An imaging method for high-throughput acquisition of three-dimensional structures of experimental animal embryonic hearts

Abstract

The present invention discloses an imaging method for obtaining three-dimensional structures of embryonic hearts of experimental animals with high throughput, comprising the following steps: (1) collecting embryonic hearts of experimental animals and fixing them with a fixing solution; (2) dehydrating or defatting the embryonic hearts fixed in step (1) and then performing a clearing treatment; (3) placing the cleared embryonic hearts into a transparent tube and then into an imaging chamber filled with a transparent solution having a refractive index of 1.41 to 1.47 for fluorescence imaging. The present invention optimizes the sample fixing and clearing methods, enabling rapid processing and achieving the purpose of obtaining three-dimensional structures of embryonic hearts with high throughput. Neither the fixing method nor the clearing method causes obvious deformation of the embryonic hearts, and they can be stored for a long time.

Description

Technical Field

[0001] The present invention belongs to the technical field of experimental animal imaging, and in particular relates to a high-throughput imaging method for acquiring the three-dimensional structure of an experimental animal embryonic heart. Background Art

[0002] Congenital heart disease (CHD) is a common congenital disease, affecting approximately 0.8% to 1.2% of fetuses. Due to the close similarity between mouse and human cardiac development, scientists often use mice as a model to study the mechanisms of CHD. Identifying phenotypic defects during cardiac development is a critical first step in mechanistic research.

[0003] Existing methods for identifying embryonic cardiac phenotypes include:

[0004] (1) Scanning laser fluorescence microscopy techniques such as confocal microscopy, light sheet imaging and two-photon imaging ([1] Esteban, I., Schmidt, P., Desgrange, A., Raiola, M., S.,Meilhac,SM,Kobbelt,L.,andTorres,M.(2022).Pseudodynamic analysis of heart tube formation in the mousereveals strong regional variability and early left–right asymmetry.NatCardiovasc Res 1,504–517.10.1038 / s44161-022-00065-1. [2] Dominguez, MH, Krup, AL, Muncie, JM, and Bruneau, BG (2023). Graded mesoderm assembly governs cellfate and morphogenesis of the early mammalian heart. Cell 186,479-496.e23.10.1016 / j.cell.2023.01.001.[3]Ivanovitch,K.Live imaging of heart tubedevelopment in mouse reveals alternating phases of cardiac differentiation and morphogenesis.);

[0005] (2) Stereological techniques such as serial tissue sections or high-resolution microscopy (HREM) ([1] De Boer, BA, Van Den Berg, G., De Boer, PAJ, Moorman, AFM, and Ruijter, JM (2012). Growth of the developing mouse heart: An interactive qualitative and quantitative 3D atlas. Developmental Biology 368, 203–213.10.1016 / j.ydbio.2012.05.001. [2] Le Garrec, J.-F., JN,Desgrange,A.,Ivanovitch,KD, E.,Bangham,JA,Torres,M.,Coen,E.,Mohun,TJ,andMeilhac,SM(2017).A predictive model of asymmetric morphogenesis from 3Dreconstructions of mouse heart looping dynamics.eLife 6,e28951.10.7554 / eLife.28951.);

[0006] (3) Tomographic imaging strategies, including micro-computed tomography (micro-CT), optical coherence tomography (OCT), and optical projection tomography (OPT) ([1]Li-Villarreal, N., Rasmussen, TL, Christiansen, AE, Dickinson, ME, and Hsu, C.-W. (2023). Three-dimensional microCT imaging of mouse heart development from early post-implantation to late fetal stages. MammGenome 34, 156–165.10.1007 / s00335-022-09976-7. [2]Wang, S., Lopez, AL, Larin, KV, Overbeek, PA, and Larina, IV (2015). Live four-dimensional optical coherence tomography reveals embryonic cardiac phenotype in mousemutant.J.Biomed.Opt.20,1.10.1117 / 1.JBO.20.9.090501.[3]Dalmasso,G.,Musy,M.,Niksic,M.,Robert-Moreno,A., -Careaga, C., Sanz-Ezquerro, JJ, and Sharpe, J. (2022). 4D reconstruction of murine developmental trajectories using sphericalharmonics. Developmental Cell 57, 2140-2150.e5.10.1016 / j.devcel.2022.08.005.).

[0007] However, each inevitably has its own limitations and cannot achieve high throughput. For example, laser fluorescence microscopy is only applicable to fluorescent hearts, confocal microscopy and two-photon imaging are time-consuming, light-sheet imaging is quick but requires long sample processing and loading, and tissue sections cannot achieve good three-dimensional reconstruction. HREM, while having sufficient resolution, is also limited by the long sample processing and sectioning time, making it difficult to achieve high throughput. Furthermore, tomographic imaging strategies generally have insufficient resolution, which makes it difficult to determine structural abnormalities in the embryonic heart.

[0008] Since the application of light sheet microscopy in the field of life sciences in 2004, it has shown extremely high efficiency in three-dimensional imaging of developmental processes (Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J., and Stelzer, EHK (2004). Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy. Science 305, 1007–1009.10.1126 / science.1100035). In recent years, many technical innovations based on the light sheet platform have been made, such as sample mounting methods (Laroche, T., Burri, O., Dubey, LK, and Seitz, A. (2019). Development of Sample-Adaptable Holders for Lightsheet Microscopy. Front. Neuroanat. 13, 26.10.3389 / fnana.2019.00026.), tissue transparent technology ([1]

[0009] H., V., A., M.,andSedmera,D.(2021).Tissue clearing and imaging methods for cardiovasculardevelopment.iScience 24,102387.10.1016 / j.isci.2021.102387.[2]Ueda,HR,Ertürk,A.,Chung,K.,Gradinaru,V., A., Tomancak, P., and Keller, PJ (2020). Tissue clearing and its applications in neuroscience. Nat Rev Neurosci 21, 61–79. 10.1038 / s41583-019-0250-1.), image processing (Gibbs, HC, Mota, SM, Hart, NA, Min, SW, Verno, AO, Pritchard, AL, Sen, A., Vitha, S., Sarasamma, S., McIntosh, AL, et al. (2021). Navigating the Light-Sheet Image Analysis Software Landscape: Concepts for Driving Cohesion From Data Acquisition to Analysis. Front. Cell Dev. Biol. 9, 739079. 10.3389 / fcell.2021.739079.), etc., have all developed rapidly. These optimizations have promoted the widespread application of light-sheet microscopy in the field of neuroscience. However, due to the small size of the embryonic heart, the difficulty in clarifying it, and the inability to achieve high throughput, light sheet microscopy has not received much attention in identifying the morphology of mouse embryonic hearts.Although its use for imaging the embryonic heart has been reported, the image quality is poor and sample mounting presents challenges ([1]Sereti, K.-I., Nguyen, N.B., Kamran, P., Zhao, P., Ranjbarvaziri, S., Park, S., Sabri, S., Engel, J.L., Sung, K., Kulkarni, RP, et al. (2018). Analysis of cardiomyocyte clonal expansion during mouse heart development and injury. Nat Commun 9, 754.10.1038 / s41467-018-02891-z. [2]Fei, P., Lee, J., Packard, R.R.S., Sereti, K.-I., Xu, H., Ma, J., Ding, Y., Kang, H., Chen, H., Sung, K., et al. (2016). Cardiac Light-Sheet Fluorescent Microscopy for Multi-Scale and Rapid Imaging of Architecture and Function. SciRep 6, 22489.10.1038 / srep22489.). Mounting methods such as clamping can affect sample stability, and using glue for mounting can obscure certain structures. Furthermore, using agarose embedding significantly increases the time required for tissue clearing and refractive index matching. Summary of the Invention

[0010] In view of the above-mentioned deficiencies in the prior art, the present invention provides a high-throughput imaging method for acquiring the three-dimensional structure of the embryonic heart of an experimental animal.

[0011] A high-throughput imaging method for acquiring the three-dimensional structure of the embryonic heart of an experimental animal, the imaging method comprising the following steps:

[0012] (1) Collect the embryonic hearts of experimental animals and fix them with fixative;

[0013] (2) Dehydrating or defatting the embryonic heart after fixation in step (1) and then performing a transparent treatment;

[0014] (3) The cleared embryonic heart was placed in a transparent tube, and then placed in an imaging chamber filled with a transparent solution with a refractive index of 1.41 to 1.47 for fluorescence imaging.

[0015] Preferably, the refractive index of the transparent solution poured into the imaging chamber is 1.45, which has the best imaging effect.

[0016] Preferably, if the embryonic heart does not express exogenous fluorescent protein, imaging is performed using a method that enhances tissue autofluorescence;

[0017] If the embryonic heart expresses an exogenous fluorescent protein, fluorescence imaging is performed using the excitation light corresponding to the exogenous fluorescent protein.

[0018] More preferably, if the embryonic heart does not express exogenous fluorescent proteins, imaging is performed using enhanced tissue autofluorescence, in which case:

[0019] In step (1), the embryonic heart of the experimental animal is from an embryo of E10.5 to E14.5, and is fixed with a formaldehyde solution first and then with a glutaraldehyde solution;

[0020] In step (1), the embryonic heart of the experimental animal is from an embryo aged E15.5 to E18.5, and is fixed with glutaraldehyde solution;

[0021] If the embryonic heart expresses an exogenous fluorescent protein, the excitation light corresponding to the exogenous fluorescent protein is used for fluorescence imaging. In this case, in step (1), formaldehyde solution is used for fixation.

[0022] For embryonic hearts that do not express exogenous fluorescent proteins, that is, do not have fluorescence, the myocardial tissue exhibits autofluorescence due to the presence of tetrapyrrohematin and myosin in myoglobin. This natural high fluorescence is generally considered a disadvantage because it may compete with the fluorescence of immunohistochemistry. However, this application utilizes this natural fluorescence. Glutaraldehyde exists in its oligomeric form and reacts with protein molecules to form cross-links, leaving many free aldehyde groups to react with amino groups in the tissue, resulting in strong fluorescence inside the tissue. We utilize the natural autofluorescence of myocardial tissue and enhance the fluorescence with glutaraldehyde, thereby improving the visualization of tissue morphology by laser light sheet imaging, making the internal structure clear and promoting the widespread adoption of this technology.

[0023] For embryonic hearts that do not express exogenous fluorescent proteins, for embryos from E10.5 to E14.5, fixation with formaldehyde first can better fix the cell morphology. For embryos from E15.5 to E18.5, since the cell morphology is no longer important in the later stages of the embryo, only the gross structure needs to be focused, formaldehyde fixation is not required at this time, saving the time required for fixation.

[0024] The main purpose of using different fixation procedures for large embryos is to speed up the process and save time, thereby achieving high throughput. Large embryos can also be fixed in formaldehyde overnight if results are not urgent, but the final results will not be significantly different because the high cell density of large embryos makes it difficult to distinguish single-cell morphology, so it is not necessary. Small embryos can also be fixed directly with glutaraldehyde instead of formaldehyde, but the cell morphology will be less clear and only the gross structure will be visible.

[0025] For embryonic hearts that have been introduced with fluorescent proteins, since the fluorescence emitted by the fluorescent proteins is strong enough, no additional treatment is required to enhance the fluorescence and the heart can be directly fixed and processed in the next step.

[0026] When using formaldehyde solution for fixation, it is generally sufficient to fix at room temperature for 2 to 3 hours. Extending the fixation time is also possible, but the fixation effect will not change much. If you need to fix overnight (usually more than 12 hours), you can place it at 4°C for fixation for at least 12 hours.

[0027] When using glutaraldehyde solution for fixation, the fixation time should be no less than 2 hours.

[0028] More preferably, if the embryonic heart does not express exogenous fluorescent protein, imaging is performed using a method of enhancing tissue autofluorescence. In this case: in step (2), the heart is first rinsed with a buffer solution, dehydrated, and then defatted and transparentized; dehydration is performed using a gradient dehydration using an alcohol solution with increasing concentrations until pure alcohol, and defatted and transparentized using a benzyl alcohol: benzyl benzoate solution (BABB organic transparent agent);

[0029] If the embryonic heart expresses an exogenous fluorescent protein, fluorescence imaging is performed using the excitation light corresponding to the exogenous fluorescent protein. In this case, in step (2), gradient defatting is performed using tetrahydrofuran solutions with increasing concentrations; and transparentization is performed using iohexol-urea solution (EZ view water-soluble transparent agent).

[0030] Further preferably, when performing gradient dehydration using an alcohol solution whose concentration is successively increased until pure alcohol, the volume concentration gradient of the alcohol is set to 50%, 75%, 90%, 100% and 100% in sequence; and the processing time for each volume concentration gradient is 30 minutes;

[0031] The volume ratio of benzyl alcohol to benzyl benzoate in the benzyl alcohol:benzyl benzoate solution is 1:2;

[0032] When performing gradient delipidation using tetrahydrofuran solutions with increasing concentrations, for embryonic hearts less than E12.5 days old, the volume concentration gradient of the tetrahydrofuran solution is set to 10%, 20%, and 30%; for embryonic hearts more than E12.5 days old, the volume concentration gradient is increased to 40%; each volume concentration gradient treatment time is 30 minutes;

[0033] The composition of the iohexol-urea solution is 5 g of iohexol and 2.625 g of urea dissolved in 2 ml of 0.03 M PBS (final refractive index of about 1.51).

[0034] Non-fluorescent embryonic hearts undergo dehydration and clearing, while fluorescent hearts undergo defatting and clearing. BABB organic clearing agent is used for clearing non-fluorescent embryonic hearts, as it is easy to prepare and provides excellent morphological imaging after clearing. EZ view water-soluble clearing agent can also be used for clearing non-fluorescent embryonic hearts. However, EZ view is difficult to prepare, requiring at least 4-5 hours to dissolve and prepare. Furthermore, the morphological imaging quality after clearing is not as good as that achieved with BABB organic clearing agent. However, fluorescent hearts require EZ view water-soluble clearing agent; BABB organic clearing agent should not be used because it quenches fluorescence.

[0035] Preferably, in order to save operation steps, the transparent tube is placed in a transparent tube during the transparent treatment, and the fluorescence imaging is performed directly after the transparent treatment. The transparent tube is preferably a glass tube.

[0036] Preferably, fluorescence imaging is performed using a ZEISS Lightsheet Z1 light sheet microscope platform, using an X5 / 0.16 air detection lens and an X5 / 0.1 illumination lens. The imaging chamber is perfused with an 87% glycerol aqueous solution. The refractive index of an 87% glycerol aqueous solution is approximately 1.45.

[0037] More preferably, during fluorescence imaging, the first magnet is fixed to the bottom end of the sample fixing rod provided with the ZEISS Lightsheet Z1 light sheet microscope platform, and the transparent tube is fixed to the sample fixing rod using a sampling rod.

[0038] The sampling rod has a certain elasticity, and a second magnet for attracting and fixing with the first magnet is provided on the top surface. The bottom has a plug section for plugging into the inner cavity of the transparent tube, and the diameter gradually decreases from top to bottom.

[0039] The refractive index of the X5 / 0.16 air detection lens is not adjustable and is fixed at 1.45 with an accuracy of 0.001, but it is not very sensitive to changes in the refractive index. In theory, for the ZEISS Lightsheet Z1 light sheet microscope X5 / 0.16 air detection lens (n ​​= 1.45), the best imaging results can be obtained when both the medium and the sample have the same refractive index (RI) of 1.45 as the detection lens. However, our experiments showed that the 5x lens can tolerate a wider range of RI variations compared to the 20x lens. Although the imaging chamber was filled with 87% glycerol (RI approximately 1.45), light also passed through the glass tube (RI approximately 1.51) and BABB (RI approximately 1.56), but this did not significantly affect the quality of embryo imaging.

[0040] Preferably, the experimental animals are mice or rats.

[0041] Beneficial effects of the present invention:

[0042] (1) By modifying the sample loading equipment and optimizing the sample fixation and clearing methods, we were able to obtain a clear three-dimensional structure image of the embryonic heart without fluorescence in as little as 5 hours after ex vivo separation, and a clear three-dimensional structure image of the heart with fluorescence in about 3 hours after fixation. The modified consumables are all convenient and easy to obtain, and all the reagents used are commercially available. The technology is stable, highly reproducible, and easy to operate, making it easy to be widely promoted. (2) Using the ZEISS Lightsheet Z1 light sheet microscope platform, we were able to obtain approximately 30 images of the three-dimensional structure of the transparent mouse embryonic heart per hour. For embryonic hearts aged E10.5-E12.5, the X and Y axis resolutions were less than 1μm, and the Z axis resolution was 1-2μm. For embryonic hearts aged E18.5, the X and Y axis resolutions were approximately 2μm, and the Z axis resolution was 5-7μm. This has achieved a high-throughput imaging method for obtaining clear three-dimensional structures of the mouse embryonic heart.

[0043] (3) Neither the fixation method nor the clearing method caused significant deformation of the embryonic heart. The heart fixed with BABB organic clearing agent could be stored for a long time, facilitating repeated photography or preservation until photography time. The embryonic heart cleared with EZ view water-soluble clearing agent could be stored for three days, facilitating experimental operation.

[0044] In summary, the method presented in this paper addresses the challenges of high throughput, poor resolution, and complex sample loading that plague existing light-sheet imaging methods. It enables rapid acquisition of the three-dimensional structure of isolated embryonic hearts; achieves high throughput without compromising image quality; and utilizes readily available, cost-effective materials suitable for a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of the structure of the fixed rod.

[0046] Figure 2 Schematic diagram of the structure of the sampling rod and glass tube.

[0047] Figure 3 Schematic diagram of the assembly structure of the glass tube, sampling rod and fixed rod.

[0048] Figure 4 This is a photo of the light sheet microscope loading device used for high-throughput imaging of embryonic heart specimens in this application, where A: commercially available hollow ear spoon; B: glass tube placed on the tip box; C: sample fixing rod; D: sampling rod; E: glass tube; F: combined loading structure; G: operating settings for imaging using ZEISS Lightsheet Z.1.

[0049] Figure 5 Representative images of the embryonic heart at different stages captured in Example 1. Rows: A1-F1 are representative 3D images; A2-F2 are representative ventral heart slices; A3-F3 are representative dorsal heart slices; and A4-F5 are 3D heart sections. Columns: A1-A5 are E10.5 days, B1-B5 are E11.5 days, C1-C5 are E12.5 days, D1-D5 are E13.5 days, E1-E5 are E14.5 days, and F1-F5 are E18.5 days. Scale bar: 100 μm.

[0050] Figure 6 For example 2, E11.5 Wnt1-Cre; Rosa26 mTmG Gross images and sections of a mouse embryonic heart. AB: 10% THF-20% THF-30% THF degreasing, half an hour per channel; CD: 10% THF-20% THF-30% THF-40% THF-50% THF degreasing, half an hour per channel; EF: 10% THF-20% THF-30% THF degreasing, 3 hours per channel. Scale bar: 200 μm.

[0051] Figure 7 Mef2C-Cre; Rosa26 photographed in Example 2 mTmG Representative images of mouse embryos. (AC): Gross images and sagittal sections of an E11.5 mouse; (DF): Gross images and sections of an E15.5 mouse; (GI): Gross images and coronal sections of an E18.5 mouse. Scale bar: 200 μm. DETAILED DESCRIPTION

[0052] In this application, fluorescence imaging was performed using the ZEISS Lightsheet Z1 light sheet microscope platform.

[0053] like Figures 1 to 3As shown, the ZEISS Lightsheet Z1 light sheet microscope platform comes with a metal sample holder rod 1. Typically, the bottom end of the rod 1 is used to connect to an adapter and then adhere to the sample being examined. In this application, this rod 1 is modified, with a magnet 2 glued to its bottom end. A sampling rod 4 is also designed, with a magnet 3 also attached to its top surface. During use, the rod 4 is attached to the magnet 3 on the bottom of the rod 2 via the magnet 3 on the top surface, making it easy to remove and place the rod 4. The bottom of the rod 4 has a plug-in section with a diameter that gradually decreases from top to bottom. This section is designed to extend into the glass tube 5 where the heart is placed, plugging into the inner wall of the tube. This secures the tube 5 to the rod 4 and allows the rod 4 to be placed beneath the rod 1 for imaging. The rod 4 can be made of wood, which has a certain degree of elasticity, facilitating the plug-in connection between the rod 4 and the tube 5.

[0054] like Figure 4 The figure shows a real-life image of the light sheet microscope loading device used for high-throughput imaging of embryonic heart specimens in this application. Figure 4 (A) A commercially available hollow ear spoon, approximately 3.8 mm wide and 158 mm long, is used to transfer dehydrated embryonic heart specimens into a glass tube filled with clearing solution. Figure 4 (B) A glass tube is placed on a pipette tip box. The glass tube is a modified Pasteur tube that can be placed in batches on a 200 μL pipette tip box commonly used in the laboratory for batch placement of embryonic heart samples. Figure 4 (C) is a sample fixing rod. The first magnet is fixed to the bottom surface of the sample fixing rod provided with the ZEISS Lightsheet Z1 light sheet microscope platform using superglue. Figure 4 (D) is a homemade sampling rod, including a wooden sampling rod at the bottom, and a second magnet is fixed to the top surface of the wooden sampling rod by superglue. Figure 4 (E) is a glass tube, which is a modified Pasteur tube with a uniformly sealed bottom and cut into 3 cm lengths. The glass tube contains an embryonic heart and is immersed in a clearing solution. Figure 4 (F) The combined loading structure integrates the fixing rod, sampling rod and glass tube, ready for the imaging process. Figure 4 (G) Imaging setup using the ZEISS Lightsheet Z.1, with a 5x / 0.16 (n = 1.45) detection system. The imaging chamber is filled with an 87% glycerol aqueous solution (refractive index approximately 1.45). Tweezers are used to easily manipulate and change samples at the locations indicated by the yellow arrows. This design significantly simplifies sample replacement and improves the efficiency of high-throughput imaging workflows.

[0055] Example 1

[0056] 1. For imaging of embryonic hearts without fluorescence, the embryonic hearts were obtained from mouse embryos aged E10.5-E14.5. The specific steps are as follows:

[0057] (1) To enhance the visualization of cell morphology, samples were first fixed with 4% formaldehyde (PFA, mass-volume ratio, 4 g formaldehyde dissolved in 100 ml PBS, pH 7.4, final concentration of PBS was 0.01 M, the same below) at 4°C overnight and then fixed with 2.5% glutaraldehyde (volume ratio, solvent is PBS, pH 7.4, the same below) at room temperature for 2 h.

[0058] (2) The embryonic hearts were rinsed twice with PBS (pH = 7.4) and then dehydrated through a gradient series of ethanol (50%, 75%, 90%, and twice 100% ethanol) (all volume concentrations, ethanol-water) for 30 minutes each. To achieve high throughput, the embryonic hearts were harvested and placed in a 96-well plate, and an automatic dehydrator (NAYO A1 / A8, Naiyou Biotechnology Co., Ltd.) was used for rinsing and dehydration.

[0059] (3) A commercially available hollow ear spoon (approximately 3.8 mm in width and 158 mm in length) was used to transfer the embryonic heart without introducing excessive alcohol into a benzyl alcohol:benzyl benzoate (volume ratio 1:2, referred to as BABB) solution for dehydration and clearing (BABB solution also has a certain degreasing effect). The dehydrated heart was transferred to a glass tube containing 100 μl of BABB solution and cleared at room temperature for 30 minutes.

[0060] (4) Using the ZEISS Lightsheet Z1 light sheet microscope platform, using homemade equipment ( Figures 1 to 3 The samples were fixed and replaced using an X5 / 0.16 air detection lens (n=1.45) and an X5 / 0.1 illumination lens. The cleared heart was immersed in BABB solution in a glass tube, and the glass tube was immersed in an imaging chamber filled with an 87% volume concentration glycerol aqueous solution (refractive index RI of approximately 1.45) for imaging. Tissue autofluorescence imaging was performed using a 561 nm laser line scanning.

[0061] (5) After imaging, Imaris 10.0 software was used for three-dimensional reconstruction and analysis of the images.

[0062] 2. For imaging of embryonic hearts without fluorescence, the embryonic hearts were obtained from mouse embryos at E18.5. The fixation steps were different from those in (1) above, but the remaining steps were the same. Specifically, the samples were fixed directly with 2.5% glutaraldehyde for 2 hours at room temperature to enhance tissue autofluorescence.

[0063] The results are as follows Figure 5 Figures 2 show representative images of embryonic hearts at different stages, captured using the aforementioned method using a ZEISS Lightsheet Z.1. A1-F1 are representative 3D images, A2-F2 are representative sections from the ventral side of the heart, showing remodeling of the outflow tract endothelial cushions, and A3-F3 are representative sections from the dorsal side of the heart, showing remodeling of the atrioventricular endothelial cushions. A4-F5 show 3D sections of the heart, and A4-E4 and A5-E5 show renderings of the endothelial cushions. The red arrows in A2 and A3 indicate the clearly visible endothelial cells on the inner side of the heart at E10.5 days, during the development of trabeculae. A4 shows direct contact between the endothelial cushions of the septal outflow tract and the ventricular septum, which develops from trabeculae, at E10.5 days. B2 and B4 show the aortic and pulmonary artery ostiae at the distal end of the outflow tract at E11.5 days, before the clear separation. B3 and B5 show the superior atrioventricular endothelial cushions (savc) and inferior atrioventricular endothelial cushions (iavc). In C2 and C4, at E12.5, the aortic and pulmonary ostia are clearly separated at the distal end of the outflow tract. In C5, the left and right atrioventricular endocardial cushions gradually develop, but their size is small. In D2 and D4, at E13.5, the outflow tract endocardial cushions fuse, morphologically without a clear boundary, and the arterial valve structure that develops from the outflow tract can be seen. In D3 and D5, the superior and inferior atrioventricular endocardial cushions fuse, and the red arrows indicate that there is still communication between the left and right ventricles. E2-E5: A fully septated four-chamber heart is formed at E14.5. F2-F5 show the complete valve structure after remodeling of the endocardial cushions at E18.5. Right ventricle (RV), left ventricle (LV), outflow tract (OFT), right atrium (RA), left atrium (LA), aorta (AO), pulmonary artery (PA), parietal outflow tract endothelial cushion (PC), septal outflow tract endothelial cushion (SC), superior atrioventricular endothelial cushion (SAVC), inferior atrioventricular endothelial cushion (IAVC), right atrioventricular endothelial cushion (RLAVC), left atrioventricular endothelial cushion (LLAVC), aortic valve (AV), mitral valve (MV), tricuspid valve (TV).

[0064] The ZEISS Lightsheet Z1 light-sheet microscope platform can capture approximately 30 images of the three-dimensional structure of the cleared mouse embryonic heart per hour. For embryonic hearts aged E10.5-E12.5, the X- and Y-axis resolution is less than 1 μm, and the Z-axis resolution is 1-2 μm. For embryonic hearts aged E18.5, the X- and Y-axis resolution is approximately 2 μm, and the Z-axis resolution is 5-7 μm. This enables high-throughput imaging of the mouse embryonic heart's clear three-dimensional structure.

[0065] Example 2

[0066] For fluorescent imaging of embryonic hearts, embryonic hearts were derived from E11.5 Wnt1-Cre; Rosa26 mice.mTmG Mouse embryos and Mef2C-Cre; Rosa26 at E11.5, E15.5, and E18.5 mTmG Mouse embryos. Mef2C-Cre mice were donated by Professor Zhang Zhen of Shanghai Jiao Tong University; Wnt1-Cre mice and Rosa26 mTmG Mice were purchased from Jackson Laboratory, USA, Wnt1-Cre; Rosa26 mTmG Mice were derived from Wnt1-Cre mice and Rosa26 mTmG Mice were obtained by mating, Mef2C-Cre; Rosa26 mTmG Mice were derived from Mef2C-Cre mice and Rosa26 mTmG Mice are obtained by mating.

[0067] The specific steps are as follows:

[0068] (1) Fix in 4% formaldehyde at 4°C overnight.

[0069] (2) The collected hearts were sequentially incubated in glass bottles containing tetrahydrofuran (THF, purchased from Millipore-Sigma, Cat. No. 186562, prepared with water at different volume concentrations) at 10%, 20%, and 30% by volume to remove lipids. For embryos older than E12.5, an additional 40% THF step was added to ensure thorough delipidation. Each step lasted 30 minutes on a shaker at room temperature.

[0070] (3) After a brief rinse with water, the sample was transferred to a specially designed glass tube containing 100 μL of EZ view solution (refractive index (RI) of approximately 1.51). The sample was incubated at room temperature for one hour to achieve optimal transparency. In this example, since BABB solution quenches fluorescence, it is not possible to use BABB to clear fluorescent samples.

[0071] The EZ view solution, also known as iohexol-urea solution, is prepared as described in the literature (Hsu, C.-W., Cerda, J., Kirk, JM, Turner, WD, Rasmussen, TL, Flores Suarez, CP, Dickinson, ME, and Wythe, JD (2022). EZClear for simple, rapid, and robust mouse whole organ clearing. eLife 11, e77419.10.7554 / eLife.77419.). It consists of 5g iohexol and 2.625g urea dissolved in 2ml of 0.03M PBS buffer, resulting in a final PBS concentration of approximately 0.01M. (Note: The EZ view reference uses 0.02M PBS, but we found that dissolution triples the volume, so we used 0.03M PBS.) The refractive index (RI) is approximately 1.51.

[0072] (4) The imaging method is the same as in Example 1, and suitable excitation light (488 nm and 561 nm) is selected for imaging.

[0073] like Figure 6 The results of different delipidation times and concentrations are shown. AF: E11.5 Wnt1-Cre; Rosa26 mTmG Gross and cross-sectional images of the mouse embryonic heart. AB: Delipidation with 10% THF-20% THF-30% THF for half an hour each, revealing normal cardiac structure after transparency. The distribution of green fluorescent cells is clearly visible. CD: Delipidation with 10% THF-20% THF-30% THF-40% THF-50% THF for half an hour each, revealing significant shrinkage of the overall cardiac structure. EF: Delipidation with 10% THF-20% THF-30% THF for three hours each, revealing significant shrinkage of the left and right atria and ventricles. Right ventricle (RV), left ventricle (LV), outflow tract (OFT), right atrium (RA), left atrium (LA), parietal outflow tract endothelial cushion (PC), septal outflow tract endothelial cushion (SC), superior atrioventricular endothelial cushion (SAVC), inferior atrioventricular endothelial cushion (IAVC). Scale bar: 200 μm. Figure 6 The CF in the middle is used as a comparison for degreasing conditions, indicating that too high a THF concentration or too long a degreasing time used for degreasing will cause the heart structure to shrink.

[0074] like Figure 7 The image shows Mef2C-Cre; Rosa26 taken with ZEISS Lightsheet Z.1 using the above method. mTmGRepresentative images of mouse embryos. AC: Gross images and sagittal sections of an E11.5 mouse. Green fluorescent cells derived from Mef2C are primarily distributed in the outflow tract myocardial wall, right ventricle, and ventricular septum. White arrows indicate that green fluorescent cells are partially distributed in the intimal cushions of the outflow tract and to a lesser extent in the intimal cushions of the atrioventricular septum. DF: Gross images and sections of an E15.5 mouse. Green fluorescent cells are almost invisible in the tricuspid and mitral valves (E, representative coronal section), but are distributed in all three leaflets of the aortic and pulmonary valves (F, representative transverse section). GI: ​​Gross images and coronal sections of an E18.5 mouse. Green fluorescent cells are abundantly distributed in the pulmonary and aortic valves, right ventricle, and ventricular septum, but are almost invisible in the mitral valve. Right ventricle (RV), left ventricle (LV), outflow tract (OFT), right atrium (RA), left atrium (LA), parietal outflow tract endothelial cushions (PC), septal outflow tract endothelial cushions (SC), superior atrioventricular endothelial cushions (SAVC), inferior atrioventricular endothelial cushions (IAVC), pulmonary valve (PV), aortic valve (AV), mitral valve (MV), tricuspid valve (TV), ventricular septum (S), left coronary valve (LC), right coronary valve (RC), noncoronary valve (NC), left-facing leaflet (LF), right-facing leaflet (RF), non-opposing leaflet (NF).

Claims

1. A high-throughput imaging method for acquiring the three-dimensional structure of the embryonic heart of an experimental animal, characterized in that: The following steps are involved: (1) collecting embryonic hearts of experimental animals and fixing them with a fixative; the experimental animals are mice; (2) Dehydrating or defatting the embryonic heart fixed in step (1) and then performing a transparent treatment; (3) placing the transparentized embryonic heart in a transparent tube, and then placing it in an imaging chamber filled with a transparent solution with a refractive index of 1.41 to 1.47, for fluorescence imaging; If the embryonic heart does not express exogenous fluorescent proteins, imaging was performed using the enhanced tissue autofluorescence method; If the embryonic heart expresses an exogenous fluorescent protein, fluorescence imaging is performed using the excitation light corresponding to the exogenous fluorescent protein; If the embryonic heart does not express exogenous fluorescent proteins, imaging can be performed using enhanced tissue autofluorescence, in which case: In step (2), the sample is first washed with a buffer solution, then dehydrated, and then degreased and transparentized; the dehydration is performed using an alcohol solution with a concentration gradually increasing until it reaches pure alcohol for gradient dehydration, and a benzyl alcohol: benzyl benzoate solution is used for degreasing and transparentization for 30 minutes; If the embryonic heart expresses exogenous fluorescent protein, the excitation light corresponding to the exogenous fluorescent protein is used for fluorescence imaging. At this time: In step (2), degreasing is performed using tetrahydrofuran solutions with increasing concentrations in a gradient manner; and transparent treatment is performed using iohexol-urea solution for 1 hour; When the dehydration is performed using an alcohol solution with a concentration increasing in sequence until pure alcohol, the volume concentration gradient of the alcohol is set to 50%, 75%, 90%, 100% and 100% in sequence; the processing time for each volume concentration gradient is 30 minutes; The volume ratio of benzyl alcohol to benzyl benzoate in the benzyl alcohol: benzyl benzoate solution is 1:2; When degreasing using tetrahydrofuran solutions with increasing concentrations for gradient degreasing, for embryonic hearts less than E12.5 days, the volume concentration gradient of the tetrahydrofuran solution is set to 10%, 20%, and 30% in sequence; for embryonic hearts more than E12.5 days, the volume concentration gradient is increased to 40%; the treatment time for each volume concentration gradient is 30 minutes; The composition of the iohexol-urea solution is 5 g of iohexol and 2.625 g of urea dissolved in 2 ml of 0.03 M PBS buffer; For fluorescence imaging, the ZEISS Lightsheet Z1 light sheet microscope platform was used for fluorescence imaging, using an X5 / 0.16 air detection lens and an X5 / 0.1 illumination lens; The imaging chamber was perfused with a glycerol aqueous solution with a volume concentration of 87%; During fluorescence imaging, a first magnet is fixed to the bottom end of the sample fixing rod of the ZEISS Lightsheet Z1 light sheet microscope platform, and a sampling rod is used to fix the transparent tube on the sample fixing rod. The sampling rod has a certain elasticity, and a second magnet for being attracted and fixed to the first magnet by opposite charges is provided on the top surface, and a plug-in section for plugging into the inner cavity of the transparent tube and with a diameter gradually decreasing from top to bottom is provided on the bottom.

2. The high-throughput imaging method for acquiring the three-dimensional structure of the embryonic heart of an experimental animal according to claim 1, characterized in that: If the embryonic heart does not express exogenous fluorescent proteins, imaging can be performed using enhanced tissue autofluorescence, in which case: In step (1), the embryonic heart of the experimental animal is from an embryo of E10.5 to E14.5, and when fixation is performed using a fixative, formaldehyde solution is first used for fixation and then glutaraldehyde solution is used for fixation; In step (1), the embryonic heart of the experimental animal is from an embryo of E15.5 to E18.5, and is fixed with a glutaraldehyde solution; If the embryonic heart expresses exogenous fluorescent protein, the excitation light corresponding to the exogenous fluorescent protein is used for fluorescence imaging. In this case, in step (1), formaldehyde solution is used for fixation.

3. The high-throughput imaging method for acquiring the three-dimensional structure of the embryonic heart of an experimental animal according to claim 2, characterized in that: When using formaldehyde solution for fixation, the fixation time shall not be less than 2 hours; when using glutaraldehyde solution for fixation, the fixation time shall not be less than 2 hours.

4. The high-throughput imaging method for acquiring the three-dimensional structure of the embryonic heart of an experimental animal according to claim 1, characterized in that: During the clearing treatment, the sample is placed in a transparent tube and fluorescence imaging is performed directly after the clearing treatment.

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