# APPLICATION NOTE  
Zebrafish observation using the Z stacking function  
Using **Celloger® Mini Plus**

## Introduction
**Zebrafish**, along with mice, is a well-known animal model widely used in biological research such as **epigenetics**¹, **clinical research**², **neuroscience research**³ and so on. Mainly, this is because it is easy to manipulate the zebrafish embryos and larvae genetically, and they are easy to observe using an optical microscope since they are small in size and have high optical clarity.

We introduce the **Celloger®** series' functions for observing zebrafish. **Celloger® Mini Plus** provides a **Z-stacking function** that automatically generates multi-layer images and the **Celloger® analysis software** offers a **merge function** to combine the multi-layered fluorescent images easily. These functions enable researchers to observe **three-dimensional (3D) structures** in a single image. We observed transgenic zebrafish (larvae) expressing **green fluorescent protein (GFP)** using these functions, as detailed in steps 1 and 2 below.

## Step 1. Z-stacking
The **3D structures**, such as tissue composed of cells, provide various information depending on the focal point; it is crucial to identify various focal points. By setting the distance between layers (**“step”**) and the number of images taken (**“N, N’”**), **Celloger®** generates multi-layer images at regular intervals (**“step”**) above and below the set position (**Z position**) ([Image placeholder: Figure 1. Z stacking]). Different settings can be entered for each point, and different Z-stacking can be performed at various points.

We utilized **Celloger® Mini Plus**, which enables **fluorescence imaging**, to create Z-stacked images of the fluorescent transgenic zebrafish. Because the acceptable focus plane varies for each part of the three-dimensional larva, we verified the appropriate focal planes by manipulating the **motorized Z stage** of the process.

1. We identified the top (**Z = 4.585**) and bottom (**Z = 4.685**) focal planes among several focal planes.
2. The coordinate (**Z = 4.635**) was set corresponding to the middle as the scan position.
3. We set to take **five pictures** at **10 µm intervals** above and below the scan position as the center ([Image placeholder: Figure 2. Procedure]).

[Image placeholder: Figure 3. Result]

Figure 3 shows the pictures captured from the top, middle, and bottom focal planes of the zebrafish. In the side view, the shape of the head was definite in the bottom focal plane, while the ventral part was clearly visible in the top focal plane.

## Step 2. Z-projection
Multi-layer imaging (**Z-stacking**) is essential for observing the **3D model**. However, interpreting the results of multiple images obtained by multi-layer imaging is laborious and time-consuming. The **Z-projection function**, which merges several layers into one image, allows one 3D sample to be observed at a glance as one image, thereby increasing the researcher’s insight.

**Projection type** is a method of integrating several **Z coordinates** located at each **X–Y position**.

* **Maximum**: integrates the brightest pixel among several Z coordinate points.
* **Average**: calculates the average brightness of several Z coordinate points.

[Image placeholder: Figure 1. Explanation of Z projection type]

In addition, **“add deviation value”**, an element that can be reinforced, was added to the **Celloger® analysis software** to obtain a clearer image.

If the images taken with Z-stacking are opened in the **Celloger® analysis software**, and the **“merge”** button on the **Z-stack tab** is clicked, a Z-projection image can be obtained ([Image placeholder: Figure 2. Procedure]). The appropriate projection type differs among samples; confirming the result for each type is recommended.

[Image placeholder: Figure 3. Result]

Figure 3 shows the result of stitching three images for projection by selecting the **“maximum”** type and **“add deviation value”** option. The head structure is clearly expressed on the bottom focal plane, and the unique ventral shape, which can be observed on the top focal plane, is expressed as a single image using the projection function.

## Conclusion
We have successfully acquired **high-quality fluorescence images** of **Zebrafish** using the **Celloger® Mini Plus** system. The **three-dimensional structure’s multiple layers of information** were successfully combined and expressed in a single image with clarity. This experiment confirms the feasibility of capturing **3D images** using various other sample types, including **spheroids** and **organoids**, which could expand the range of research scope available to researchers.

## Reference
1. Balasubramanian, S., Raghunath, A., & Perumal, E. (2019). Role of epigenetics in zebrafish development. *Gene*, 718, 144049. https://doi.org/10.1016/j.gene.2019.144049  
2. Kinth, P., Mahesh, G., & Panwar, Y. (2013). Mapping of zebrafish research: a global outlook. *Zebrafish*, 10(4), 510–517. https://doi.org/10.1089/zeb.2012.0854  
3. Stewart, A. M., Braubach, O., Spitsbergen, J., Gerlai, R., & Kalueff, A. V. (2014). Zebrafish models for translational neuroscience research: from tank to bedside. *Trends in Neurosciences*, 37(5), 264–278.