Posted by
Julio Vazquez on
Jul 18, 2008; 5:12pm
URL: http://confocal-microscopy-list.275.s1.nabble.com/Quantifying-fluorescence-help-tp593618p593633.html
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:
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...
Handling/analyzing digital microscopy images can be difficult, intimidating, and fraught with potential hazards (as you may be aware if you have followed the thread on "image manipulation"). There are many ways to process/analyze images, which are hard to efficiently learn on a forum like this one if you are a complete beginner (we can't give you a class on image analysis). If you have a confocal facility at your institution, make sure you use their expertise in this area.
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024
= On Jul 18, 2008, at 6:25 AM, Maria Mazzillo wrote:
Search the CONFOCAL archive at
I am a PhD student at Auburn University just getting started into my thesis work. I am
looking for a reliable method for quantifying fluorescence.
My project deals with marine dinoflagellates (zooxanthellae) that reside intracellularly in
hosts (usually cnidarians). I am working with cultures or isolates of only the dinoflagellates
for my confocal work. I have an antibody that was created against the surface secretions
of mucilage (secreted as part of a daily cycle by the alga) from one strain of zooxanthellae.
I am attempting to use this antibody to label various strains to identify differences in
mucilage between them. Thus far, I have seen that the strain the antibody was created
against labels around the cell fairly brightly. Most samples either show this or a complete
lack of labeling. However, a few samples show a faint fluorescence lifted off the cell
surface. I would like to be able to quantify this fluorescence in comparison to either the
control strain that the antibody was created against or against a known fluorescence.
Any help on ideas for this, or places to look for ideas would be greatly appreciated.
Thanks!
Maria Mazzillo