Posted by
Reece, Jeff (NIH/NIDDK) [E] on
URL: http://confocal-microscopy-list.275.s1.nabble.com/widefield-getting-better-images-than-spinning-disk-tp7585177p7585218.html
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I was wondering how long it would take before Guy responded. There have been discussions on the listserv in years past about the effect of pinhole on brightness, and Guy has always stressed this basic concept. I can't find those previous discussions, but there is a nice graph here that illustrates the concept (scroll down to Figure 6, the graph on the right):
http://www.leica-microsystems.com/science-lab/super-resolution-on-a-heuristic-point-of-view-about-the-resolution-of-a-light-microscope/ (disclaimer: this is the only place I could find it on the web; thanks Leica)
which makes sense if you look at Figure 3 on the same page, just visually estimating how much of the total light from the psf must be inside the Airy Disk.
So, another way to state the same concept: only 16% of in-focus light is thrown away when the pinhole is ~1 AU (i.e. "confocal" in the general sense).
If anyone has the original references that show the graph of integrated intensity vs AU, or another place it might be on the web for free, I would be interested. Perhaps those links are with the previous discussion on the listserv that I can't find.
With the 10x/0.45 lens, and the pinhole at ~3 AU, you are collecting more like 94% of the in-focus light.
And since the FWHM z-resolution is ~20 microns for that pinhole and lens (assuming 500nm as the emission wavelength), then you are collecting ~47% of the out-of-focus light that originates from 10 microns away from the focal plane.
All theoretical of course, but usually not far off when the system is aligned properly. ;-)
Hope that helps.
Cheers,
Jeff
-----Original Message-----
From: Guy Cox [mailto:
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Sent: Sunday, May 22, 2016 1:55 AM
To:
[hidden email]
Subject: Re: widefield getting better images than spinning disk
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Repeat 100 times:
CONFOCAL ONLY THROWS OUT
OUT OF FOCUS LIGHT!
The reason spinning disk systems lose light is to attain speed, and has nothing to do with the confocal principle.
Guy
Guy Cox, Honorary Associate Professor
School of Medical Sciences
Australian Centre for Microscopy and Microanalysis, Madsen, F09, University of Sydney, NSW 2006
-----Original Message-----
From: Confocal Microscopy List [mailto:
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Sent: Sunday, 22 May 2016 10:45 AM
To:
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Subject: Re: widefield getting better images than spinning disk
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I would recommmend setting the exposure time to 100 ms or however long an exposure it takes to get a good image. If the image gets bright but the contrast still looks worse than wide field, then maybe there's an alignment issue. But I suspect your sample is just too dim to image with confocal at 10 ms per frame. Not many samples are bright enough for that.
Remember that confocal is designed to throw out light in order to improve optical slicing.