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-tp2242987p2354550.html

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Sorry... one (major) correction:

The formulas for widefield and confocal fluorescence will be different, since in single point scanning confocal,  the excitation light is also focused into a PSF and therefore affects resolution, while in widefield fluorescence, illumination is uniform across the field of view, and therefore only the emission wavelength should matter. 

According to the Olympus/FSU web site I listed:

 r (lateral) = 0.6 lambda (em) /NA for widefield fluorescence  

r (lateral) = 0.4 lambda / NA for confocal fluorescence (with lambda = average of Ex and Em, or maybe the geometrical average (square root of sum of squares))

and I expect the formula for luminescence to be the same as the one for widefield florescence (because in both cases you are imaging light emitting points)

However, in the Zeiss paper, they claim that lateral confocal resolution only depends on the excitation wavelength (and the pinhole aperture), and their formulas are more complex. 

I think your formula for brightfield is the generally accepted one.

My understanding is that these formulas are all approximations based on certain assumptions and definitions. 

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org

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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