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Hi John,
I wouldn't recommend using PMT for single molecule fluorescence microscopy.
The QE of PMTs is not high enough, and you also have to boost
up the gain so background takes over, making it an highly inefficient way to do single molecule detection.
Most people I know (myself included) use an APD with confocal,
or EMCCD with TIRF or epi.

Hope this helped.

Eli

---- Original message ----
>Date: Thu, 26 Jul 2007 19:13:01 -0400
>From: John Oreopoulos <[log in to unmask]>  
>Subject: Re: Single-molecule imaging with an Olympus Fluoview confocal microscope?  
>To: [log in to unmask]
>
>   Search the CONFOCAL archive at
>   http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>   But is that normal for single-molecule imaging to
>   use the Kalman filter? Is this how other people do
>   it? Or are they just using home-built
>   single-molecule confocal microscopes with APDs? I
>   guess my big question is whether or not a commercial
>   confocal microscope with a PMT can be used for
>   single molecule imaging instead of a confocal with
>   APDs.
>   John
>   On 26-Jul-07, at 7:09 PM, Sarah Kefayati wrote:
>
>     Search the CONFOCAL archive at
>     http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>     John,
>     you may find it useful,try kalman averaging in
>     your fluoview,it highly reduces background noise!
>      
>     cheers
>
>      
>     On 7/26/07, John Oreopoulos
>     <[log in to unmask]> wrote:
>
>       Search the CONFOCAL archive at
>       http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>       Hi,
>        
>       I'm wondering if anyone has had experience
>       imaging single fluorescing
>       molecules immobilized on a surface using a
>       standard laser-scanning Olympus confocal
>       microscope (Fluoview - any of the models).
>       Someone came to me today with a sample of  a
>       dilute solution of proteins that had been
>       labeled with 1-10 Oregon Green and Texas Red
>       fluorochormes per protein. Using TIRF and an
>       EMCCD camera, it was very easy to see bright
>       little spots spread around the surface. 
>       There are a few reasons we'd like to switch to
>       using our confocal however for this imaging
>       situation. Does anyone know if the PMTs on an
>       Olympus confocal are sensitive enough to
>       image immobilized single-molecules with these
>       dyes? What settings should I aim for in the
>       software?
>       I tried imaging once, and I was able to see
>       bright spots, but only after driving the gains
>       of the PMTs very high. It was a very noisy
>       image, and the spots bleached fairly quickly
>       unfortunately, and so I can't be sure if what I
>       was seeing with the confocal was the protein
>       or fluorescent junk in the sample. I also had
>       to open the pinhole up all the way. Is there any
>       way I can optimize this ie: reduce the
>       background and increase the signal?
>       We're using a 1.45 NA oil objective.
>        
>       Thanks in advance!
>
>       John Oreopoulos, BSc,
>       PhD Candidate
>       University of Toronto
>       Institute For Biomaterials and Biomedical
>       Engineering
>       Centre For Studies in Molecular Imaging
>       Tel: W:416-946-5022
>