 3D visualization and high-content cellular analyses of the lymphoma TME.
Results: Several antibodies, including CD3, CD8, CD20, CD68, CD163, CD14, CD15, FOXP3 and Ki67, were screened for labeling the TME in lymphoma tumors. The 3D imaging results of the TME from three types of lymphoma, reactive lymphocytic hyperplasia (RLN), diffuse large B-cell lymphoma (DLBCL), and angioimmunoblastic T-cell lymphoma (AITL), were quantitatively analyzed, and their cell number, localization, and spatial correlation were comprehensively revealed.
Conclusion: We present an advanced imaging-based method for efficient 3D visualization and high-content cellular analysis of the lymphoma TME, rendering it a valuable tool for tumor pathological diagnosis and other clinical research.
... Sample trimming LSFM coupled with tissue clearing is often advertised as the best solution to image large biological samples ("large" meaning several millimeters or up to a few centimeters, depending on what the system can accommodate). Protocols for whole-mount imaging of nearly every organ in small vertebrates are easily found in the literature (Alessio and Zhang, 2021;Epp et al., 2015;Frenkel et al., 2023;Kolesová et al., 2021;Maldonado et al., 2020;Nehrhoff et al., 2016;Wu et al., 2021). Strikingly, several research teams even reported successful clearing and imaging of entire rodent bodies at single-cell resolution (Cai et al., 2019;Kubota et al., 2017;Mai et al., 2023;Pan et al., 2016;Tainaka et al., 2014). ...
In parallel with the development of tissue-clearing methods, over the last decade, light sheet fluorescence microscopy has contributed to major advances in various fields, such as cell and developmental biology and neuroscience. While biologists are increasingly integrating three-dimensional imaging into their research projects, their experience with the technique is not always up to their expectations. In response to a survey of specific challenges associated with sample clearing and labeling, image acquisition, and data analysis, we have critically assessed the recent literature to characterize the difficulties inherent to light sheet fluorescence microscopy applied to cleared biological samples and to propose solutions to overcome them. This review aims to provide biologists interested in light sheet fluorescence microscopy with a primer for the development of their imaging pipeline, from sample preparation to image analysis. Importantly, we believe that issues could be avoided with better anticipation of image analysis requirements, which should be kept in mind while optimizing sample preparation and acquisition parameters.
... Nonetheless, tissue clearing cannot fully address the problem of differences in composition among cardiac tissue, resulting in a lower imaging quality at deeper imaging depths [4,7]. In addition, dense muscular tissues limit the depth of penetration of clearing agents, and the limited penetration depth constrains the use of clearing reagents on mature hearts; therefore, researchers have primarily applied tissue-clearing techniques on embryonic hearts [8]. ...
The use of combined optical imaging and tissue sectioning has potential for use in visualizing heart-wide fine structures at single-cell resolution. However, existing tissue preparation methods fail to generate ultrathin cavity-containing cardiac tissue slices with minimal deformation. This study developed an efficient vacuum-assisted tissue embedding method to prepare high-filled, agarose-embedded whole-heart tissue. Utilizing optimized vacuum parameters, we achieved 94% filled whole-heart tissue with the thinnest cut slice of 5 µm. We subsequently imaged a whole mouse heart sample using vibratome-integrated fluorescence micro-optical sectioning tomography (fMOST) with a voxel size of 0.32 µm × 0.32 µm × 1 µm. The imaging results indicated that the vacuum-assisted embedding method enabled whole-heart tissue to withstand long-term thin cutting while ensuring that slices were consistent and of high quality.
... There are many available tissue-clearing methods that can be used for various purposes. Each of these methods has advantages and disadvantages that were thoroughly reviewed byKolesová et al. (2021). Among the most common families of tissue clearing are Disco, CUBIC, and Clarity. ...
Sympathetic efferent axons regulate cardiac functions. However, the topographical distribution and morphology of cardiac sympathetic efferent axons remain insufficiently characterized due to the technical challenges involved in immunohistochemical labeling of the thick walls of the whole heart. In this study, flat‐mounts of the left and right atria and ventricles of FVB mice were immunolabeled for tyrosine hydroxylase (TH), a marker of sympathetic nerves. Atrial and ventricular flat‐mounts were scanned using a confocal microscope to construct montages. We found (1) In the atria: A few large TH‐immunoreactive (IR) axon bundles entered both atria, branched into small bundles and then single axons that eventually formed very dense terminal networks in the epicardium, myocardium and inlet regions of great vessels to the atria. Varicose TH‐IR axons formed close contact with cardiomyocytes, vessels, and adipocytes. Multiple intrinsic cardiac ganglia (ICG) were identified in the epicardium of both atria, and a subpopulation of the neurons in the ICG were TH‐IR. Most TH‐IR axons in bundles traveled through ICG before forming dense varicose terminal networks in cardiomyocytes. We did not observe varicose TH‐IR terminals encircling ICG neurons. (2) In the left and right ventricles and interventricular septum: TH‐IR axons formed dense terminal networks in the epicardium, myocardium, and vasculature. Collectively, TH labeling is achievable in flat‐mounts of thick cardiac walls, enabling detailed mapping of catecholaminergic axons and terminal structures in the whole heart at single‐cell/axon/varicosity scale. This approach provides a foundation for future quantification of the topographical organization of the cardiac sympathetic innervation in different pathological conditions.
... In recent years, studies examining vascular architectures and their adaptive changes in various biological tissues under pathological conditions have been numerous. 4,5 Traditional medical imaging techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET), can capture macrovessels within whole organs; however, their limited resolutions render the effective identification of a single capillary impossible. ...
Significance:
Visualization of intact vasculatures is crucial to understanding the pathogeneses of different neurological and vascular diseases. Although various fluorescent vessel labeling methods have been used in combination with tissue clearing for three-dimensional (3D) visualization of different vascular networks, little has been done to quantify the labeling effect of each vessel labeling routine, as well as their applicability alongside various clearing protocols, making it difficult to select an optimal combination for finely constructing different vasculatures. Therefore, it is necessary to systematically assess the overall performance of these common vessel labeling methods combined with different tissue-clearing protocols.
Aim:
A comprehensive evaluation of the labeling quality of various vessel labeling routines in different organs, as well as their applicability alongside various clearing protocols, were performed to find the optimal combinations for 3D reconstruction of vascular networks with high quality.
Approach:
Four commonly-used vessel labeling techniques and six typical tissue optical clearing approaches were selected as candidates for the systematic evaluation.
Results:
The vessel labeling efficiency, vessel labeling patterns, and compatibility of each vessel labeling method with different tissue-clearing protocols were quantitatively evaluated and compared. Based on the comprehensive evaluation results, the optimal combinations were selected for 3D reconstructions of vascular networks in several organs, including mouse brain, liver, and kidney.
Conclusions:
This study provides valuable insight on selecting the proper pipelines for 3D visualization of vascular networks, which may facilitate understanding of the underlying mechanisms of various neurovascular diseases.
... In contrast, both solvent-based methods cleared the samples within hours but resulted in transparency below 50%. Changes in sample size occurred as expected: hydrophilic methods led to sample expansion, which might be an advantage when one is interested in increasing imaging resolution; solventbased protocols resulted in sample shrinkage, which can be advantageous when imaging large samples [18]. ...
Mesenchymal stromal cells (MSCs) injected intravenously are trapped in the capillaries of the lungs and die within the first 24 h. Studying the biodistribution and fate of labelled therapeutic cells in the 3D pulmonary context is important to understand their function in this organ and gain insights into their mechanisms of action. Optical tissue clearing enables volumetric cell tracking at single-cell resolution. Thus, we compared three optical tissue-clearing protocols (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC), modified stabilised 3D imaging of solvent-cleared organs (s-DISCO) and ethyl cinnamate (ECi)) to evaluate their potential to track the biodistribution of human umbilical cord MSCs expressing the tdTomato fluorescence reporter and investigate how they interact with host cells in the mouse lung. The results showed that although CUBIC clearing is the only method that enables direct imaging of fluorescently labelled MSCs, combining s-DISCO or ECi with immunofluorescence or dye labelling allows the interaction of MSCs with endothelial and immune cells to be studied. Overall, this comparative study offers guidance on selecting an optical tissue-clearing method for cell tracking applications.
... Despite a plethora of advanced cleaning methods, most of them are designated for brain and nervous tissues and lack the consideration of the nature of the heart (Kolesová et al., 2021). Unlike the brain, the heart is enriched in connective tissues and autofluorescence (Sands et al., 2022). ...
... Kolwsová and colleagues tested different clearing protocols for imaging whole embryos, embryonic, and adult hearts with GFP genetic fluorescence. The results pointed out that DBE (dibenzylether) did not preserve the GFP signals and triggers the shrinking of tissue; CLARITY improved the clearing effect but compromise the GFP signals; SCALE clearing led to good clearing until E12.5 but failed to function well in large-scaled samples in later stages; CUBIC showed better performance of clearing with imaging although adult hearts took a longer time (7-14 days) (Kolesová et al., 2021). ...
Biological tissues are naturally three-dimensional (3D) opaque structures, which poses a major challenge for the deep imaging of spatial distribution and localization of specific cell types in organs in biomedical research. Here we present a 3D heart imaging reconstruction approach by combining an improved heart tissue-clearing technique with high-resolution light-sheet fluorescence microscopy (LSFM). We have conducted a three-dimensional and multi-scale volumetric imaging of the ultra-thin planes of murine hearts for up to 2,000 images per heart in x-, y-, and z three directions. High-resolution 3D volume heart models were constructed in real-time by the Zeiss Zen program. By using such an approach, we investigated detailed three-dimensional spatial distributions of two specific cardiomyocyte populations including HCN4 expressing pacemaker cells and Pnmt+ cell-derived cardiomyocytes by using reporter mouse lines Hcn4DreER/tdTomato and PnmtCre/ChR2−tdTomato. HCN4 is distributed throughout right atrial nodal regions (i.e., sinoatrial and atrioventricular nodes) and the superior-inferior vena cava axis, while Pnmt+ cell-derived cardiomyocytes show distinct ventral, left heart, and dorsal side distribution pattern. Our further electrophysiological analysis indicates that Pnmt + cell-derived cardiomyocytes rich left ventricular (LV) base is more susceptible to ventricular arrhythmia under adrenergic stress than left ventricular apex or right ventricle regions. Thus, our 3D heart imaging reconstruction approach provides a new solution for studying the geometrical, topological, and physiological characteristics of specific cell types in organs.
... CUBIC is often considered the method of choice. The main advantage is that CUBIC chemicals elute endogenous chromophores (e.g., hemoglobin), reducing blood autofluorescence Kolesová 2016Kolesová , 2021. In addition, CUBIC preserves fluorescence, allowing the protocol's performance after the immunolabeling, especially regarding whole-mount GFP cardiac mouse samples Kolesová 2016Kolesová , 2021. ...
... The main advantage is that CUBIC chemicals elute endogenous chromophores (e.g., hemoglobin), reducing blood autofluorescence Kolesová 2016Kolesová , 2021. In addition, CUBIC preserves fluorescence, allowing the protocol's performance after the immunolabeling, especially regarding whole-mount GFP cardiac mouse samples Kolesová 2016Kolesová , 2021. CUBIC protocol was optimized to optically clear mouse bones and detect cancer metastasis (e.g., lung, kidney) in different mouse models, for instance, by imaging the triplepositive breast cancer metastasis in bones and brain (Kubota 2017;Tainaka 2018;Takahashi 2020). ...
... CUBIC was also applied in preclinical models to detect the vessel architecture before and after kidney injury (Hasegawa 2019). However, the main downside lies in the long clearing period for larger specimens (Kolesová 2021). ...
Understanding the inner morphology of intact tissues is one of the most competitive challenges in modern biology. Since the beginning of the twentieth century, optical tissue clearing (OTC) has provided solutions for volumetric imaging, allowing the microscopic visualization of thick sections of tissue, organoids, up to whole organs and organisms (for example, mouse or rat). Recently, tissue clearing has also been introduced in clinical settings to achieve a more accurate diagnosis with the support of 3D imaging. This review aims to give an overview of the most recent developments in OTC and 3D imaging and to illustrate their role in the field of medical diagnosis, with a specific focus on clinical applications.
Graphical abstract
