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

Image￫Adjust ￫ 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

观察不同细胞结构域荧光强弱的变化

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.