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3.1 Segmentation of zebrafish organs based on deep learning
Deep learning was applied to segment and quantify the organs of zebrafish collected by Mueller matrix OCT. Dice-Loss function[31] was used to prevent the imbalance problem of positive and negative data samples, as shown in Fig. 6(a). Similarly, as shown in the Fig. 6(a), Dice-loss causes obvious shock in the decline of the loss curve. The MPA score of this model is 92.34 as shown in Fig. 6(b). 
Using the trained model, five organs of zebrafish, such as the body, yolk sac, stomach, eyes and spine were successfully segmented. The segmentation results provide a basis for quantitative analysis of organ development in zebrafish.
Fig.7 shows the segmentation results of a single B-scan. The body, eyes, yolk sac, swim bladder and spine of the zebrafish were successfully segmented. (a) is the whole zebrafish, (b)-(f) are the body, eyes, spine, soft yellow sac and swim bladder of zebrafish. Follow the principle of complete organ count. The volume of zebrafish is the sum of body, eyes, spine, yolk sac and swim bladder according to the result of multi-organ segmentation of zebrafish by single B-scan in Fig.7. Similarly, the volume of yolk sac is the sum of yolk and swim bladder.

3.2 The developmental volume of zebrafish embryos
The images of zebrafish embryo development from 1dpf to 3dpf by microscopic and Mueller matrix OCT are shown in Fig. 8. From the Figure, it can be seen that the area of embryo outline and the yolk sac decrease gradually with time. In particular, the area decreased obviously on the third day. In addition, the eyes and spine grow clearly from Fig. 8 (c) and (f).
Fig. 9(a) is the area of embryo and yolk sac by microscope, while Fig. 9(b) displays the volume of embryo and yolk sac from 1dpf to 3dpf development by Mueller matrix OCT. From Fig. 10(a), the volume decreases with time during the development process, which is consistent with the actual situation of embryonic development[32, 33]. While the area of the embryo increases on the 3rd day of development from Fig. 9(a), which is inconsistent with what is observed in Fig. 8. Result illustrates that the volume from Mueller matrix OCT is more accurate than the area from microscope in quantitative analysis the embryo development. Then, the proportion of yolk sac to embryo in volume obtained by Mueller matrix OCT and area proportion obtained by microscope were quantified and shown in Fig. 10. From Fig. 10, the overall trend of embryonic development shows a gradual decline, which is consistent with the general development trend[32]. The results show the feasibility of quantitative method.
3.3 The developmental volume of zebrafish organs
Similarly, the volume of multiple organs of zebrafish from 4dpf to 19 dpf were observed and calculated. The specific method for calculating the total volume of zebrafish are introduced in Section 2.5. The volume of zebrafish during the development process were quantitatively calculated and displayed in Fig. 11. It can more intuitively and clearly analyze the organ development of zebrafish at 4-19 dpf stage. From Fig. 11, the volume development of zebrafish shows a gradually increasing trend. This growth trend is consistent with the normal growth law of fish under normal feeding conditions[33, 34].
The volume of three organs such as swim bladder, eyes, and spine of zebrafish were calculated respectively, as shown in Fig. 12. From Fig. 12, the development of the spine stabilizes within 16 days, while the development of eyes showed an obvious increased trend. And the swim bladder volume fluctuates and is unstable during the growth process. The swim bladder is the organ of fish to maintain balance in the water. It contracts and expands the smooth muscles and striated muscles of the swim bladder wall to control the volume of gas in the water according to the level of water. Smooth muscles, striated muscles and blood vessels plays a key role in the flow of gas to and from the swim bladder[35]. So, the swim bladder fluctuates in size as it grows. As shown in Fig. 12, the minimum (4dpf) and maximum(19dpf) values of fish swim bladder are 2.0×〖10〗^(-3)mm³, and 5.6×〖10〗^(-3)mm³. From the comparison of the volume between 4dpf and 19dpf, the volume of fish swim bladder of zebrafish gradually increases during the 16 days of growth, even if there are factors affecting the shrinkage and expansion of the volume.
The yolk sac is mainly composed of lipids and proteins. The yolk sac of zebrafish is an important nutrient supply organ during the development of 1-7dpf, so it tends to be gradually absorbed. The yolk sac development of 1-7dpf in zebrafish was analyzed in our study. During the first 5 days, the only source of nutrients absorbed is the yolk sac, and on the fifth day, the digestive tract develops to feed. However, at 5-6 days, the yolk sac is not completely depleted. So at this stage, there are not only endogenous nutrients, but also exogenous nutrients. On the seventh day, the yolk sac is completely depleted of nutrients. After this, nutrient absorption is mainly dependent on exogenous feeding [36, 37]. Therefore, it can be seen in Fig. 13 (a) that the volume of yolk sac increases on the fifth day, and such fluctuations are caused by the sudden intervention of exogenous feeding. After the seventh day, the volume of yolk sac begins to rise linearly (Fig. 13 (b)). From this stage, the volume development has nothing to do with its own nutrient supply.
Generally, area is taken as the measurement index in the microscope, the body length and yolk sac area of zebrafish grow to a certain node in the process of development and maintain a constant state [38]. However, this does not mean that the volume of each organ is constant. Similar to people of the same height and different weights, volume monitoring is particularly important for developmentally deformed or diseased organs when they are deformed from the z direction. In the microscope, this problem can only be solved by moving the zebrafish around and observing it through multiple scans during imaging. Since this is a subjective judgment, there is some inaccuracy. In addition, the operation of multiple scanning also reflects the shortcomings of tedious experiment and time consuming. Therefore, our experimental results prove that the Mueller matrix OCT quantitative characterization of multiple organ volume of zebrafish effectively solves the above problems.
Similar to the analysis of zebrafish embryos in the previous section, the growth data of organs of zebrafish were analyzed by quantifying the proportion of each organ. Fig. 14 shows the proportion of the volume of eyes, spine, yolk, and swim bladder to the volume of body development of zebrafish at each time point of development, represented by P1, P2, P3, P4 (description in section 2.6). P3 in Fig. 14 also reflects the volume fluctuation of yolk sac caused by feeding of zebrafish at 5dpf. The curves of P2 and P4 do not seem to change in relation to their absolute volumes. This is because the two organs are small in size, fluctuating between 1% and 2% relative to the body, so the proportion data changes relatively steadily.

3.4 Mathematical model of zebrafish organs development
Logistic function was employed to fit the data of multiple organs of zebrafish to confirm the feasibility of our method and the reliability of the result[38]. Except for the body volume and 1-7dpf yolk volume, the maximum ordinate of the fitting curve of the organ development is uniformly adjusted to 0.06mm³, which is convenient for unified observation. Fig. 15 displays the Logistic function fitting results of multiple organ volume data of zebrafish. It can be seen from Fig. 15(a)and (b), the fitting results show that the actual volume of zebrafish body and eyes have a good fitting effect with the theoretical data. Such fitting results also prove the validity of the data in statistics of the body and eyes in section 3.3. From Fig. 15(c) and (d), there was also a subtle increase in the fit of the spine and swim bladder. The fitting results were consistent with the statistics in Fig. 12. 
The yolk sac, as a special organ, is continuously absorbed from 1dpf to 7dpf due to its role in providing nutrients, and begins to absorb exogenous food to grow after the 7dpf. Therefore, the development of yolk sac volume was divided into two stages in the function fitting. Fig. 15 (e) and (f) show the fitting of yolk sac degradation in the first 7 days of development and growth with external food absorption after 7 days, respectively. As can be seen from the Fig. 15, the fitting curve of multi-organs is consistent with the experimental results, which reflects the effectiveness of the experiment. 
	Conclusion
A multi-classification analysis method of zebrafish organs by combining deep learning and Mueller matrix OCT was proposed in this paper. The volume and growth trend of zebrafish organs were quantified from 3D perspective by Mueller matrix OCT. The proportion of partial organs were quantified. Results demonstrate that the overall growth trend of zebrafish increased gradually within 16 days, although the spine development was slow, but also confirmed that the growth was stable with subtle changes. Finally, logistic function was employed to fit the data of multiple organs of zebrafish to confirm the feasibility of our method. The fitting effect of Logistic function once again demonstrate the feasibility of Mueller matrix OCT method in analyzing the growth and development trend of multiple organs of zebrafish. 
Although the study is on a small number of samples, these results indicate that the organs volume and growth trend of zebrafish during the development process. It can provide more abundant analytical means for toxicology, developmental biology and other disciplines in the analysis of organ developmental malformations.