Re: reducing size of illumination in epi-fluorescence

Posted by Raghu Parthasarathy on
URL: http://confocal-microscopy-list.275.s1.nabble.com/reducing-size-of-illumination-in-epi-fluorescence-tp593586p593604.html

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

Hi,

   First, you should certainly be able to get a bleached FRAP spot *far* smaller than 60 microns using the setup you've described.  (I've done this routinely, on lipid bilayers, on the same sort of microscope.)  So I suspect something else is wrong.  Think about this: your stated bleaching objective lens magnification is 100X, and your camera has approx. 10 micron pixels, so a 60 micron spot should be 600 pixels wide on your image -- i.e. about half the total field of view (for a 1200x1200 px camera)!  If your spots really are this wide, either your field diaphragm isn't closing, or it's not much of a diaphragm!

   Second, even if your spot is 60 microns wide (which I stress it should not be) it's incorrect to conclude that you necessarily need 1hour to see diffusion. The edges of the spot should blur well before this, and you can extract the diffusion coefficient from how the shape of the whole spot changes with time.  If your sample really needs 1 hour to recover, then it's not fluid.

best wishes,

Raghu

 

--
Raghuveer Parthasarathy
[hidden email]

Assistant Professor
Department of Physics
1274 University of Oregon
Eugene, OR 97403-1274
http://physics.uoregon.edu/~raghu/

----- Original Message ----
From: Badri Ananth <[hidden email]>
To: [hidden email]
Sent: Monday, July 21, 2008 2:48:28 PM
Subject: reducing size of illumination in epi-fluorescence

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

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