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PUPAID:一个R + ImageJ管道,用于彻底和半自动处理和分析多通道免疫光数据

Paul Régnier^1,2, Camille Montardi³, Anna Maciejewski-Duval^1,2

¹Immunology-Immunopathology-Immunotherapy (i3) Laboratory, INSERM UMR-S 959, Sorbonne Université, Paris, France.

|September 19, 2024

概括

PUPAID是一个新的R + ImageJ工作流程,用于半自动化免疫光分析. 它比现有方法识别更多的单元,并将数据导出为FCS文件,以实现广泛的兼容性.

科学领域:

计算生物学计算生物学
图像分析图像分析
免疫光显微镜显微镜

背景情况:

多通道免疫光产生复杂的数据集.
精确的细胞细分和信号提取对于分析至关重要.
现有的方法可能会在高密度细胞群体中扎.

研究的目的:

引入PUPAID,用于免疫光数据处理的半自动化工作流.
为了实现强大的细胞细分和光信号提取.
为研究人员提供一个用户友好和兼容的分析工具.

主要方法:

使用R和ImageJ语言开发工作流程.
在感兴趣的区域内实施半自动电池细分 (感兴趣的区域) 和信号提取.
可选地集成R Shiny应用程序以提高可用性.
导出单细胞数据作为标准化的FCS文件.

主要成果:

PUPAID成功处理了多通道免疫光数据.
工作流准确地从细分的细胞中提取光信号.
与StarDist和Cellpose相比,PUPAID识别的细胞数量显著增加,特别是在密集的区域.
导出的FCS文件确保与各种细胞计分析软件的兼容性.

结论:

PUPAID为免疫光数据分析提供了一种强大且易于使用的解决方案.
该工作流提高了细胞检测灵敏度,特别是在具有挑战性的高密度样本中.
标准化的FCS文件输出促进了科学界的数据共享和整合.

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Immunofluorescence Microscopy

Immunofluorescence Microscopy

A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.

Super-resolution Fluorescence Microscopy

Super-resolution Fluorescence Microscopy

Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...