Re: reducing size of illumination in epi-fluorescence

Posted by Raghu Parthasarathy on
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   About the size of a FRAP spot for microscopy:  The comments from Julio Vazquez may certainly be more correct than my memory of the Nikon aperture size, since for a while now we've been simply using our own set of field apertures -- simply pieces of metal with holes drilled in that slide into the appropriate slot on the microscope.  This is simple to do, and gives considerable freedom.  (I had forgotten about this, but it is still certainly the case that I was still able to happily do FRAP with Nikon's field diaphragm for years before these home-made apertures.)

   About extracting diffusion coefficients from the recovery of a spot that's large enough so that only the edges are effectively blurred in a reasonable time:  The diffusion of the entire irregular spot doesn't follow any nice function, but does obey Fick's laws (by assumption!) and can be modeled as such.  So, you can take a picture, take another picture later, and ask what diffusion coefficient would cause the first picture to be transformed into the second over the time between the two acquisitions.  This is a very brief sketch of the method I've employed for FRAP analysis.  A slightly longer one can be found on the last page of this paper: http://pubs.acs.org/cgi-bin/abstract.cgi/jacsat/2007/129/i17/abs/ja067819i.html .  A description of the 1d version of this sort of treatment can be found in Howard Berg's excellent book, "Random Walks in Biology," in one of the appendices.  And, I'm happy to write more later.

   Good luck!

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: Julio Vazquez <[hidden email]>
To: [hidden email]
Sent: Tuesday, July 22, 2008 9:42:29 AM
Subject: Re: reducing size of illumination in epi-fluorescence

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On our Nikon E800, with the 100x objective, the fully closed field diaphragm (~1 mm diameter) gives a spot of about 40 microns, so not too far off from the 60 microns reported by Badri.  This is actually a quite small spot when compared to the full field of view (but significant compared to the area covered by the camera, which is much smaller).

I agree with Raghu's second point though... you can indeed measure the diffusion properties of your sample by looking at a small region near the edge (or anywhere for that matter) of the bleached area, not necessarily the entire bleached spot, so it doesn't really matter which size or shape your bleached area is... the calculations may be a bit more difficult though, because an arbitrary region will be located asymmetrically inside your bleached area. Maybe then the simplest would be to tape a square piece of foil to your field diaphragm holder, on the outside, so that half of your field is bleached. You can then look at recovery in the center (near the bleached edge), where you can approximate your situation to a two vessel system separated by a wall. This would be even more true if using a lower power objective.

--

Julio Vazquez, 

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org/

On Jul 22, 2008, at 7:24 AM, Raghu Parthasarathy wrote:

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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

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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