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Jeremy Adler wrote:

> (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.

Why?  Opening the pinhole is only going to let in photons you
don't want (outside the focal plane) and all your deconvolution
will do is try with greater or lesser succcess to get rid of them.

I've said this many times before but I guess it bears repeating.
The reason confocal images are short of photons has nothing to
do with the pinhole (it doesn't lose any in-focus photons) and
has everything to do with dwell time.  Aquiring an image for
one second with a wide-field system (CCD camera) captures photons
from each pixel for one second.  Acquiring an image for one second
on a confocal (512x512 pixels) captures photons from each pixel
for 4 microseconds.  You can't increase light intensity by
250,000 times to compensate since you'll just saturate the
fluorochrome.

> Given that image quality requires both resolution and contrast is this
> strategy viable ?.

I think my views on this will be clear .... which isn't to say
that there will never be a situation where opening the pinhole
might help.  If you have an optical condition which makes spherical
aberration unavoidable, for example, you'll need to open the
pinhole just to get all the in-focus light.  A good enough
deconvolution system might then correct the optical effects
of the SA.

> (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.

In this case taking a stack just to deconvolve one slice would
be madness.  (In multiphoton, where you don't bleach outside the
focal plane, it might be a different story).  But the million
dollar question here is why do you need confocal at all?  Is there
something very distracting / interfering outside the plane you
need to image?  If not, why not just use widefield and a CCD?

Guy

--
Associate Professor Guy Cox
Electron Microscope Unit,
University of Sydney,
NSW 2006, Australia

Phone:+61 2 9351 3176    Fax:+61 2 9351 7682
http://www.guycox.net