MOE_Golgi_Analyzer_v1

Description of the folder content : 1) The macro in .ijm format. Suited for analysis of 3-channel confocal fluorescence microscopy images of mammalian cells (~200*200µm). Requires ImageJ v1.4 with Bio-render plugin. Images should be as .nd2 format but it can easily be changed, simply search & replace all occurences of ".nd2" with your format in the macro code. Images should be organized with every replicate of a same test-condition in a unique folder. The macro will analyze the whole folder at once and will create a folder in it to save results. 2) A folder named "example_data", it contains 3 representative images that can be used to test the macro. It also contains a results folder with representative data obtained via the analysis of these representative images with the macro (see Description of the macro for description of the results obtained) ____________________________ Description of the macro : input : 3-channel image with C1 = nucleus labeling (e.g. DAPI, Hoechst, etc.) C2 = signal of interest, the one you want to measure in whole cells & in the region of interest C3 = region of interest (ROI) (e.g. an antibody directed against a particular organelle, in our case Golgi apparatus) this macro will : count the cells according to C1 (user input of threshold values for C1) create ROI(s) according to C3 (user input of threshold values, or manual setting of each image for C3) measure signal of C2 (mean min max grey values, integrated density, area) in whole cells (user input of threshold values for C2) measure signal of C2 in ROI(s) save results as a .csv file it will also create several .png images for each analyzed one : C1+nucleusROI (to assess correct cell counting) C3+ROIC3 (to assess correct creation of ROI(s) from C3 signal) C2 (glow LUT) + ROIC3 (to assess correct thresholding of C2 signal) C2+ROIC3 merge C1+C2+C3

文件夹内容说明：
1) 格式为.ijm的宏脚本：适用于哺乳动物细胞（约200×200μm）的三通道共聚焦荧光显微图像分析，需使用搭载Bio-render插件的ImageJ v1.4。图像格式默认需为.nd2，但可灵活修改：只需在宏脚本代码中全局搜索并替换所有.nd2字段为您所用的图像格式即可。图像需按实验条件分组，同一测试条件下的所有生物学重复需置于单独文件夹中。本宏脚本可一次性批量分析整个目标文件夹，并在其中创建结果保存子文件夹。
2) 名为"example_data"的示例数据文件夹：内含3张可用于宏脚本功能测试的代表性图像，同时附带一个结果文件夹，存储了使用本宏脚本对这些示例图像分析后得到的代表性结果（结果详情请参见宏脚本说明部分）
____________________________
宏脚本说明：
输入要求：三通道图像，各通道定义如下：
C1为细胞核标记通道（如DAPI、Hoechst等）；
C2为目标信号通道，即需在全细胞及目标区域内定量的信号；
C3为目标区域（ROI，Region of Interest）标记通道（例如针对特定细胞器的抗体染色，本案例中为高尔基体）。
本宏脚本可实现以下分析功能：
- 依据C1通道计数细胞（需用户输入C1通道的阈值参数）；
- 依据C3通道生成目标区域（ROI）（需用户输入C3通道的阈值参数，或手动为每张图像设置ROI）；
- 定量全细胞内的C2通道信号，包括灰度均值、最小值、最大值、积分光密度及面积（需用户输入C2通道的阈值参数）；
- 定量ROI内的C2通道信号；
- 将所有分析结果保存为.csv格式文件。
此外，本宏脚本还会为每张已分析的图像生成以下.png格式的可视化结果，用于验证分析流程的正确性：
- C1通道与细胞核ROI叠加图（用于验证细胞计数的准确性）；
- C3通道与基于C3信号生成的ROI叠加图（用于验证ROI生成的正确性）；
- 应用热度色阶（glow LUT）的C2通道图像与C3 ROI叠加图（用于验证C2通道阈值设置的合理性）；
- C2通道图像与C3 ROI的叠加图；
- C1、C2、C3三通道合并图