## 3 Results
Figure 1: Cross-sectional pictures of CE (HH25-HH27 embryonic stage) with various counterstaining: (**A**): no stain, (**B**): 1% silver proteinase, (**C**): 1% eosin, (**D**): 1% I2 KI, (**E**): 5% silver proteinase, (**F**): 5% eosin, (**G**): 3% I2 KI, (**H**): 1% PMA, (**I**): 1% PTA. The upper four rows are profiles of radiopacity: no distinct peaks (1*), brain (1), heart (2), liver (3), sclerotome or neural canal (4). The lower four rows are 3D isosurface renderings. Separate images in high resolution can be found in Supplementary (Figures S1–S9). When utilizing I2 KI, PMA, and PTA stains, 1% PMA staining for 24 h at both 25 \({}^{\circ}\)C and 40 \({}^{\circ}\)C resulted in the biggest visualized embryo volume relative to no stain, whereas 3% I2 KI staining resulted in the least viewable embryo volume relative to no stain. This is a significant drawback of I2 KI and reflects the findings of other researchers [27] about tissue deformation and excessive shrinkage when iodine-based compounds are used as a contrast. Analysis of the X-ray density of CE organs and tissues stained with 3% IKI showed that heating has no impact on the amount of staining, However, keeping for 48 h improves  
Figure 2: Representative microCT cross-sectional images of CE (HH25-HH27 embryonic stage) with different counterstraining and histological section with hematoxylin-eosin staining (magnification \(\times\)2) for comparison. MicroCT cross-sectional images: no stain (**A**), 3% I2 KI (**B**), 1% PMA (**C**), 1% PTA (**D**). Radiopacity profiles: no specific peaks (1*), brain (1), heart (2), liver (3), sclerotome or neural canal (4). Histological section: brain (1), heart (2), liver (3), sclerotome or neural canal (4). Separate images in high resolution can be found in Supplementary (Figures S1 and S7–S9). overall contrast by 2-2.5 times. Although the X-ray density of the tissue was enhanced, it was found that 3% I2 KI staining considerably reduced the distance between CE organs, decreased the amount of differential contrast, and weakened the clarity of the borders between internal organs (Figure 2, Tables 1 and 2). Researchers found the similar shrinkage in human embryos, particularly in relation to fictitious skin calcification. However, it appears that the primary cause of this impact is not related to a change in stain osmolarity, as originally thought by researchers [37], but rather to the I2 KI solution's progressive acidification, which has also been validated in subsequent studies [36]. The results of X-ray density calculation for CE organs and tissues stained with 1% PMA contrast indicate that there is no pronounced effect of exposure time on the quality of visualization. At the same time, heating to 1% PMA staining to 40 \({}^{\circ}\)C results in a significant increase in general and differentiated CE organs contrast by 20% and histograms alignment (grey value). Importantly, that differentiated contrast between CE organs was well expressed at 40 \({}^{\circ}\)C after 24 h exposure and was 2395.9 \(\pm\) 148 in brain, 5714.7 \(\pm\) 176 in heart, 11,255 \(\pm\) 559 in liver and 5744.5 \(\pm\) 212 in sclerotome or neural canal (Table 3). All of the data acquired, in our opinion, point to the application of 1% PTA at 40 \({}^{\circ}\)C for 24 h as the most ideal and effective way of CE staining throughout the early stages of growth. We next applied this strategy for staining CE from 4 to 8 days (HH22-HH34) of embryogenesis with identification of internal organs after microCT imaging in the following step of our research. The findings obtained are shown in Figure 3 and Table 4, illustrating a high degree of visibility of CE organs and tissues from 4 to 8 days (HH22-HH34) when utilizing the technique of 1% PTA staining at 40 \({}^{\circ}\)C heating and exposure for 24 h. Filters Al 0.5 mm, Al 1 mm, and Al + Cu were utilized with a passing radiation level in most CE planes in the range of 30-50% while scanning CE at all phases of development, allowing visible separation of the surrounding backdrop from the CE structures.