http://confocal-microscopy-list.275.s1.nabble.com/widefield-getting-better-images-than-spinning-disk-tp7585177p7585203.html
I'm still trying to wrap my head around the light loss situation. So I
I am fairly certain the camera is focused correctly on the pinholes. But,
> *****
> 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.
> *****
>
> 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-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.
> >
> >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.
>