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.
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