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使用Image J进行自动荧光定量分析(二) 细胞膜荧光定量方法

已有 32803 次阅读 2014-2-5 02:04 |个人分类:活色生香de生物科学|系统分类:科研笔记| 荧光定量分析

Quantificationof fluorescence intensity by ImageJ for HEK cell

细胞膜区域荧光定量分析

http://www.unige.ch/medecine/bioimaging/tricks/imagejtutorials/Quantification.pdf

Wang H, Sugiyama Y, Hikima T, Sugano E, Tomita H, Takahashi T, Ishizuka T, and Yawo H. Molecular determinants differentiating photocurrent properties of two channelrhodopsins from chlamydomonas. J Biol Chem 284: 5685-5696, 2009.


File → Open →’.zvi’

Choose the single plane with interested cell 选择最佳的单层图片

Image →duplicate

Range: X (number of the single plane) X为层数

Uncheck the “duplicate stack”->OK

Draw a region of interest (ROI) around yourobject with one of the drawing tools (in the toolbar)

选择单个细胞

Image →duplicate (this will only copy the ROI area)

Image →duplicate

ImageAdjust → Threshold→ apply  转换为二进制图

Process-> Image Calculator->Operation: AND -> OK (original image AND Binary image)


取原图和二进制图同不为0的部分



得出的新图



Analyze → Analyzeparticles  choose the binary image这一步可以得到索要测量区域的面积.

only write down the Total area, this will calculator the black area


Analyze → Measure测量 这一部可以得到荧光强度值


荧光强度=measure值/面积area



 


Add pseudo color to images

将单色图转换为16色图(基于荧光强度的不同)
File →Open →


Image →duplicate


Image →Types →8 bits

调节最佳对比度Image →Adjust→ Brightness/contrast→apply

转换 Image →Lookuptables →16 colors


 

Plot of intensity values across the image

察不同细胞结构域荧光强弱的变

Analyze → Plot profile


Select “live”



reference:

Wang H, Sugiyama Y, Hikima T, Sugano E, Tomita H, Takahashi T, Ishizuka T, and Yawo H. Molecular determinants differentiating photocurrent properties of two channelrhodopsins from chlamydomonas. J Biol Chem 284: 5685-5696, 2009.

 




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