Re: Calculating voxel size in wide field system

Posted by Daniel James White on May 10, 2010; 8:46am
URL: http://imagej.273.s1.nabble.com/vesicle-distribution-in-cell-population-tp3688365p3688368.html

Hi Yehuda.

make sure you dont confuse physical sampling voxel spacing with optical resolution.

the optical resolution is limited by diffraction and the N.A.  of the objective lens.
Voxel dimensions are simply whatever you set the hardware up to do for spatial sampling.

If you tell the stage to do 5 micron z steps then thats the z resolution of the images,
but the optical resolution in z is probably nothing like 5 microns.... in this case you might well miss the info in between the z slices.

Anon:
"The point is, the image of a point is not a point"

Its the point spread function, which for a high NA light microscope objective is about 2-3x larger in z than in xy.

you can see what shape it is in 3D by calculating it approximately using Bob Ds ImageJ plugin for that
http://www.optinav.com/Diffraction-PSF-3D.htm

It does not have sharp edges, since its of a size in the order of the wavelength of the light, so that would be impossible.
(Where is the photon exactly?)

But, you can measure the full with at half maximum,
and get some idea of the extent in z direction of the PSF.

This extent is related to the z resolution.

for the xy case, its the famous combination of Abbes equation :
d = lambda / 2N.A.
(there is also an equation for the z case... can you find it?)
and the (arbitrary) Rayleigh criterion for distinguishing 2 point light sources.

Wikipedia will help you out here!

cheers

Dan


Begin forwarded message:

>
> Date:    Sun, 9 May 2010 05:57:49 -0700
> From:    yehuda brody <[hidden email]>
> Subject: Calculating voxel size in wide field system
>
> Hello,
> Is there a way to calculate the accurate voxel size (in the Z section) in a wide-field system?
>
> Yehuda Brody
> PhD candidate
> Institute of Nanotechnology and Advanced Materials,
> The Mina & Everard Goodman Faculty of Life Sciences
> Bar-Ilan University
> Ramat-Gan 52900
> Israel
>

Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

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