Re: Deconvolution Algorithms Continued
Posted by
Lutz Schaefer on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Re-Deconvolution-Algorithms-Continued-tp1593543p1593637.html
Neeraj,
a confocal PSF (or generally, any incoherent
PSF of an aplanatic microscope system) is the product of the excitation and
emission distributions. The exitation PSF could be approximated by a widefield
PSF and the emission PSF by a widefield PSF (using the emission wavelength) but
convolved by the pinhole geometry. You need to be careful with sampling and
scaling issues. Other than that its not a big deal. The approximation with
widefield vs point-scanner had been okay in nearly all cases that came
across my work. For the calculation of widefield PSF's I suggest the classic
Sarah Gibson and Frad Lanni's paper "Experimental test of an analytical model of
aberration in an oil immersion objective lens used in three dimensional light
microscopy" JOSA V.9 N.1 1992 (which was to the best of my knowledge also
implemented in XCOSM). But of course, you may also use the slightly
more accurate vector wave model.
I hope this was of some help
Lutz
____________________________________
Lutz
Schaefer
Advanced Imaging Methodology Consultation
16-715 Doon Village
Rd.
Kitchener, Ontario, N2P 2A2
Phone/Fax: (519) 894
8870
Email: [hidden email]
Website: http://home.golden.net/~lschafer/
____________________________________
----- Original Message -----
Sent: Saturday, November 29, 2008 5:25
PM
Subject: Re: Deconvolution Algorithms
Continued
I would like to thank everyone who replied to the
Deconvolution Algorithm questions. Once I Get back to the lab on Monday we
will start comparing the suggested algorithms. Thanks Brian for your
comments as well, the commercial product we are comparing against is the
Autoquant module in the Nikon NIS elements software.
I do have a question for all of you regarding
calculating the point spread function. For some of the open source
algorithms we are using we have to input the PSF in the deconvolution
algorithm. For this we using a an open source software which calculates
theoretical PSF after you input imaging parameters such as, magnification, NA
etc. but this software does not have Laser Scanning Confocal microscopy as an
imaging modality option, it only has wide field fluorescence. Is there a
formula for theoretically calculating PSF in which you can input
the Confocal pin hole as a parameter, or would it be easier to determine
the PSF experimentally using fluorescent beads?
Best Regards,
Neeraj.
Neeraj
V. Gohad, PhD
Postdoctoral
Fellow,
Okeanos Research
Group
Department of Biological
Sciences
132
Long Hall,Clemson University
Clemson,
SC-29634
864-656-3597
[hidden email]