uncaging of ATP with 2P
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Search the CONFOCAL archive at
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Dear List
Members,
one of my users
would like to use caged ATP and to preferably uncage it using 2 Photon
excitation followed by fast imaging. We are in the process of
evaluating confocal MP systems for this purpose, but since our samples are not
jet ready to use for testing we were wondering if anybody out there has
experience with the same issue.
For the fast
scanning the resonance scanner from Leica seems to perform really well, however
we are not sure if the power brought on the sample in this short time is enough
for the uncaging process. If it is not a dual scan system like the Zeiss Duo
Scan or the Olympus SIM scanner would be clearly preferable. Another approach
might be the use of channelrhodopsin, were the same concerns
apply.
Any comments
and experiences are highly appreciated.
Cheers,
Karin
------------------------
Ing. Karin Aumayr
Chief of Microscopy and
Imaging
BioOptics Department
Institute of Molecular
Pathology
Dr.Bohr-Gasse 7
1030 Wien, Austria
Tel:
+43-1-797 30 - 3701
Fax: +43-1-798 71 53
E-Mail:
[hidden email] |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
If you are using a Ti:Saph laser for your 2-photon system you will probably have a lot of excess power that you are not using. Depending on your microscope, you may be able to divert this extra power into a lamp port or some other access path. You could then image a region, then open a shutter in the auxiliary beam path to allow more power in to do the uncaging. It wouldn't be as precise as aiming with a scanning system, but it should be relatively simple and inexpensive to implement. I guess it will just depend on how small a region you need to uncage.
Craig On Tue, Aug 19, 2008 at 10:25 AM, Karin Aumayr <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
 
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Jerry Sedgewick-2 |
Re: uncaging of ATP with 2P and 2 photon/MPE in multiuser facilities
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In reply to this post by Karin Aumayr
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal If you are willing and have the heart to invest the time, and if this does not violate patent laws in Europe, you might consider building a custom system with a resonant mirror with instructions provided on Mike Sanderson and Ian Parker's websites: http://users.umassmed.edu/michael.sanderson/mjslab/confocal_microscopy.htm This isn't for the faint of heart, and it may well be a year investment, but in our lab we have constructed this unit, which can now image at 15, 30, 60 and 120 fps. The greatest frustration in the building process comes with the time it takes for the components to arrive. Troubleshooting the electronics, no matter how trivial the connections, can also be time consuming and maddening. In the end, however, it is a unit you will know thoroughly and, for better or worse, it is something that can be fixed and aligned by the person who built it without having to wait for what can too often be a frustrating visit after a technician didn't solve the alignment (or other) problem. Furthermore, components can be added fairly easily along the light path. Oh, and an additional component should be ordered for every part of the system as components change over time. At any rate, the costs are much less than with commercial units even when purchasing 2 of each major component. Which takes me to the issue of a multiphoton in a multiuser facility. These are not turn key systems. Lasers go out of alignment and these need re-aligning, which isn't trivial. To my way of thinking, the alignment is best done when there are holes (made by the laser) in the wall for aligning. Those who have constructed these systems can relate, I'm sure. Regardless, however, of whether these are commercial or custom units, someone has to check these systems periodically for misalignment and other issues. Our webpage is http://www.bipl.umn.edu. Cheers, Jerry Karin Aumayr wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Dear List Members, > > one of my users would like to use caged ATP and to preferably uncage > it using 2 Photon excitation followed by fast imaging. We are in the > process of evaluating confocal MP systems for this purpose, but since > our samples are not jet ready to use for testing we were wondering if > anybody out there has experience with the same issue. > For the fast scanning the resonance scanner from Leica seems to > perform really well, however we are not sure if the power brought on > the sample in this short time is enough for the uncaging process. If > it is not a dual scan system like the Zeiss Duo Scan or the Olympus > SIM scanner would be clearly preferable. Another approach might be the > use of channelrhodopsin, were the same concerns apply. > > Any comments and experiences are highly appreciated. > Cheers, > Karin > > ------------------------ > Ing. Karin Aumayr > Chief of Microscopy and Imaging > BioOptics Department > Institute of Molecular Pathology > Dr.Bohr-Gasse 7 > 1030 Wien, Austria > > Tel: +43-1-797 30 - 3701 > Fax: +43-1-798 71 53 > E-Mail: [hidden email] -- Jerry (Gerald) Sedgewick Program Director, Biomedical Image Processing Lab (BIPL) Department of Neuroscience, University of Minnesota 312 Church St. SE, 1-205 Hasselmo Hall Minneapolis, MN 55455 (612) 624-6607 [hidden email] http://www.bipl.umn.edu Author: "Scientific Imaging with Photoshop: Methods, Measurement and Output." Rawlight.com (dba Sedgewick Initiatives) 965 Cromwell Avenue Saint Paul, MN 55114 [hidden email] (651) 308-1466 http://www.quickphotoshop.com http://www.heartFROMstone.com http://www.rawlight.com --- Get FREE High Speed Internet from USFamily.Net! -- http://www.usfamily.net/mkt-freepromo.html --- |
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In reply to this post by Craig Brideau
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hello, another system you may want to
consider is the Prairie Ultima http://www.prairie-technologies.com/
as it comes with two light paths and is therefore engineered for un-caging. You
could also buy two 2P lasers if you really want to break the bank. The Prairie
with the AOD can image as fast as you want, depending on your ROI, but can
realistically go 25fps at 512x512. I have seen the imaging produced by the Ian Parker
2P scope at UCI and I must say that it is very impressive. You can also get a
list of components from the UCSF group for building your own scope. On our
Zeiss LSM 510 scopes (in our multi-user facility J) we use a Coherent 2P
laser and split the beam (polarizing beam splitter and ¼ wave plate) so that
half goes to each scope. However, then each beam goes through its own AOM which
controls the percent power to the scope. We operate the laser with an
independent computer. In short this system is not perfect but works pretty
well. It is still unclear to me how much GVD is created by the AOMs. However, the
comparison between 2P imaging on the 510s and the Prairie is like night and day.
Cheers, Brian D Armstrong PhD Light Microscopy Core Manager Beckman Research Institute City of 626-359-8111 x62872 http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx From: Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal If you are using a
Ti:Saph laser for your 2-photon system you will probably have a lot of excess
power that you are not using. Depending on your microscope, you may be
able to divert this extra power into a lamp port or some other access
path. You could then image a region, then open a shutter in the auxiliary
beam path to allow more power in to do the uncaging. It wouldn't be as
precise as aiming with a scanning system, but it should be relatively simple
and inexpensive to implement. I guess it will just depend on how small a
region you need to uncage. On Tue, Aug 19, 2008 at 10:25 AM, Karin Aumayr <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear List Members, one of my users would like to use caged ATP and to
preferably uncage it using 2 Photon excitation followed by fast
imaging. We are in the process of evaluating confocal MP systems for this
purpose, but since our samples are not jet ready to use for testing we were
wondering if anybody out there has experience with the same issue. For the fast scanning the resonance scanner from Leica seems
to perform really well, however we are not sure if the power brought on the
sample in this short time is enough for the uncaging process. If it is not a
dual scan system like the Zeiss Duo Scan or the Olympus SIM scanner would be
clearly preferable. Another approach might be the use of channelrhodopsin,
were the same concerns apply. Any comments and experiences are
highly appreciated. Cheers, Karin ------------------------ Ing. Karin Aumayr Chief of Microscopy and Imaging BioOptics Department Institute of Molecular Pathology Dr.Bohr-Gasse 7 1030 Tel: +43-1-797 30 - 3701 Fax: +43-1-798 71 53 E-Mail: [hidden email]
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