reducing size of illumination in epi-fluorescence

Posted by Badri Ananth on
URL: http://confocal-microscopy-list.275.s1.nabble.com/reducing-size-of-illumination-in-epi-fluorescence-tp593586.html

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Hi all,

I have a question regarding epi-fluorescence microscopy and I'm hoping to
get an answer here - I apologize for posting a question that is not related
to confocal microscopy.  I am a graduate student at UCSB and my technical
knowledge of microscopy is quite basic so I'd appreciate any guidance you
can give me.

I want to do FRAP (fluorescence photobleaching) experiments on lipid
bilayers.  Currently we use our microscope - an old one, a Nikon Eclipse
TE300 inverted microscope with a TE-FM epi-fluorescence attachment, to image
lipid bilayers (and also for cellular immunofluorescence work).  This is
hooked up to a Coolsnap ES2 cooled-CCD imaging system.  

I use a 100x objective to bleach a small spot in the lipid bilayer using
light from a 100w Mercury lamp.  I use a 10x objective to monitor the
recovery in a wider field.  My problem is that even with the field diaphragm
fully stopped down, the smallest field of illumination I can achieve is ~60
microns in diameter.  Since the diffusion coefficient of the lipids is of
the order of 1 sq. micron/sec., the recovery time is extremely long -
approx. 1 hr.  I would like to reduce this by bleaching smaller spots.

Is there a way to reduce the size of the illuminated spot, e.g. by using a
pinhole in the light path?  Where and how would I place this pinhole?  

Thanks in advance - I will also try to contact Nikon technical support for
help with this, but I suspect they'll try to sell me a new microscope instead.
regards,
badri