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Mark,

That's a good question.  We attached a coverslip with a small drop of 
oil to the objective as a way to deal with immersion objectives.  I 
don't know what it means that the "sample" is in air; if some light 
is then totally internally reflected back into the coverslip, which 
could be a spectral issue due to dispersion.

Using two matched objectives is probably a better method, but I 
imagine considerably more difficult due to alignment constraints and 
obtaining matched objectives.  The objectives we had obeyed the 'sine 
condition', which I believe means that there are fewer high-NA rays 
than low NA rays- this would help mitigate our experimental error as well.


At 04:19 PM 9/2/2007, you wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hi Andrew
>
>While I can see that method would work for lenses designed to work 
>in air, wouldn't total internal reflection be a problem for lenses 
>with an NA over 1.0? The only way I can think of doing it is to use 
>two objectives of the same type looking at each other at a common 
>focal point with the right immersion medium between them. Then the 
>overall transmission would be half that of a single lens...
>
>Cheers Mark
>
>Andrew Resnick wrote:
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>We've done similar measurements, it's not too difficult.
>>
>>The main trick is handling the large NA lenses.  Our setup was 
>>light source -> objective -> integrating sphere -> 
>>spectrometer.  After normalizing to the source, we obtained really 
>>good data.  It's pretty easy, actually.
>>
>>Andy
>>
>>
>>At 08:45 AM 8/31/2007, you wrote:
>>>Search the CONFOCAL archive at
>>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>>Dear all,
>>>
>>>one of the main differences of objectives is their transmission 
>>>efficiency at certain wavelengths. One way to compare this is the 
>>>trial-and-error method, however, this is not straight forward.
>>>
>>>My idea is to use a combination of spectrophotometer and a lamp 
>>>with a more or less even spectra (i.e. Xenon) on some kind of 
>>>optical bench. This would make the setup independent from the 
>>>manufacturer. Beside the distance between the light source and the 
>>>detector, there are obviously more things to consider: different 
>>>diameter of the back focal plane, different focal lenghts...
>>>
>>>I would like to hear about your opinion about how to measure 
>>>objective transmission. Have you ever done this in your lab? Did 
>>>you find a setup that worked for you?
>>>
>>>cheers,
>>>Michael
>>
>>Andrew Resnick, Ph. D.
>>Instructor
>>Department of Physiology and Biophysics
>>Case Western Reserve University
>>216-368-6899 (V)
>>216-368-4223 (F)
>
>Andrew Resnick, Ph. D.
>Instructor
>Department of Physiology and Biophysics
>Case Western Reserve University
>216-368-6899 (V)
>216-368-4223 (F)