I did an immunofluorescence on 293 cells for BrdU incorporation. I counted all cells and made a statistical measurement of the positive cells.There's a different incorporation of BrDU between my cells, but I'd like to measure the intensity of each positive cell with a tool. Does anyone know a method by which I can measure the intensity of fluorescence by Volocity?
First take a picture by using the exactly same setting-- the same exposure time, the same gain, the same contrast, the same brightness, and no gamma correction. Any imaging software for microscopes could work, but photoshop is also usable. For example, check the histogram (maybe you can open from window or edit tab depending on version you are using). The window would show the mean of the intensity (I) of selected or whole area and the pixel size (P) of the area. So the total intensity =I*P. You might also select areas showing no signal to know the background intensity. The histogram window would show the mean of background intensity (Ib). You might want to subtract the background and the formula is: (I-Ib)*P.
If you count cell number of the area, you could know the average intensity per cell.
It is good idea to use black-white images rather than color images.
If you are worried about quantification by fluorescent images, ELISA is also available.
I've done this kind of thing using the open source scientific image analysis package ImageJ (or rather the Fiji distribution of this). You can download ImageJ at http://rsbweb.nih.gov/ij/ or Fiji (which comes bundled with a large number of extras) at http://fiji.sc/. It's very good for proper scientific measurements (unlike photoshop which I would strongly argue against for scientific imaging) and has a very helpful and knowledgable user community. The easiest way to use it is to record macros and run these to go through all your images.
There are a couple of things to do to get valid results:
1. make sure you use the same exposure settings for all your images and DON'T OVEREXPOSE! best to use a 14- or 16-bit monochrome camera for this.
2. make sure your illumination is even (use shading correction)
3. make sure you don't quench your signal by looking too long at your samples before taking the picture. Long lived/bleaching resistant fluorophores such as Alexa fluors work best.
4. measure only the signal from the nuclei. for this use a DAPI nuclear stain. Image both DAPI and BrdU. Identify nuclei by a simple threshold on the Dapi image, use this as a mask to measure the intensity on the signal in the nuclei.
If you have no or very little experience in image analysis, ask this question (with an example image) on the ImageJ user forum and people will help you. If you send me an example image I would be able to tell you if the imaging is good enough and I could do you a simple macro for ImageJ.
Most of the details of image processing in photoshop is done through a "black box"; you don't really know what the program does under the hood. remeber Photoshop was designed to make visualy pleasing images, not to analyze image data.
Thank you for your help! I've already used Image J and I know how to use it. I need to acquire again al my images using the same exposure settings.
- Badges
- Science topic