Name: Confocal and Light Sheet Fluorescence Microscopy (LSFM) Imaging of Cleared Mouse Kidneys
Uploaded: 2024-02-09T13:30:00
Description: Watch this Scientific Journal Video about Confocal and Light Sheet Fluorescence Microscopy LSFM Imaging of Cleared Mouse Kidneys at JoVE.com

confocal and light sheet fluorescence microscopy (lsfm) imaging of cleared mouse kidneys

透明化小鼠肾脏中肾小球的荧光标记用于显微镜检查

The number and volume of glomeruli are very important for the diagnosis and treatment of various kidney diseases1,2,3,4,5. The golden standard of glomeruli number estimation is the physical dissector/fractionator combination. However, this method requires special reagents and equipment, making it slow and expensive6,7,8,9. Biopsy provides a wealth of information, but obviously, this method is only suitable for rough estimations10,11. Medical imaging technologies, including magnetic resonance imaging (MRI), computed tomography (CT), and X-ray, are also widely used in glomerular detection12,13,14,15, but such technologies require bulky instruments. New methods, such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometer16 or the thick and thin section method17, have also been used in glomerular detection, though they remain tedious and laborious.
With the help of transparency technologies, it is possible to observe deeper depths and obtain richer and more complete information from thick tissues or even whole organs18,19,20,21,22,23. Therefore, transparency technologies have been widely used in kidney research24. The observation and detection of glomeruli in the cleared kidneys are also involved. However, these published articles either only briefly referred to glomerular detection25 or used difficult-to-achieve labeling methods such as transgenic animals26, self-produced dyes13, or high-concentration antibody incubation27 to label the glomeruli. In addition, although studies had analyzed glomeruli in cleared kidneys, the analyses were always limited13 or relied on analysis algorithms established by the authors themselves26.
We have previously demonstrated a more convenient way to label the glomeruli in mice kidneys28. By using Imaris, we found that glomeruli count, frequency, and volume could be quickly obtained. Thus, here we present a more accessible, comprehensive, and simplified set of methods to label and analyze the glomeruli of mice kidneys.

作者聚焦：通过标记透明化组织中的肾小球来辅助肾脏生物学研究 

一种高效、快速的小鼠肾小球标记和分析方法

Labeling and counting glomeruli using conventional methods can be complicated, tedious, time consuming, and bulky instrument are required. Our method offers an instant and intuitively accurate labeling method for analysis of the glomeruli within the entire kidney. Our technique is a quick, easy, accurate, and cheap method to analyze glomeruli in the entire kidney.It does not require any special equipment, so it can be carried out in any laboratory. This is a groundbreaking, rapid, and simple method to label glomeruli, allowing for an intuitive observation of all the glomeruli within the entire kidney. It holds great potential to assist researchers to make significant breakthroughs in the study of the kidney by providing information such as glomeruli quantity, volume, distribution, and more.On one hand, we're exploring the application of this glomeruli labeling method in clinical kidney disease research. On the other hand, we'll keep working on enhancing methods to label biotargets like blood vessels or specific proteins in transparent tissues, adjusting them to meet our research needs.

Watch this Scientific Journal Video about Confocal and Light Sheet Fluorescence Microscopy LSFM Imaging of Cleared Mouse Kidneys at JoVE.com

Confocal and Light Sheet Fluorescence Microscopy (LSFM) Imaging of Cleared Mouse Kidneys  

共聚焦和光片荧光显微镜 （LSFM） 清除小鼠肾脏的成像

首先，准备透明的肾组织样本。然后打开共聚焦显微镜，首先激活激光功率，然后接合共聚焦控制开关。接下来，启动计算机，启动显微镜并启动共聚焦硬件。运行自检后，启动共聚焦软件。选择合适的镜头。从立方R试剂中取出样品并将其放入共聚焦皿中。盖上盖子以防止立方 R 试剂干燥。将样品移动到视场中心并调整焦点，直到样品对焦。对于 3D 重建，在一个方向上调整缩放平面，直到看不到荧光，将此位置定义为顶部。然后以相反的方向调整缩放平面，直到看不到荧光，将此位置定义为底部。单击对话框右下角的运行按钮以启动扫描。对于大图像重建，将视场定位在样品的中间，并将其确定为中心。选择二乘二的视场。在ND多功能界面中，选择Z堆栈以重建大图像。根据需要调整面板中的各种选项，然后按运行按钮开始扫描。用胶水将透明肾脏粘附在样品固定适配器上，然后等待肾脏牢固连接。然后将肾脏浸入样品箱中，并将样品箱滑入显微镜系统。密封舱口。在定位界面中，将样品调整到适当的观察位置。切换到采集接口，设置光路。选择 488 纳米激光器。选择 405-488-561-640 的光束滤光片，然后将光分光滤光片块设置为 SBS LP 490。选择相机二并将伪颜色更改为绿色。输入从通道子菜单获得的左 Z 偏移量，然后输入右 Z 偏移量。选择最小的变焦并对其进行微调以获得光学清晰度。确定整个器官的 XY 边界和 Z 范围。然后将激光强度和曝光时间调制到小于 10 毫秒。单击“开始实验”以启动该过程。如果组织过大，请使用显微镜软件将两张或多张图像与图像拼接相结合。打开捕获的文件，然后选择“处理”。然后是方法和拼接，然后是方法参数。单击“应用”以完成图像拼接。使用血管内注射的fitindex链标记肾小球。在清除的肾脏中，可以通过使用光片显微镜或共聚焦显微镜清楚地观察到肾小球。

Research

JoVE Journal

Biology

Confocal and Light Sheet Fluorescence Microscopy (LSFM) Imaging of Cleared Mouse Kidneys

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological ...