Re: Resolution for fluorescence, brightfield and luminescence
Posted by
Julio Vazquez on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Live-cell-Imaging-with-fluoview-1000-tp2242987p2354214.html
Hi Monique,
I won't attempt to give you a definite answer on this, but point you to some literature on the topic:
plus a couple of points:
for fluorescence, I believe the relevant wavelength generally used in the formula would be the average of the peak excitation and peak emission values, although I'm sure the real value would be a more complex one (if your emission as a tail in the longer wavelengths, these will spread your PSF, thus reducing resolution)
The second point that I would stress (and some will probably disagree with this) is that the definition of the minimum resolvable distance is a somewhat arbitrary one. Namely, if you are imaging two diffraction limited spots of similar intensity, their images will be two PSFs, and the minimum resolvable distance will be the distance between the center of the two PSFs so that when you do an intensity profile you see a dip in contrast that you deem is sufficient for you to know with a certain degree of certainty that you have two spots (about 75% of max peak values). Obviously, this depends also on the contrast and noise in your image, and also depends on the fact that you have a priori knowledge that you are looking at two diffraction limited spots. In a real situation, you don't necessarily know that, and therefore you criteria for "certainty" may be a bit different...
Julio.
--
Julio Vazquez, PhD
Director of Scientific Imaging
Fred Hutchinson Cancer Research Center
1100 Fairview Ave. N., mailstop DE-512
Seattle, WA 98109-1024
Tel: Office: 206-667-1215/ Lab: 206-667-4205
FAX: 206-667-6845
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On Feb 19, 2009, at 8:10 AM, Vasseur Monique wrote:
Dear all,
My question is:
On the same microscope, same objective, same immersion medium and same
sample, is there a difference in resolution depending of the microscopy
method I am using (fluorescence, luminescence or brightfield) since the
lightpath is not the same?
Is it correct to consider the following?
For brightfield: r = (1.22 * illumination wavelenght)/(NA objective +
NA condenser)
For fluorescence and confocal: r = (0.61 * excitation (?) wavelenght) /
NA objective
For luminescence: r = (0.61 * emission (?) wavelenght) / NA objective
Thanks in advance,
monique