Jan,

The back and forth about your problem seems to have muddied things a bit.

About the first question below, WF systems generally speaking have 
much higher light throughput and more efficient light sensors for a 
higher overall quantum detection efficiency. Thus, less exposure for 
the same S/N is usually available using WF. It is not a question of 
simply using the same microscope; it is the entire light path that it 
is at issue. Only way to be certain comparing two systems is to use a 
robust sample (no photobleaching, e.g., fast decay time phosphor 
grains).

Deconvolution is a requirement for standard WF 3D imaging. It more 
than has a place, it is essential.

If you are limited to a confocal and you need more signal, opening 
your pinhole and applying deconvolution is reasonable. For whatever 
pinhole size you choose, you will need to acquire a 3D PSF. I believe 
you first asked what PSF to use; opinions will vary but my preference 
is to acquire bead/reference object PSF data appropriate to the 
objective and wavelengths you are using; at minimum, match the 
Nyquist criterion in x,y,z. From the reference data for each 
objective estimate the theoretical 3D PSF function and use that to 
deconvolve your images. (I prefer using the theoretical since it 
won't directly add noise to the decon. process)

To actually get any benefit from deconvolution you have to sample 
your specimen using Nyquist spacing. It doesn't matter whether you 
are using WF or large pinholes. Except for speed, 2D deconvolution is 
practically worthless (sorry I am being a little harsh); it is no 
better than an Unsharp filter. Actually, using a Fourier filter and 
"notching out the very lowest frequencies" does a pretty good job of 
removing haze and greatly improves contrast, and it is quite fast.

Mario

>Thanks for all the comments. What would be the advantages of using 
>WF over wide-pinhole confocal (assuming it's the same microscope)?
>
>Off list Vincent suggested using 2D deconv. with a theoretical PSF, 
>which might be the way to go. It's my understanding that 
>deconvolution has a place in WF imaging (which I guess is close to 
>what I'm doing), am I correct?
>
>Thanks again,
>
>Jan
>
>....


-- 
________________________________________________________________________________
Mario M. Moronne, Ph.D.

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