Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Thanks all for the enthusiastic and very informative responses.
Although it seems like 'analysis methods for colocalization studies are a
field of contention and enigma' and 'an issue afflicted with ambiquity and
inconsistency'(from the excellent review from Bolte and Cordelieres, 2006,
suggested by Jens), no one argues that it should be done.
Surely, editorial staff in biological journals and anyone doing microscopy
should get a glimpse of that excelent thread.
Marc
Marc Thibault, Ph.D.
Canada Chair in Cartilage Tissue Engineering
École Polytechnique, Montreal
-----Original Message-----
From: Patrick Van Oostveldt [mailto:[log in to unmask]]
Sent: 11 février 2007 17:27
To: [log in to unmask]
Subject: Re: colocalisation without software
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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