http://confocal-microscopy-list.275.s1.nabble.com/widefield-getting-better-images-than-spinning-disk-tp7585177p7585207.html
light from elsewhere. If your specimen is 30 microns thick, and your
compared to widefield. So yes, it'll be a lot darker just because you
> *****
> 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.
> *****
>
> Hi all,
>
> 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.
>
> Thanks,
> Rafael
>
> On Sun, May 22, 2016 at 3:26 PM, Andrea Latini <
[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.
>> *****
>>
>> 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.
>>