Re: reducing size of illumination in epi-fluorescence

Posted by Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/reducing-size-of-illumination-in-epi-fluorescence-tp593586p593609.html

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Depending on how tight the focus is at the field diaphragm you might need to use an additional lens to focus the lamp light onto your pinhole. A tighter focus on the pinhole will help it pass more light; otherwise you may lose too much power going through the pinhole.

Craig

On Mon, Jul 21, 2008 at 5:24 PM, Julio Vazquez <[hidden email]> wrote:

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Badri, 

You can try to remove the field diaphragm insert, place a disc of aluminum foil over the diaphragm, and drill a pinhole in the foil.  The diaphragm fully closed has a diameter of about 1-2 mm. With a pinhole, you could probably get a bleached field maybe ten times smaller. I'd be very careful not to damage the diaphragm!

A more elegant option would be to have a piece of aluminum or steel that would fit in the slot occupied by the field diaphragm, and drill pinholes at the right location (or a series of pinholes of different sizes, so you could change the bleached area by sliding the metal piece (or using different ones). This may be more costly to manufacture, but might be doable if you have a good machine shop. 

--

Julio Vazquez, 

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org

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On Jul 21, 2008, at 2:48 PM, Badri Ananth wrote:

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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