*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy*****
Hi Don,
From my calculatons it seems rather that the popular Yokogawa spinnig disk seems to err on the side of higher light throughput rather than confocality. My quick and dirty calculations suggest that for GFP the 50 micron diameter pinhole of the Yokogawa corresponds to about 1.25 Airy units with a 100/1.4 objective, 2 Airy units with a 60x/1.4, and approximately 3 Airy units with a 40x/1.3.
The Olympus DSU offers interchangeable disks with different pinhole or slit sizes, and the Prairie swept field has a plate with a choice of slits and pinhole arrays ranging (I cite from memory, so could be a bit off) from 22 to 60 microns, therefore allowing to work closer to 1 Airy unit with more objectives.
Regarding laser power, 5 mW is definitely a lot on a single point scanning confocal, where the light is focused to a point on the specimen. We would only go near those power levels if we actually wanted to bleach the sample (FRAP, etc...). On a Yokogawa disk, the same 5 mW of nominal power at the fiber would be distributed over the 1,000 points illuminated at any given time (minus about 30% loss through the disk), so the power density per area at the sample would be 1,000-2,000 times less than on a single point confocal. On a spinning disk without micro lenses, the power per area unit would drop maybe another 30-50 fold, and on a wide field, where the intensity is spread over the whole FOV, intensity per area unit would be millions of times less than on a single point scanning confocal.
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024
http://www.fhcrc.org----- Original Message -----
From: "Donald O'Malley" <
[hidden email]>
To:
[hidden email]
Sent: Sunday, May 20, 2012 6:10:35 AM
Subject: Pin-hole issue in disk vs. line scanning confocal
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy*****
The point was made that 5 mW is a lot of laser power. With live specimens it is extremely useful to trade off some optical sectioning by gradually opening the "pinhole" aperture-- this allows one to operate at much lower light intensities, and can easily be done in the course of live imaging experiments with most line-scanning confocals (i.e. aperture can be adjusted without disturbing the specimen on the microscope stage). With the fixed pinhole size of disk scanners, which are designed to maximize spatial resolution, a large laser power is required, which is not ideal for extended live cell/animal imaging experiments.
My question is if commercial disk scanners have a set of disks allowing one to switch to larger apertures, and if this trade-off can be made "on-line", i.e. in between successive acquisitions on a sample? I would especially appreciate a pointer to any peer-reviewed publications on this topic.
Best Regards,
Don
Don O'Malley
Assoc. Professor
Dept. Biology
[hidden email]<mailto:
[hidden email]>
617-373-2284
Locked
