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John,
obviously there is a difference. Besides the different wavelengths, when
using a mirror the image formation in the detection path is almost coherent.
With a fluorescent object on the other hand, it is incoherent. The
difference between these two forward problems lies in the way how the final
intensities on the camera are produced. In the coherent case, (complex)
amplitudes of elementary sample waves add up (causing the typical
constructive/destructive interference) before they become subject to
convolution with the observation PSF. Finally, the camera will sense the
absolute intensity. In the incoherent case, no interference takes place. The
forward problem can be modeled as the sum of the intensities before
convolution. This is the fundamental difference! In practice you will also
see that the mirror images will show depth varying transverse fringe
patterns when your mirror has a good quality, indicating partial coherence.
This is clearly not the case when you are using a SIP chart. To me, adding
amplitudes and adding intensities are different quantities after convolution
that you can't really compare against each other better than approximately.
On that subject is plentiful literature available, one contribution that
comes immediately to mind is Norbert Streibl's thesis from 1984.
Best Regards
Lutz
__________________________________
L u t z S c h a e f e r
Sen. Scientist
Mathematical modeling / Image processing
Advanced Imaging Methodology Consultation
16-715 Doon Village Rd.
Kitchener, ON, N2P 2A2, Canada
Phone/Fax: +1 519 894 8870
Email: [log in to unmask]
Website: http://home.golden.net/~lschafer/
___________________________________
--------------------------------------------------
From: "John Oreopoulos" <[log in to unmask]>
Sent: Sunday, July 03, 2011 12:25
To: <[log in to unmask]>
Subject: Using a mirror for axial resolution testing
> *****
> 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=
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