Posted by
Johannes Helm on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Acceptor-Photobleaching-vs-Sensitized-Emission-FRET-results-tp4828946p5632659.html
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Bonsoir, Louis,
that's one of the frequent questions I am being asked by "my" users.
The thing is:
Your microscope objective and condenser will provide a certain resolution,
officially dependent solely on numerical apertures. In case of a
transmitted light image, it will be dependent on the numerical apertures
of both, the condenser and the objective (proper Koehler illumination
assumed). The LATERAL microscope resolution in this case is
d = (1.2 * lambda) / (NA obj + NA cond.)
(at which, in case of day light filter operation, lambda is a somewhat
undefined thing, assume 500nm and / or use a panchromatic green filter to
reduce wavelength bandwidth).
In case of epi-illumination, the objective will also be the condenser, so
that the denominator reduces to (2*NA obj) though, in some special cases,
you might nevertheless end up with different ray paths for the
illumination and the detection, anyway, since the illumination ray path in
very special cases is a light cylinder around the "real" objective"
mirrored onto the preparation (annular illumination).
("Axial resolution" resp. "depth of field" in the wide field case is a
complicated and sometimes "debated" issue.)
You might call "d" the size of a "resel" (resolution element). Which is
different from a "pixel" (picture element).
Multiply the size of that resel with the magnification factor of all the
optics between the object and the chip of the camera. If you are lucky,
you have an adjustable zoom optics which allows you to adjust the
magnfication so that the image on the chip is magnified by a factor, which
makes sure the Nyquist theorem is fulfilled, at least. So: One resel
imaged onto the chip covers at least 2 pixels of the chip, preferrably
more (but not too much so that you do not oversample too much, loosing a
lot of light).
There is, in other words, to my mind not a straight forward answer to your
question. Unless you simply like to divide the dimensions of your camera
ship, which you can find in the manual of that camera, by the total
magnification factor. Then, you have the pixel size in the image, although
this might not really help you unless you compare it to the resel size as
mentioned above.
Best wishes,
Johannes
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>
> Bonjour à tous,
>
> I have a tiff image, 1392 x 1040 - 4.65 um square pixels acquired with a
> camera interline Sony , 1.4 megapixel, color(7.6mm x 6.2mm array). I use
> a 40 x objective mounted on a table microscope Leica DME.
>
> Can I find the pixel size in hte image?
>
> Thanks ,
>
> Louis
>
--
P. Johannes Helm, M.Sc. PhD
Seniorengineer
CMBN
University of Oslo
Institute of Basic Medical Science
Department of Anatomy
Postboks 1105 - Blindern
NO-0317 Oslo
Voice: +47 228 51159
Fax: +47 228 51499
WWW: folk.uio.no/jhelm