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Visualisation of FPs by Inverted Microscope

Beat Ludin beat.ludin at LIS.CH
Fri Feb 12 02:45:41 EST 1999


Dr Robert Gay wrote:

>Dear GFPers
>
>We're looking for a microscope system for the visualisation of
>EBFP/EGFP/EYFP directly in culture flasks.
>
>At the momment we can visualise cells expressing FPs when cultured
>directly on coverslips and viewed using an Olympus BH2 microscope with
>the Reflected Light Fluorescence attachment.  Does anyone know of a
>similar system set up for an inverted microscope which allows direct
>visualisation in the culture flasks ?

Any inverted fluorescence microscope should work. What you need is an 
objective combining long working distance, as high as possible N.A., and 
preferably a correction collar for coverslip thickness (e.g. Leica's 20x 
NA0.4 or 40x NA0.55 N PLan L Corr, 63x NA0.70 PL Fluotar L Corr, or 
something similar from Zeiss, Nikon, or Olympus). Don't expect the same 
image quality you get with a dedicated high-NA coverslip-corrected 
objective though. And it's going to be very hard or even impossible to 
visualize the weak EBFP fluorescence through plastic, unless you have 
very high expression levels, because cell culture plastic is slightly 
autofluorescent and the lower-NA objectives don't gather light very 
effictively. Last but not least, make sure to use a culture medium with 
low riboflavin concentration (DMEM is very bad, Ham's F12 is 10x better, 
we use a special DMEM LR, with low riboflavin).

Cheers,

          Beat


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