http://confocal-microscopy-list.275.s1.nabble.com/widefield-getting-better-images-than-spinning-disk-tp7585177p7585208.html
I would break down how the two technologies work in very small time fragments. In a 10 ms exposure the spinning disc is still adding together a huge number of small point exposures. In fact in that time a specific fluorescent molecule will get exited a few times at best for maybe a couple microseconds at a time. To counteract the inevitable noise problem from such short exposures you do a ton of averaging, in other words exposing for long enough for the disc to spin many times. The excitation power at each point is somewhat brighter than widefield because of the focused laser, but it cannot be orders of magnitude brighter or you will rapidly destroy your sample by causing a ton of triplet state excitation. Resonant point-scanning confocals have a similar limitation, so people generally use averaging in resonant mode unless speed is of utmost importance.
In a widefield, if you expose the image for 10 ms then the detector sees every fluorescent molecule for the full 10 ms. That means each pixel has had a chance to see a lot more emitted photons than a pixel in a spinning disc that has been integrating over the same period of time.
University of Pittsburgh Dept. of Developmental Biology
I'm still trying to wrap my head around the light loss situation. So I should expect to collect the majority of in-focus light, but overall only ~5% light is collected compared to widefield? For excitation, only 1.6% of light is transmitted through the disk to the specimen?
I am fairly certain the camera is focused correctly on the pinholes. But, I am not sure about the other knob on the top of the Crest, that is one of the things I am looking into now.
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> Post images on
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> *****
>
> dear Jeff,
> collection efficiency CF/WF is 5% to 6% in our systems with 70um
> pinholes (fill factor 8.2%). 4.6-4.8% with 60um (fill factor 5.8%).
> about 3-3.5% with 40um pinholes (fill factor 4.2%). values measured at
> 500nm.
>
> hence, my proposal to double check for proper system alignment
> (pinholes to detector focusing and excitation).
>
> regards
> Andrea
>
> Andrea Latini
> President
> CrestOptics Srl
>
>
> On Sun, 22 May 2016 18:03:48 +0000, Reece, Jeff (NIH/NIDDK) [C]
> <
[hidden email]> wrote:
>
> >*****
> >To join, leave or search the confocal microscopy listserv, go to:
> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> >Post images on
http://www.imgur.com and include the link in your posting.
> >*****
> >
> >I was wondering how long it would take before Guy responded. There
> >have
> been discussions on the listserv in years past about signal as a
> function of confocal pinhole, 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-he> uristic-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.
> >
> >My understanding of the Crest Disk, from the info I see on their web
> >site,
> is that throughput on the excitation side is only 1.6%, compared to
> widefield illumination (disk pulled out). So if you increase the
> camera exposure time by a factor of ~70 on the confocal image, the
> signal levels of the in-focus portion should be similar to the
> widefield image, if everything is aligned properly. Theoretically.
> >
> >Hope that helps.
> >Cheers,
> >Jeff
> >
> >
> >-----Original Message-----
> >From: Guy Cox [mailto:
[hidden email]]
> >Sent: Sunday, May 22, 2016 1:55 AM
> >To:
[hidden email]
> >Subject: Re: widefield getting better images than spinning disk
> >
> >*****
> >To join, leave or search the confocal microscopy listserv, go to:
> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> >Post images on
http://www.imgur.com and include the link in your posting.
> >*****
> >
> >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:
[hidden email]]
> On
> Behalf Of Sam Lord
> >Sent: Sunday, 22 May 2016 10:45 AM
> >To:
[hidden email]
> >Subject: Re: widefield getting better images than spinning disk
> >
> >*****
> >To join, leave or search the confocal microscopy listserv, go to:
> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> >Post images on
http://www.imgur.com and include the link in your posting.
> >*****
> >
> >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.
>