Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I would add to this excellent summary that you should become 
"quantitatively" aware of fading issues with your fluorphores.  
Although it is possible to reduce the degree of fading with various 
mounting media, the rate does not go to zero.  For that reason, you 
may have to standardize your observation time so that you capture 
images only after x amount of exposure.

I would also stress Julio's point that the images should be captured 
BELOW saturation.  Once you saturate an image, all bets are off.

You might also ask yourself what level of precision you need.  Are 
you looking for changes of 10%, 50% etc.  

Best of luck,

Joel


> 
> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-
> bin/wa?S1=confocal 
> =
> Hi Maria,
> 
> It's difficult to give a precise answer without knowing how much you 
> already know or don't know. But basically:
> 
> 
> 1. Prepare all samples in the same manner; ideally at the same time
> 
> 2. Get good images (maximize the dynamic range with your microscope 
> system): on a confocal, try to get the background close to, but not 
> equal to zero, and get your highest signals close to the max 
> intensity limit, but not saturated.
> 
> 3. Collect all images under the same conditions ; ideally, collect 
> them back to back on the same day, so as to minimize any instrument 
> (and operator) variability. Adjust settings for the brightest sample, 
> and use the same for all other samples. If images obtained this way 
> are not good and you need to change imaging parameters between 
> samples, you should either include reference standards (e.g. beads), 
> or do the appropriate calculations to normalize results (but this may 
> be complicated, and not necessarily accurate)
> 
> 
> 4. Use your favorite software to measure the pixel intensities 
> (total, average, intensity profile, etc, depending on what you need 
> to know) in your regions of interest; ImageJ is great as a start:
> 
> http://rsbweb.nih.gov/ij/
> 
> If you want to measure labeling intensities in the entire 
> microorganisms, then you should image the whole cells in 3-D,using 
> proper sampling in x, y, and z, and use some 3-D imaging software to 
> get the numbers. You can also do that with ImageJ by analyzing all 
> relevant sections (or projections of all relevant sections)
> 
> 
> If you can make it this far, then you should use the additional 
> suggestions (such as Jim Pawley's 39 steps...)
> 
> If you can make it that far but still have difficulties, let us know 
> what the specific problem is...
> 


--
Joel B. Sheffield, Ph.D.
Biology Department, Temple University
1900 North 12th Street
Philadelphia, PA 19122
[log in to unmask]  
(215) 204 8839, fax (215) 204 0486
http://astro.temple.edu/~jbs