High-Performance Microcomputing Tomography of Chick Embryo in the Early Stages of Embryogenesis
by
, , and
Igor Rzhepakovsky
1
,
Sergei Piskov
1,
Svetlana Avanesyan
1,
Magomed Shakhbanov
1,
Marina Sizonenko
1,
Lyudmila Timchenko
1,
Mohammad Ali Shariati
2
,
Maksim Rebezov
3,4 and
Andrey Nagdalian
5,*
1
Faculty of Medicine and Biology, North-Caucasus Federal University, Pushkina Street 1, 355000 Stavropol, Russia
2
Scientific Department, Semey Branch of Kazakh Research Institute of Processing and Food Industry, Gagarin Avenue 238G, Almaty 050060, Kazakhstan
3
Department of Scientific Research, V. M. Gorbatov Federal Research Center for Food Systems, Talalikhin Street 26, 109316 Moscow, Russia
4
Faculty of Biotechnology and Food Engineering, Ural State Agrarian University, Karl Liebknecht Street 42, 620075 Yekaterinburg, Russia
5
Laboratory of Food and Industrial Biotechnology, North-Caucasus Federal University, Pushkina Street 1, 355000 Stavropol, Russia
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(19), 10642; https://doi.org/10.3390/app131910642
Submission received: 16 August 2023
/
Revised: 20 September 2023
/
Accepted: 22 September 2023
/
Published: 25 September 2023
(This article belongs to the Special Issue Advances in Molecular Imaging and Its Biomedical Application)
Abstract
:X-ray contrast techniques were tested on the chick embryos in early periods of embryogenesis. For contrast stain, reagents with radiopacity in various concentrations were used: silver proteinate, eosin, Lugol’s solution (I2KI), phosphomolybdic acid and phosphotungstic acid under heating at 25 °C and 40 °C and exposure for 24 and 48 h. The use of silver proteinate, eosin and I2KI in various concentrations in the contrast of the chick embryo in the early period of embryogenesis did not make it possible to obtain microtomographic results that provide reliable microstructural analysis. The most optimal and effective method of embryo staining at the HH22–HH34 embryonic stages reliably determined the staining of 1% phosphotungstic acid at 40 °C heating and exposure for 24 h. Taking into account the size of the chick embryos and their structures at the HH22–HH34 embryonic stages, the features of the development, location of organs, and the minimum permissible parameters of microtomography for obtaining high-quality and reliable results were determined by the isometric spatial resolution of 8.87 μm, X-ray voltage 50 kV, X-ray current 500 μA, and the use of filters started from Al 0.5 mm. Microtomographic results were obtained, characterized by the appearance of the chick embryo at the HH22–HH34 embryonic stages, and they visualized the locations and structures of the chick embryo organs and provided calculation of their volume and X-ray density. The results of the work open up significant prospects for using the chick embryo at the early embryonic period of embryogenesis as an alternative model for screening teratogenicity.
1. Introduction
For many years, scientists have been interested in using the chick embryo (CE) as a model for experimental pharmacology, medicine, and embryology. The growing CE possesses a variety of characteristics that make it an efficient biological model on which teratogenic effects on all significant organ systems in embryogenesis may be studied with a high level of accuracy. It has a short incubation period, is economically favorable, is of a suitable size, is accessible for a variety of experimental manipulation techniques, is unaffected by the metabolism of the mother organism, and is available [1,2,3].
The CE model is particularly helpful for researching the teratogenicity of novel drugs and materials during the development stage when scientists obtain a large number of experimental samples. In terms of high costs, difficult experiments, lengthy durations, labor intensity, and a large number of animals, the model of pregnant mammals, which is the principal one for assessing teratogenicity prior to preclinical research, is far from ideal. Professional experience has shown that this is particularly important when the market for new pharmaceuticals is growing quickly and when viral pandemics require the rapid development of vaccines, medications, and other treatments, the majority of which lack data demonstrating the safety of use during pregnancy [4,5]. In this regard, the CE model may be a good substitute, but there is still a need for this animal model to be more consistently applied [6].
Morphological characteristics in this context are crucial aspects for investigating the processes of development and its diseases, since embryonic development is a very complicated process involving dynamic temporal changes in many structures. The morphological information that is now available is also scarce and ambiguous. The primary cause of this is the difficulty of utilizing conventional histological examination techniques to investigate the three-dimensional anatomy of embryos, particularly during the early stages of development, which are characterized by their tiny size and intricate spatial features [7,8].
The use of three-dimensional visualization techniques is crucial in this regard [9,10,11,12,13]. Among these, the X-Ray microtomography (microCT) technique is gaining popularity for researching both healthy embryogenesis and developmental abnormalities [14,15,16]. This is a non-destructive method of visualizing the internal structure of biological objects. Its benefits include high investigation speed, high resolution, accurate quantitative assessment of the size and morphology of tissues, the possibility of full-volume visualization, and analysis of inter-organ relations of the object with its saving for other types of research [17,18].
The low X-ray density of embryonic tissues, particularly in the early stages of development, limits the broad adoption of the approach, despite the benefits of microCT in providing quantitative 3D images. The ability of the microCT approach to acquire high-resolution structural information is constrained by the poor inherent contrast of CE tissues [19]. The neurological system and thoraco-abdominal organs are laid down and differentiated during the embryonic phase, which makes it a crucial time for teratogenic alterations.
In order to collect data sets with high contrast and high resolution for complete embryos and particular organ systems, contrast agents rich in X-ray radiation are used [14]. Due to the features of the substances, tissues, diffusion, and sample sizes, various staining agents have varying impacts on the quality of microCT images of organs [20,21]. Currently, a few contrast chemicals have been found for using in microCT images of soft tissues in embryos. At the moment, distinct approaches for visualizing structural components in microCT exist [22,23]. Reduced X-ray density in the picture backdrop allows for different microCT imaging techniques that enable the non-contrast visualization of the embryo’s shape and its development of organs [14,24]. For instance, synchrotron radiation-based microCT may boost the contrast-to-noise ratio by up to 200 times, but it is realistically only possible with synchrotron light sources and systems that use precise focus X-ray tubes [25]. However, none of them are sufficient for extensive teratogenicity screening. The main drawbacks are prolonged exposure to the contrast medium, high contrast toxicity, substantial sample shrinkage after contrast staining, and ambiguous organ visual separation [26,27].
The relevance of this study is determined by the lack of clear recommendations on the use of particular contrast agents suitable for microCT imaging of CE, particularly at the early stages of embryogenesis as a substitute model for screening teratogenicity and in the aspect of comparative morphology. In light of this, the goal of this work was to create an X-ray contrast technique that was both ideal and efficient for microCT imaging of CE during the early stages of embryogenesis.
2. Materials and Methods
2.1. Chemicals
Chemicals were obtained from the following sources: formalin solution, neutral buffered 10%, isopropyl alcohol ≥ 99.7%, ethyl alcohol 95%, medical paraffin Histomix, hematoxylin, eosin (Biovitrum, St. Petersburg, Russia); phosphotungstic acid hydrate 99.99%, phosphomolybdic acid ≥ 99.99, silver proteinate ~8% Ag, potassium iodide ≥ 99.0%, iodine ≥ 99.9% (Sigma-Aldrich, St. Louis, MO, USA).
2.2. Embryo Preparation
Fertilized chicken eggs “Hysex Brown” (Agrokormservice Plus, Adygea, Russia) were used for the experiment. Eggs were incubated for periods from 4 to 8 days (from HH22 to HH34) at 37.5 °C and 50% relative humidity in a digital incubator Rcom Maru Deluxe Max 380 (Autoelex Co., Gyeongsangnam-do, Korea). Embryos were sacrificed by chilling (<4 °C for 4 h) for each day of incubation (between 4 and 8 days), in accordance with the CE’s recommendations for compassionate euthanasia [28,29].
The eggs were cracked starting at the blunt end. A little part of the inner membrane and the shell were cut away to reveal the embryo. The use of CE for subsequent imaging was avoided when the membrane bled or the CE exhibited visible structural flaws [30]. For 72 h, the retrieved embryos were preserved in a 10% buffered formalin solution.
2.3. Stain
The following radiopaque reagents were employed to assess contrast staining on CE (5 day, HH25-HH27): phosphomolybdic acid (PMA, 1%), phosphotungstic acid (PTA, 1%), eosin (1%), eosin (5%), silver proteinate (1%), and silver proteinate (5%). The selection of radiopaque reagents was influenced by the fact that they are utilized in other imaging techniques and can, in some cases, offer precise biological targeting, uniform and thorough staining, rapid tissue penetration without producing artifacts, like diffusion rings, staining of bulk and dense tissues, and compatibility with histopathological examination [27,31,32].
The CE fixed in a 10% buffered formalin solution were washed under running water for 12 h, dehydrated in replaceable portions of ethanol 30% (2 h), 50% (2 h), 70% (12 h) and placed in solutions of radiopaque stains at 1:20 (V of embryo to V of solution), and were kept for 24 and 48 h at 25 °C and 40 °C (Table 1).
2.4. MicroCT Imaging Systems
Test tubes containing CE samples were used to convey them to the Skyscan 1176 microtomograph (Bruker, Kontich, Belgium), where foam retainers were used to hold the tubes in position. An 11-megapixel camera (4000 × 2672 pixels) was rotated 180° (0.3°/step) to perform CE scanning. Three images were averaged for an 8.87 m isometric spatial resolution throughout each phase. The main variable factor set in the Skyscan 1176 Control software (Bruker, Kontich, Belgium) was the thickness of the absorber (filter), which depends on the level of X-ray contrast and the thickness of the object. On the majority of CE flights, the passing radiation level should be between 30 and 50%. In this case, the intensity of X-ray radiation was determined automatically, depending on the chosen filter, to represent the overall and distinct contrast of the object’s parts in the best way; all images were included in the histogram’s contrast zone (minimum and maximum gray values). To obtain the best image quality with minimal scanning time, the voxel size and scans averaged parameters were constant for all samples. The reconstruction parameters, ring artifact correction, beam hardening correction, and the minimum and maximum for CS to image conversion, were selected depending on the output image and the histogram of distribution of gray values of all images. A wider filter highlighted the embryo in respect to the surroundings and raised the overall level of object contrast.
The analysis and reconstruction of grouped images, commonly known as picture stacks, into 3D data sets, was performed using the NRecon 1.7.4.2 program from Bruker in Kontich, Belgium. It took around two hours to prepare each sample tube. For post-processing, alignment, orientation in space (x, y, and z), mapping of X-ray contrast profiles, and highlighting certain areas of reconstructed materials, DataViewer 1.5.6.2 software (Bruker, Kontich, Belgium) was utilized. Three-dimensional images were viewed using the CTvox 3.3.0r1403 program (Bruker, Kontich, Belgium).
Using the CT-analyser 1.18.4.0 software (Bruker, Kontich, Belgium) and our own procedures reported in other works [33,34]. we performed morphometry and evaluated the X-ray density of various CE structures in Hounsfield units (HU). MicroCT scan settings used in the experiment are presented in Table 2.
2.5. Histological Preparation
To assess the quality of CE organs differentiation on microCT images, following the example of other researchers [35], their verification with appropriate histological sections was carried out. This was accomplished by dehydrating a 5-day CE in isopropyl alcohol and encasing it in Histomix medical paraffin, which was provided by Biovitrum (a Saint Petersburg, Russia-based company). A rotary microtome NM 325 (Thermo Fisher Scientific, Waltham, MA, USA) was used to create histological sections with a thickness of 6 m in the sagittal plane. Hematoxylin and eosin were used to stain the sections. The Axio Imager 2 (A2) research class microscope from Carl Zeiss Microscopy (Oberkochen, Germany), along with a dedicated AxioCam MRc5 camera and Zen 2 Pro software, were used to capture images of the micropreparations.
2.6. Statistical Data Processing
For each contrast method indicated in Table 1, 10 samples were used. For visualization of 2D and 3D structures, as well as for visualization of radiopacity profiles, the most characteristic representative materials were used. Segmentation and quantification were carried out according to the recommendations of Kim et al. [26].
The assessment of individual differences in the samples was carried out using statistical analysis using ANOVA, followed by post hoc testing using p < 0.05 as a significance threshold.
3. Results
The outcomes, as shown in Figure 1 and Supplementary (Figures S1–S9)—images of Figure 1 in high resolution, demonstrate how the staining process affected the visualized quantities of CE. Silver proteinate (1% and 5%) staining had a layer of the stain on the external tissues of CE, which increased its natural size and distorted the outlines in addition to having weak differential contrast. Figure 1 also shows a low level of general and differential contrast at staining with 1% silver proteinate and eosin solutions. Perhaps these results originate from an inadequate amount of stain concentration. The key limiting element in this aspect, according to Busse et al. [32], is the low concentration of eosin employed for soft tissue contrast. However, in our case, staining with eosin, particularly at 5% concentration, caused deformations in the embryo tissues as well as a significant overall reduction in the volume of the tissues, which, in the opinion of some [36], may cause artifacts that are mistaken for anomalies of embryonic development.
The relative rise in general contrast level of CE when stained with 1% I2 KI and 5% silver proteinate and eosin solutions does not allow for obvious differentiations of organs and tissues. All of the techniques of coloring described above permitted the use of filters that only contained a high degree of transmitted radiation or scanning without a filter at all, which increased the number of picture defects and decreased image quality. Simultaneously, the exposure duration and temperature had little effect on the outcome.
Staining with 3% I2KI and 1% solutions of PMA and PTA (Figure 2) made it possible to achieve a high level of general contrast of CE at embryonic stage HH25 (5 day) and gave more histologically interpreted data. This is evidenced by both the use of 0.5–1 mm Al filters (Table 2) during scanning and the low histogram (grey value) profile of the area around the CE.
When utilizing I2KI, PMA, and PTA stains, 1% PMA staining for 24 h at both 25 °C and 40 °C resulted in the biggest visualized embryo volume relative to no stain, whereas 3% I2KI staining resulted in the least viewable embryo volume relative to no stain. This is a significant drawback of I2KI 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 overall contrast by 2–2.5 times. Although the X-ray density of the tissue was enhanced, it was found that 3% I2KI 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, Table 1 and Table 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 I2KI 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 °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 °C after 24 h exposure and was 2395.9 ± 148 in brain, 5714.7 ± 176 in heart, 11,255 ± 559 in liver and 5744.5 ± 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 °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 °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. Simultaneously, the histogram (grey value) peaks defining the several CE organs were at a high level.
Figure 3 shows distinct contrast and numerous organs highlighted in three projections. The applied technic allowed us to obtain a high-quality 3D visualization of isosurfaces, as also can be seen from Figure 3. Furthermore, we assessed the X-ray density of several CE structures created using the recommended approach (Table 4).
Table 3 shows that during growth and development from the fourth to eighth days (HH22–HH34 embryonic stages), the total X-ray density of CE tissues prepared with the suggested method decreases. This is due to both an increase in size and a decrease in the permeability of the contrasting substance, and a change in the ratio of stroma and parenchyma in organs. From the fourth to fifth days, there was a pronounced decrease in the X-ray density of the meninges and the eye by more than two times. The total X-ray density of the embryo, as well as the structures of the sclerotome (neural canal), mesonephros and stomach also decreased by 20–30%. The X-ray density of the heart and liver structures increased to 20%. From the fifth to sixth days, the change in both the total X-ray density and individual organs and structures does not change significantly, apparently due to minor morphological changes, unlike an increase in the CE by 5–6 times, as well as with the active laying and growth of individual organs from the fourth to fifth days of incubation. From the sixth to seventh days, the general X-ray density of CE and X-ray density of almost all CE organs and tissues, except the brain, are particularly pronounced. From the seventh to eighth days, the X-ray density of the studied structures does not change significantly, except for an increase in the brain, which is associated with an increase in the volume of the meninges in its structure, the appearance of the inflection point of the lateral ventricle on the sixth day [38], as well as with a possible increase in cell density, which according to some data [35] may contribute to better stain penetration.
The computation of the volumes of the structures to be stained, which is shown in Table 5, is a crucial component of structural analysis in microCT, in addition to X-ray density.
The visualized volume of CE generated using the recommended approach grows by 595% from day 4 to day 5, 131% from day 5 to day 6, 267% from day 6 to day 7, and 155% from day 7 to day 8 as the embryo develops. According to the findings, the brain, together with the eyes and heart, occupy over 20% of the cerebellum between the fourth and sixth days of development. The liver, excretory, digestive, and reproductive systems start actively growing on day seven. On the beginning on the sixth day of embryogenesis, the lungs may be seen. All of the data met the criteria for the growth of the embryonic phases under study.
4. Conclusions
The early phases of embryogenesis (HH22–HH34) could not be contrasted with silver proteinate, eosin, or Lugol’s solution (I2KI) without obtaining unreliable microCT data for trustworthy microstructural investigation. According to our findings, using 1% phosphotungstic acid at 40 °C heating for a 24 h exposure time is the most ideal and productive way to stain CE at the HH22–HH34 embryonic stages.
Given the size of CE and their structures at the HH22–HH34 embryonic stages, the peculiarities of the development and location of organs, the minimum permissible parameters of microCT for obtaining reliable, high-quality results are an isometric spatial resolution of 8.87 μm, X-ray voltage 50 kV, X-ray current 500 μA and the use of filters starting from Al 0.5 mm upwards. The MicroCT results enabled us to characterize the appearance of the CE at the HH22–HH34 embryonic stages to visualize the locations, structures of CE organs and to calculate volume and X-ray density. This article confirms once again the value of CE as an animal model and the importance of its standardization in the aspect of morphological analysis. The results obtained open up significant prospects for the use of CE in the early embryogenesis as an alternative model for teratogenicity screening.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app131910642/s1, Figure S1: Representative cross-sectional images of CE (HH25-HH27 embryonic stage), no stain (A). Radiopacity profile: no specific peaks (1*); Figure S2: Representative cross-sectional images of CE (HH25-HH27 embryonic stage), 1% silver proteinate. Radiopacity profiles: brain (1), heart (2), liver (3), sclerotome or neural canal (4).; Figure S3: Representative cross-sectional images of CE (HH25-HH27 embryonic stage), 1% eosin. Radiopacity profiles: brain (1), heart (2), liver (3), sclerotome or neural canal (4): Figure S4: Representative cross-sectional images of CE (HH25-HH27 embryonic stage), 1% I2KI. Radiopacity profiles: brain (1), heart (2), liver (3), sclerotome or neural canal (4); Figure S5: Representative cross-sectional images of CE (HH25-HH27 embryonic stage), 5% silver proteinate.. Radiopacity profiles: brain (1), heart (2), liver (3), sclerotome or neural canal (4); Figure S6: Representative cross-sectional images of CE (HH25-HH27 embryonic stage), 5% eosin. Radiopacity profiles: brain (1), heart (2), liver (3), sclerotome or neural canal (4); Figure S7: Representative cross-sectional images of CE (HH25-HH27 embryonic stage), 3% I2KI. Radiopacity profiles: brain (1), heart (2), liver (3), sclerotome or neural canal (4); Figure S8: Representative cross-sectional images of CE (HH25-HH27 embryonic stage), 1% PMA. Radiopacity profiles: brain (1), heart (2), liver (3), sclerotome or neural canal (4); Figure S9: Representative cross-sectional images of CE (HH25-HH27 embryonic stage), 1% PTA. Radiopacity profiles: brain (1), heart (2), liver (3), sclerotome or neural canal (4).
Author Contributions
Conceptualization, I.R. and L.T.; methodology, S.P. and S.A.; software, I.R.; validation, I.R. and S.P.; formal analysis, A.N. and M.R.; investigation, I.R., S.P., S.A., M.S. (Magomed Shakhbanov) and M.S. (Marina Sizonenko); resources, A.N.; data curation, I.R., S.P. and S.A.; writing—original draft preparation, I.R., S.P. and M.S. (Marina Sizonenko); writing—review and editing, L.T., A.N., M.A.S. and M.R.; visualization, I.R. and S.P.; supervision, L.T.; project administration, I.R.; funding acquisition, I.R. and A.N. All authors have read and agreed to the published version of the manuscript.
Funding
The study was funded by a grant from the Russian Science Foundation No. 23-24-00282, https://rscf.ru/en/project/23-24-00282/ (last accessed 7 May 2023).
Institutional Review Board Statement
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of North Caucasus Federal University (protocol code 003 from 3 August 2023).
Informed Consent Statement
Not applicable.
Data Availability Statement
All raw data are available upon request from the corresponding authors.
Acknowledgments
The authors are open for collaboration in the field of microcomputing tomography of different objects.
Conflicts of Interest
The authors declare no conflict of interest.
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Figure 1.
Cross-sectional pictures of CE (HH25–HH27 embryonic stage) with various counterstaining: (A): no stain, (B): 1% silver proteinate, (C): 1% eosin, (D): 1% I2KI, (E): 5% silver proteinate, (F): 5% eosin, (G): 3% I2KI, (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).
Figure 1.
Cross-sectional pictures of CE (HH25–HH27 embryonic stage) with various counterstaining: (A): no stain, (B): 1% silver proteinate, (C): 1% eosin, (D): 1% I2KI, (E): 5% silver proteinate, (F): 5% eosin, (G): 3% I2KI, (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).
Figure 2.
Representative microCT cross-sectional images of CE (HH25-HH27 embryonic stage) with different counterstaining and histological section with hematoxylin-eosin staining (magnification ×2) for comparison. MicroCT cross-sectional images: no stain (A), 3% I2KI (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).
Figure 2.
Representative microCT cross-sectional images of CE (HH25-HH27 embryonic stage) with different counterstaining and histological section with hematoxylin-eosin staining (magnification ×2) for comparison. MicroCT cross-sectional images: no stain (A), 3% I2KI (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).
Figure 3.
Representative cross-sectional images of CE (HH22–HH34 embryonic stages) counterstained with 1% PTA (left side) and isosurface 3D renderings (right side). The following organs are marked on cross-sectional images of CE: brain (1), eye (2), heart (3), liver (4), sclerotome (spine and neural canal) (5), mesonephros (6), stomach (7), lungs (8).
Figure 3.
Representative cross-sectional images of CE (HH22–HH34 embryonic stages) counterstained with 1% PTA (left side) and isosurface 3D renderings (right side). The following organs are marked on cross-sectional images of CE: brain (1), eye (2), heart (3), liver (4), sclerotome (spine and neural canal) (5), mesonephros (6), stomach (7), lungs (8).
Table 1.
Compositions and protocols for counterstaining CE (HH25-HH27 embryonic stage), n = 10, M ± m.
Table 1.
Compositions and protocols for counterstaining CE (HH25-HH27 embryonic stage), n = 10, M ± m.
| # | Stain | Exposure Time, h | Temperature, °C | Visualized Volume Relative to no Stain, mm3 (%) |
|---|---|---|---|---|
| 1 | No stain (70% ethanol) | - | - | 196.0 ± 4.0 (100) |
| 2 | Silver proteinate, 1% (70% ethanol) | 24 | 25 | 147.3 ± 3.1 (75.2) |
| 3 | Silver proteinate, 1% (70% ethanol) | 24 | 40 | 91.8 ± 3.9 (48.8) |
| 4 | Silver proteinate, 1% (70% ethanol) | 48 | 25 | 160.0 ± 4.5 (61.2) |
| 5 | Silver proteinate, 1% (70% ethanol) | 48 | 40 | 84.3 ± 3.1 (43.0) |
| 6 | Eosin, 1% (70% ethanol) | 24 | 25 | 90.1 ± 3.2 (46.0) |
| 7 | Eosin, 1% (70% ethanol) | 24 | 40 | 93.7 ± 3.5 (47.8) |
| 8 | Eosin, 1% (70% ethanol) | 48 | 25 | 104.7 ± 4.2 (53.4) |
| 9 | Eosin, 1% (70% ethanol) | 48 | 40 | 81.2 ± 2.9 (41.4) |
| 10 | I2KI, 1% (70% ethanol) | 24 | 25 | 106.6 ± 4.1 (54.4) |
| 11 | I2KI, 1% (70% ethanol) | 24 | 40 | 92.8 ± 3.8 (47.4) |
| 12 | I2KI, 1% (70% ethanol) | 48 | 25 | 98.0 ± 3.6 (50.0) |
| 13 | I2KI, 1% (70% ethanol) | 48 | 40 | 101.3 ± 4.1 (51.7) |
| 14 | Silver proteinate, 5% (70% ethanol) | 24 | 25 | 128.1 ± 5.0 (65.4) |
| 15 | Silver proteinate, 5% (70% ethanol) | 24 | 40 | 109.4 ± 4.9 (55.8) |
| 16 | Silver proteinate, 5% (70% ethanol) | 48 | 25 | 166.2 ± 6.1 (84.8) |
| 17 | Silver proteinate, 5% (70% ethanol) | 48 | 40 | 213.3 ± 7.2 (108.8) |
| 18 | Eosin, 5% (70% ethanol) | 24 | 25 | 66.0 ± 3.3 (33.7) |
| 19 | Eosin, 5% (70% ethanol) | 24 | 40 | 53.8 ± 3.1 (27.5) |
| 20 | Eosin, 5% (70% ethanol) | 48 | 25 | 34.7 ± 2.1 (17.7) |
| 21 | Eosin, 5% (70% ethanol) | 48 | 40 | 32.1 ± 2.8 (16.4) |
| 22 | I2KI, 3% (70% ethanol) | 24 | 25 | 55.3 ± 3.1 (28,2) |
| 23 | I2KI, 3% (70% ethanol) | 24 | 40 | 47.0 ± 2.2 (24,0) |
| 24 | I2KI, 3% (70% ethanol) | 48 | 25 | 51.6 ± 2.1 (26.3) |
| 25 | I2KI, 3% (70% ethanol) | 48 | 40 | 45.0 ± 2.5 (23.0) |
| 26 | PMA, 1% (70% ethanol) | 24 | 25 | 146.0 ± 4.1 (74.5) |
| 27 | PMA, 1% (70% ethanol) | 24 | 40 | 152.0 ± 5.6 (77.6) |
| 28 | PMA, 1% (70% ethanol) | 48 | 25 | 98.0 ± 3.1 (50.0) |
| 29 | PMA, 1% (70% ethanol) | 48 | 40 | 116.3 ± 6.2 (59.3) |
| 30 | PTA, 1% (70% ethanol) | 24 | 25 | 105.2 ± 6.5 (53.7) |
| 31 | PTA, 1% (70% ethanol) | 24 | 40 | 105.3 ± 5.1 (53.7) |
| 32 | PTA, 1% (70% ethanol) | 48 | 25 | 92.4 ± 4.1 (47.1) |
| 33 | PTA, 1% (70% ethanol) | 48 | 40 | 92.0 ± 3.2 (46.9) |
Table 2.
MicroCT scan settings for contrast stain CE (HH25-HH27 embryonic stage), n = 10.
| Stain | Exposure Time, h | Tempe-Rature, °C | Filter | X-ray Voltage, kV | X-ray Current, μA | Scans Averaged | Voxel Size (μm) | Ring Artifact Correction | Beam Hardening Correction (%) | Minimum for CS to Image Conversion | Maximum for CS to Image Conversion |
|---|---|---|---|---|---|---|---|---|---|---|---|
| No stain (70% ethanol) | - | - | Al 0.2 mm | 45 | 550 | 3 | 8.87 | 10 | 20 | 0.001 | 0.03 |
| Silver proteinate, 1% (70% ethanol) | 24; 48 | 25; 40 | no filter; Al 0.2 mm | 40; 45 | 550; 600 | 3 | 8.87 | 10 | 0 | 0.006–0.01 | 0.02–0.03 |
| Eosin, 1% (70% ethanol) | 24; 48 | 25; 40 | no filter | 40 | 600 | 3 | 8.87 | 10 | 0 | 0.01 | 0.03 |
| I2KI, 1% (70% ethanol) | 24; 48 | 25; 40 | Al 0.2 mm; Al 0.5 mm | 45; 50 | 500; 550 | 3 | 8.87 | 10 | 30 | 0.002–0.005 | 0.017–0.025 |
| Silver proteinate, 5% (70% ethanol) | 24; 48 | 25; 40 | no filter; Al 0.2 mm | 40; 45 | 550; 600 | 3 | 8.87 | 10; 20 | 21 | 0.002–0.004 | 0.025–0.035 |
| Eosin, 5% (70% ethanol) | 24; 48 | 25; 40 | Al 0.2 mm | 45 | 550 | 3 | 8.87 | 10 | 21 | 0.002–0.003 | 0.025 |
| I2KI, 3% (70% ethanol) | 24; 48 | 25; 40 | Al 0.5 mm; Al 1 mm | 50; 65 | 380; 500 | 3 | 8.87 | 10 | 41 | 0.002–0.003 | 0.06–0.09 |
| PMA, 1% (70% ethanol) | 24; 48 | 25; 40 | Al 0.5 mm; Al 1 mm | 50; 65 | 380; 500 | 3 | 8.87 | 10 | 41 | 0.001–0.002 | 0.07–0.09 |
| PTA, 1% (70% ethanol) | 24; 48 | 25; 40 | Al 1 mm | 65 | 380 | 3 | 8.87 | 10 | 41 | 0.001 | 0.075–0.1 |
Table 3.
X-ray density of various structures of CE (HH25-HH27 embryonic stage), n = 5.
| Stain, Exposure Time, Temperature | X-ray Density, HU | ||||
|---|---|---|---|---|---|
| The Chick Embryo | Brain | Heart | Liver | Sclerotome (Neural Canal) | |
| I2KI (3%), 24 h, 25 °C | 1089.1 ± 50 | 1526.4 ± 75 | 3503.4 ± 129 | 6972.4 ± 264 | 2991.6 ± 140 |
| I2KI (3%), 24 h, 40 °C | 839.6 ± 47 | 957.3 ± 43 | 3152.4 ± 166 | 6979.3 ± 348 | 3347.9 ± 273 |
| I2KI (3%), 48 h, 25 °C | 2845.1 ± 235 | 3307.6 ± 201 | 7228.8 ± 301 | 15,525.0 ± 699 | 8181.1 ± 465 |
| I2KI (3%), 48 h, 40 °C | 2483.8 ± 178 | 2084.4 ± 156 | 6570.1 ± 435 | 13,575.0 ± 621 | 7785.9 ± 289 |
| PMA (1%), 24 h, 25 °C | 384.9 ± 29 | 759.8 ± 56 | 1629.0 ± 91 | 3603.6 ± 188 | 1680.4 ± 110 |
| PMA (1%), 24 h, 40 °C | 635.1 ± 42 | 1349.2 ± 72 | 2120.9 ± 99 | 4565.9 ± 245 | 2080.9 ± 130 |
| PMA (1%), 48 h, 25 °C | 472.8 ± 29 | 810.5 ± 44 | 1825.1 ± 102 | 4138.9 ± 203 | 1907.0 ± 98 |
| PMA (1%), 48 h, 40 °C | 828.7 ± 37 | 1544.0 ± 128 | 2953.4 ± 144 | 5685.5 ± 276 | 2344.3 ± 175 |
| PTA (1%), 24 h, 25 °C | 1652.7 ± 95 | 1962.4 ± 108 | 3992.9 ± 264 | 8030.0 ± 301 | 4716.8 ± 234 |
| PTA (1%), 24 h, 40 °C | 2065.5 ± 116 | 2395.9 ± 148 | 5714.7 ± 176 | 11,255.0 ± 559 | 5744.5 ± 212 |
| PTA (1%), 48 h, 25 °C | 1790.0 ± 102 | 2258.2 ± 190 | 4026.8 ± 134 | 8611.1 ± 301 | 5653.6 ± 267 |
| PTA (1%), 48 h, 40 °C | 2362.3 ± 157 | 2626.2 ± 114 | 6185.8 ± 312 | 10,816.0 ± 489 | 5716.2 ± 229 |
Table 4.
X-ray density of various structures of CE (HH22-HH34 embryonic stages) stained with 1% PTA solution for 24 h at 40 °C, n = 5.
Table 4.
X-ray density of various structures of CE (HH22-HH34 embryonic stages) stained with 1% PTA solution for 24 h at 40 °C, n = 5.
| Structures | X-ray Density, HU | ||||
|---|---|---|---|---|---|
| 4 Days (HH22–HH24) | 5 Days (HH25–HH27) | 6 Days (HH28–HH29) | 7 Days (HH30–HH32) | 8 Days (HH33–HH34) | |
| The chick embryo | 3115.9 ± 111 | 2065.5 ± 116 | 2447.7 ± 125 | 1517.2 ± 84 | 1807.9 ± 103 |
| Brain | 5284.4 ± 202 | 2395.9 ± 148 | 2752.9 ± 112 | 2393.7 ± 135 | 3238.3 ± 193 |
| Eye (left) | 7514,4 ± 257 | 3780.2 ± 180 | 3255.0 ± 177 | 1693.4 ± 95 | 1897.2 ± 98 |
| Heart | 4923.2 ± 226 | 5714.7 ± 176 | 5401.1 ± 245 | 3513.8 ± 164 | 3563.9 ± 198 |
| Liver | 9244.0 ± 378 | 11,255.0 ± 559 | 12,155.0 ± 589 | 5763.6 ± 243 | 6154.7 ± 302 |
| Sclerotome (spine and neural canal) | 6978.4 ± 320 | 5744.5 ± 212 | 6339.1 ± 369 | 2658.6 ± 139 | 2775.2 ± 173 |
| Mesonephros (left) | 10,091.0 ± 461 | 8785.3 ± 342 | 9659.7 ± 401 | 4302.4 ± 157 | 4382.3 ± 189 |
| Stomach | 13,873.0 ± 598 | 10,064.0 ± 497 | 9785.7 ± 389 | 4233.1 ± 187 | 4298.8 ± 194 |
| Lungs (left) | not defined | not defined | 10,038 ± 450 | 3624.5 ± 143 | 2911.0 ± 157 |
Table 5.
Visualized volume of CE and CE organs stained with 1% PTA solution for 24 h at 40 °C with (HH22-HH34 embryonic stages), n = 5, M ± m.
Table 5.
Visualized volume of CE and CE organs stained with 1% PTA solution for 24 h at 40 °C with (HH22-HH34 embryonic stages), n = 5, M ± m.
| Structures | Visualized Volume, mm3 | ||||
|---|---|---|---|---|---|
| 4 Days, (HH22–HH24) | 5 Days, (HH25–HH27) | 6 Days, (HH28–HH29) | 7 Days, (HH30–HH32) | 8 Days, (HH33–HH34) | |
| The chick embryo | 17.7 ± 1.6 | 105.3 ± 5.1 | 137.6 ± 8.9 | 367.6 ± 23.2 | 571.3 ± 38.0 |
| Brain | 3.7 ± 0.2 | 22.3 ± 1.9 | 27.9 ± 1.7 | 54.5 ± 3.4 | 80.8 ± 4.7 |
| Eye (left) | 0.31 ± 0.04 | 3.94 ± 0.5 | 6.54 ± 0.5 | 33.3 ± 1.9 | 66.0 ± 3.2 |
| Heart | 0.65 ± 0.05 | 2.33 ± 0.1 | 2.91 ± 0.2 | 6.6 ± 0.5 | 10.5 ± 0.9 |
| Liver | 0.08 ± 0.006 | 0.74 ± 0.04 | 1.1 ± 0.05 | 5.1 ± 0.3 | 10.1 ± 0.8 |
| Mesonephros (left) | 0.06 ± 0.004 | 0.44 ± 0.02 | 0.74 ± 0.03 | 2.3 ± 0.1 | 3.27 ± 0.2 |
| Stomach | 0.06 ± 0.005 | 0.44 ± 0.01 | 0.75 ± 0.03 | 2.24 ± 0.1 | 6.23 ± 0.3 |
| Lungs (left) | - | - | 0.09 ± 0.006 | 0.36 ± 0.03 | 1.46 ± 0.06 |
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Rzhepakovsky, I.; Piskov, S.; Avanesyan, S.; Shakhbanov, M.; Sizonenko, M.; Timchenko, L.; Shariati, M.A.; Rebezov, M.; Nagdalian, A. High-Performance Microcomputing Tomography of Chick Embryo in the Early Stages of Embryogenesis. Appl. Sci. 2023, 13, 10642. https://doi.org/10.3390/app131910642
AMA Style
Rzhepakovsky I, Piskov S, Avanesyan S, Shakhbanov M, Sizonenko M, Timchenko L, Shariati MA, Rebezov M, Nagdalian A. High-Performance Microcomputing Tomography of Chick Embryo in the Early Stages of Embryogenesis. Applied Sciences. 2023; 13(19):10642. https://doi.org/10.3390/app131910642
Chicago/Turabian StyleRzhepakovsky, Igor, Sergei Piskov, Svetlana Avanesyan, Magomed Shakhbanov, Marina Sizonenko, Lyudmila Timchenko, Mohammad Ali Shariati, Maksim Rebezov, and Andrey Nagdalian. 2023. "High-Performance Microcomputing Tomography of Chick Embryo in the Early Stages of Embryogenesis" Applied Sciences 13, no. 19: 10642. https://doi.org/10.3390/app131910642
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