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The Green HeNe laser by comparison tends to be very weak irrespective of which brand confocal you use. If the excitation max is a fair distance away from your laser, then these two problems will compound. Opening the pinhole as you have done seems like a great idea. I would also look at the objective you are using. To maximise signal you must use the highest NA available, and match the refractive index of your specimen to the lens. Are you using a water, oil or glycerine lens? You should also look at using a lens with an adjustable collar, I like to adjust these by trial and error looking at the sharpness of widefield fluorescence by eye. You need to adjust the collar then the fine focus, and repeat over until the sharpest image is reached. When you do this (spherical aberration correction) much more light will go through the pinhole. 
 
Also it is no secret that there will be some signal loss when using spectral detectors.
Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008 Royal Melbourne Hospital
Parkville, Victoria,      3050
Australia
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From: Confocal Microscopy List on behalf of A. Hamdoun
Sent: Fri 08/08/2008 9:53 AM
To: [log in to unmask]
Subject: Confocal of membrane proteins.



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I am attempting to capture the distribution of membrane proteins on microvilli using a
Zeiss LSM 510 Meta. I find that although I have what seems to be fantastic expression of
mCherry or eGFP tagged protein (as determined by epi-fluor) the signal on the confocal
seems excessively photon-starved.

I am wondering what else could be causing this beyond the usual limitation of confocal
pinhole. For instance could this is related to the suboptimal wavelength of the HeNe laser
(543 vs. the excitation of 587 for mCherry)? The limit of the detector signal:noise? Or 
just some problem in how I am using the confocal?

I find that I have to image the membrane at 40x, zoomed in and using channel mode
rather than lambda (i.e long pass emission collection across many of the spectral
channels of the meta PMT), and that I have to open up the pinhole to 2 Airy disk units.

This also leads to second question which is how realistic is spectral detection if it further
reduces the signal that can be captured from any given FP. Has anyone here had any
experience with spectral detection on this scope using multiple FPs and membrane
proteins? Is there a physical limitation for imaging imposed  by how many FPs are in the
membrane (as compared to cytosolic proteins)?

thanks in advance for any advice.