Z intensity

[Sarah Kefayati]

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Hello everyone and hope you all are doing well!

 

 

I am checking the intensity lost along the z axiso f our lenses which are 40x/.8 water dipping lens and 60x/1.2 w lens;by collecting the z stack from a homogeneous fluorescent sample ,I have noticed the max intensity obtained from 60x lens is dropping faster than the 40x dipping lens.I am just wondering this fact is true in general or it's because I'm using a dipping lens?

 

best

Sarah

[James Pawley]

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>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>Hello everyone and hope you all are doing well!
>
>
>I am checking the intensity lost along the z axiso f our lenses
>which are 40x/.8 water dipping lens and 60x/1.2 w lens;by collecting
>the z stack from a homogeneous fluorescent sample ,I have noticed
>the max intensity obtained from 60x lens is dropping faster than the
>40x dipping lens.I am just wondering this fact is true in general or
>it's because I'm using a dipping lens?
>
>best
>Sarah

If your homogenous sample is plastic, then it is unlikely to have the
same RI as the water for which the objectives were designed. Hence
more SA and more signal loss. In the case of the 1.2 lens, you MUST
have a coverslip between the objective and the fluorescent specimen.

If the fluorescent specimen is a water solution, then SA is unlikely
to be the problem and the loss of signal with depth may be related to
the dye concentration being too high and absorbing the excitation
beam as it moves into the specimen.

Jim P.

--
               **********************************************
Prof. James B. Pawley,                          Ph.  608-263-3147
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               "If it ain't diffraction, it must be statistics." Anon.

[Julio Vazquez]

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Higher NA lenses are likely to be more sensitive to small differences in coverslip thickness, and/or refractive index mismatch (see for instance: http://www.microscopyu.com/articles/formulas/formulascoverslipcorrection.html ); therefore, under sub-optimal imaging conditions, I would expect the light intensity to drop faster with the 1.2 NA than with the 0.8 NA dipping lens. Also, your 40/0.8 dipping lens may or may not be corrected for a coverslip (check for a "0.17"," "-, or "0" mark)), and either objective may have a correction collar to compensate for differences in coverslip thickness. Whether the setting matches the actual coverslip will also affect the degree of spherical aberration, and therefore the degree of light loss deep in your sample. 

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org/

=

On Feb 7, 2008, at 2:51 PM, Sarah Kefayati wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello everyone and hope you all are doing well!

 

 

I am checking the intensity lost along the z axiso f our lenses which are 40x/.8 water dipping lens and 60x/1.2 w lens;by collecting the z stack from a homogeneous fluorescent sample ,I have noticed the max intensity obtained from 60x lens is dropping faster than the 40x dipping lens.I am just wondering this fact is true in general or it's because I'm using a dipping lens?

 

best

Sarah