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

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

Dear,

I think that you can also expect some interference from moving objects. A
deltavision system can record at higher speed and hance collects more photons
from a point during its movement in the field of view.
Have you ever tested this by blocking any possible movement in the image. I can
imagine that small obejcts will be invisible in the confocal or disapear due to
accumulation if they move or shake faster that the sampling rate.

Bye

Patrick

 Quoting "Locknar, Sarah A" <[log in to unmask]>:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Julio and others-
>
> I've been trying to figure out the reason some of my users say that the
> can see small things better with the deltavision (widefield-
> camera-based system) than the confocal.  I think maybe your argument
> below about the beam profile may answer it.  I've had several people say
> they prefer the deltavision.  One looks at punctate vesicle-type
> staining and the other was looking at sub-resolution beads (190 nm I
> think).  The person looking at the beads said he couldn't see them at
> all with the confocal (in another facility- so I'm just going on what
> they said).  I thought it was just that with a camera-based system you
> can keep the shutter open for as long as it takes to acquire enough
> photons while with confocal the dwell time is not very adjustable.  I
> would think with averaging or photon-counting, the two techniques should
> give similar images.  Any thoughts on this?  Do any people use confocals
> for single-molecule work, for instance?
> Thanks-
> Sarah
>
>
>  Keep in mind, however, that even if you oversample like a madman, your
> reading at any given pixel, on a confocal,  is still the intensity of
> the fluorescence of the spot excited by your focused laser beam, whose
> dimensions are equal to an Airy disc. Therefore, if you sampled five
> 50-nanometer pixels, you would still be roughly imaging the same
> 250-nanometer spot, and spreading your "zone of confusion". Clearly, the
> intensity profile of the laser beam is a Gaussian, so this is not
> absolutely true, and the intensity would be higher when you hit your
> fluorescent speckle dead-on, but in real life confocal microscopy, I
> would say the approximation is about correct.
>
> On a CCD camera, however, if you oversampled like crazy, and you had an
> amazing signal to noise, I guess in theory you could determine the exact
> location of the center on intensity of your Airy image with almost
> unlimited accuracy. But a very small fluorescent speckle will have a
> finite intensity, and by spreading this intensity over more pixels, your
> signal to noise goes down, and in practice again, you probably wouldn't
> gain too much.
> --
> Julio Vazquez,
> Fred Hutchinson Cancer Research Center
> Seattle, WA 98109-1024
>
>
> http://www.fhcrc.org/
>
>


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