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

Hi Jeremy,

You may be interested in the paper by Jeff Larson referenced below that
shows results for blind deconvolution of 2D confocal images with
different pinhole sizes.

Opening the pinhole will increase the number of photons and the haze,
however, the signal-to-noise ratio will be improved, which benefits the
deconvolution algorithm.  I don't believe it is necessary to collect a
3D stack - just a single 2D image or 2D time-lapse sequence and run the
deconvolution.  The extra photons that are collected will have come from
close to the plane of interest.

There is always a balance between signal and resolution, and if you have
bleaching issues it would be better to sacrifice some resolution (which
the deconvolution can attempt to recover) for improved signal quality
(IMHO).

Reference:

        J.M. Larson, "2-D and 3-D deconvolution of confocal fluorescence
images by maximum
                likelihood estimation", Proc. SPIE 2641, 86-94 (2002). 

Please contact me off the list if you need any assistance processing
your data.

Cheers,
David
---
David Biggs
Senior Research Scientist
AutoQuant Imaging, Inc.
[log in to unmask]
www.aqi.com


-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Jeremy Adler
Sent: Thursday, December 02, 2004 7:33 AM
To: [log in to unmask]
Subject: confocal pinhole size and deconvolution

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

(1) Since confocal images are generally short of photons, the option of
acquiring more photons by opening the pinhole and then deconvolving the
image set to restore the quality, seems attractive.
Given that image quality requires both resolution and contrast is this
strategy viable ?.

(2) Since deconvolution ideally requires a Z series that covers the
likely size of a PSF and, if only single image is really required, is it
better to acquire the Z series and deconvolve or to use the same
acquisition time to improve the quality (acquiring more photons) from a
single image plane ?.  This choice arises when the fluorophore of
interest bleaches rapidly in single photon confocal imaging.

--
Dr Jeremy Adler
Dev. Biol.
Wenner-Gren Inst.
Stockholm University
S-106 91 Stockholm
Sweden

+46 (0)8 16 1568

--