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Hello again,

A few years ago I sent a question to the confocal listserver about the differences in measuring confocal axial resolution with sub-diffraction-sized beads or mirrors and fluorescent sheets (see messages attached below). At the time, one of the most informative responses came from Brad Amos (response message also below). Since then, I've been doing these types of measurements on various confocal instruments and can confirm with certainty that the two test specimens (fluorescent beads vs. fluorescent sheets) yield different results when measuring the FWHM of the axial profile. So my question now is: Which specimen should we be using to report instrument performance in terms of axial resolution? I've been to quite a few microscopy courses and the conventional wisdom seems to be that fluorescent beads are best, but Brad's book chapter that he linked to made an argument that fluorescent sheets are better and that it's incorrect to use beads to report axial resolution. In case anyone is interested, in my hands, fluorescent sheets consistently yield an axial resolution that is a few hundred nm bigger than what the bead measurement says for a given instrument with a particular set of optics. I'm hoping Brad might chime in again here or anyone else who has something to say about this.

John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca



On 2011-07-25, at 3:31 PM, Brad Amos wrote:

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> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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> 
> The equations for resolution in z are different for a plane reflector, a
> thin fluorescent lamina and a subresolution bead. The commercial story on
> this is a mess of unattributed equations of obscure provenance. You may find
> useful information in the following article, which is a permitted preprint
> of an article about confocal microscopy to be edited and published in the
> Elsevier online 'Comprehensive Biophysics', where Tony Wilson, Gail
> McConnell and I have tried to make sense of it all :
> http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf


*****
On 2011-07-03, at 12:25 PM, John Oreopoulos wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> 
> I have a question regarding the use of a mirror for measuring the axial resolution of a confocal imaging system (laser scanning or spinning disk). 
> 
> Jim Pawley's handbook has a chapter that describes a few useful tests that can be done across different wavelengths to assess your microscope's axial resolution using a mirror. I have wondered for quite some time if there is any difference between using a mirror and the excitation wavelengths or a fluorescent object (microsphere) to test axial resolution. I could only find one posting on this in the archive:
> 
> http://lists.umn.edu/cgi-bin/wa?A2=ind0106&L=CONFOCALMICROSCOPY&D=0&P=8438
> 
> So I'm curious, what is the consensus out there right now? Is there a difference or not? I haven't actually had a chance to test it yet and see if the results obtained with a mirror (with laser illumination) match those obtained with a sub-diffraction sized fluorescent microbead.
> 
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca