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Hi Guy,

On Nov 1, 2011, at 6:04 AM, CONFOCALMICROSCOPY automatic digest system wrote:

> Date:    Mon, 31 Oct 2011 22:20:34 +1100
> From:    Guy Cox <[log in to unmask]>
> Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)
> 
> Daniel White wrote:
> 
> " in a confocal you throw away most of the signal, as its out of focus.
> So as a result the images are often very noisy.  "
> 
> This is often stated but IT IS TOTALLY UNTRUE.  What is out of focus is
> noise, not signal.

Its not really "noise" ... rather its out of focus "signal".
A confocal pinhole physically rejects most of that signal as its out of focus,
thats the idea of the confocal pinhole. 

In widefield deconvolution microscopy we use that out of focus "signal" 
and get better contrast and signal:noise( meaning shot noise mainly, but also background noise... not the same thing)

>  If you have no SA (and, honestly, if you are
> seriously interested in high-resolution imaging that should be a given)
> then a confocal microscope with the pinhole set at 1 Airy diameter
> throws away no signal at all.  

it does throw away out of focus signal... but thats what its supposed to do. 
And it does still let through some amount of out of focus signal, 
so its not perfect at excluding blur. There is still a PSF. 

Where there is still blur there is a chance to deconvolve 
(so long as the PSF is still bigger then 1 voxel) 
and at the same time suppress the noise and increase the contrast. 

So deconvolution of confocal images IS a good idea a large % of the time. 

> So why are confocal images often noisy?
> Well, it's just statistics.  If you take a wide-field image with a 1
> second exposure each point is exposed for one second.  If you take a
> confocal image at 512 x 512 for 1 second then each point is exposed for
> ~4 microseconds.  The difference is rather substantial ...

Absolutely right Guy, its a huge difference. About 5 or 6 orders of magnitude in pixel dwell / exposure time. 
Worse still, new modern PMTs are still not as sensitive as a good EMCCD camera..
so you lose even more signal.... so you have to put in much much much more light. 

So here is the crunch:
People bang on about confocal being some kind of "Gold Standard"
for high resolution biological fluorescence imaging.

This is not true. There are different kinds of microscopes that are suited to different situations. 
They all have pretty similar resolution (as defined by Abbe / Rayleigh / etc.)
but they vary greatly in contrast. Without good contrast you cant see the resolution thats there. 

In short:
1) Confocal:
 Single point laser scanning confocal is slow for a large field of view but can be very fast for a very small field of view, 
and required a large amount of energy for illuminating the sample per pixel as the pixel time is very short. 
Spinning disc confocals get a  fast frame rate over a large field of view -  and illuminate the sample with less intensity 
Much better for living stuff. 

Single laser point scanning Confocal is really good for: Going really fast over a tiny field of view, and imaging difficult samples 
where the scattering and optical propertied of the sample mean that widefield deconvolution wont work
because the PSF is not the same all over the sample, and/or scattering is a problem. 

2) Widefield systems plus deconvolution:
can get away with much much less illumination light power and get a very high  
signal:noise and dynamic range compared to confocal systems, and be much faster for a large field of view
But you only get really good images
if the PSF is the same all over the sample, and you know the PSF shape, and there is little scattering. 

3) Take home messages. 
a)Fixed, dead, thin samples? Optically well behaved samples?
Widefield deconvolution is probably a good choice. 
Confocal is not the best tool here.... it would be like driving a 300 million dollar military tank in a mortor bike grand prix race. 
You will definitely finish the race, slowly... but you might not win... even though you have the most expensive "bike"

b) Difficult, thicker, scattery, optically naughty samples, in a small field of view?
Bring out the hammer - use the point scanning confocal or even 2 photon. 
Otherwise, consider more efficient alternatives. 

c) If the only tool you have is a hammer, every problem resembles a nail. 
We have lots of tools and lots of different kinds of samples, 
so we need to choose the right one, 
and not always use point scanning confocal because someone misinformed said its the "Gold Standard"


Dr. Daniel James White BSc. (Hons.) PhD

Leader - Image Processing Facility,
Senior Microscopist,
Light Microscopy Facility.

Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

+49 (0)15114966933 (German Mobile)
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+49 (0)351 210 1078 (Fax MPI-CBG LMF)
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