FCS + two photon?
Locked
123
123
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We've been quite happy with our chambers from In Vivo Scientific. Kurt -----Original Message----- From: Confocal Microscopy List on behalf of B. Prabhakar Pandian Sent: Thu 12/6/2007 1:59 PM To: [hidden email] Subject: Microscope enclosure chambers Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello, We are looking for a microscope enclosure chambers that will control temperature, humidity and gas with feedback for long term studies. I am looking for enclosures that will also allow ports for tubing to come out of the chamber. The tubing is connected to a syringe pump outside the enclosure. Any suggestions with feedback on companies to checkout will be great. Thanks, -Prabhakar |
 
|
Edrun Andrea Schnell |
Re: Deep penetration in tissue with two photon
|
|
In reply to this post by Maria Smedh
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Maria, we are trying to find out how deep it is possible to go, and especially the difference between the visible lasers and the 2P. Just to know what kind of experiments we can do... In my latest experiments I could image around 30-40 um with the argon laser, 45-50 um with 2P, and 60-70 with 2P + NDD. I think that both the depth and the difference are a bit to small compared to what I have seen elsewhere. Thanks for all your answers everyone! We will discuss them in my group, and I might have some more comments/questions later. Regards, Edrun -- Edrun Andrea Schnell Divisional engineer, Dept. of Physics, NTNU Hogskoleringen 5, 7491 Trondheim Tel.: +47 73 59 36 69 On Fri, 7 Dec 2007, Maria Smedh wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi Edrun, > > How deep are you trying to go? At least our old C-Apochromat 40x/1.2 W > objectives only have a working distance of ~0.3 mm (which have been debated > here on the list whether it is counted from the top lens element or from the > cover glass)... > > /Maria > > > > > > ~~~~~~~~~~~~~~~~~~ > > Maria Smedh, Ph.D. > > > > Centre for Cellular Imaging > The Sahlgrenska Academy, Göteborg University > Visiting address: Medicinareg. 7A, 413 90 Göteborg > Postal address: Box 435, 40530 Göteborg > Delivery address: Medicinaregatan 1G, 413 90 Göteborg, Sweden > > Phone: + 46 (0)31 786 3704 > Mobile: + 46 (0)70 298 3424 > E-mail: [hidden email] > > Webpage: http://www.cf.gu.se/ > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On > Behalf Of Edrun Andrea Schnell > Sent: den 7 december 2007 12:20 > To: [hidden email] > Subject: Re: Deep penetration in tissue with two photon > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > We use the C-Apochromat 40x/1.2 W objective, which is supposed to be the > best for two photon, and yes, we do have an AOM. We don't use any mounting > medium since the spheroids are in solution (PBS, RPMI etc) in glass wells. > > -Edrun > > -- > > Edrun Andrea Schnell > Divisional engineer, > Dept. of Physics, NTNU > Hogskoleringen 5, 7491 Trondheim > Norway > > > On Fri, 7 Dec 2007, Nuno Moreno wrote: > > > Search the CONFOCAL archive at > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > > Witch objective and mounting medium are you using? Is the laser passing > > through an AOM? > > > > Edrun Andrea Schnell wrote: > > > Search the CONFOCAL archive at > > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > > > > Dear list, > > > > > > it is a known fact that a two photon laser will penetrate deeper into > > > tissue, and we have done several experiments to show this, but without > > > success. We have used spheroids labeled with several dyes, and imaged > > > with our Zeiss LSM510 with 1) visible laser and descanned detector, 2) > two > > > photon laser and descanned detector and 3) two photon laser and > > > non-descanned detector. We have only found a difference of about 10-20 > um > > > from exp. 1) to exp. 3), whereas it should be around 100 um. > > > > > > So I'm wondering if anyone else have done this kind of experiment and > have > > > any tips as to how to image this increase in penetration depth? Type of > > > sample and dye etc.? > > > > > > Thanks a lot! > > > > > > Regards, > > > Edrun A. Schnell > > > > > > -- > > > > > > Edrun Andrea Schnell > > > Divisional engineer, > > > Dept. of Physics, NTNU > > > Hogskoleringen 5, 7491 Trondheim > > > Norway > > > > > > > -- > > Nuno Moreno > > Cell Imaging Unit > > Instituto Gulbenkian de Ciência > > http://uic.igc.gulbekian.pt > > http://www.igc.gulbekian.pt > > phone +351 214464606 > > fax +351 214407970 > > > |
|
|
Andrew Resnick |
Re: Microscope enclosure chambers
|
|
In reply to this post by B. Prabhakar Pandian
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Ours was made by Solent Scientific, for an upright Leica DM 6000. It's designed specifically for what you are talking about (which is also, coincidentally, the type of studies we perform). Andy At 04:59 PM 12/6/2007, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hello, > We are looking for a microscope enclosure chambers that > will control temperature, humidity and gas with feedback for long term studies. >I am looking for enclosures that will also allow ports for tubing to >come out of the chamber. The tubing is connected to a syringe pump >outside the enclosure. > >Any suggestions with feedback on companies to checkout will be great. > >Thanks, > >-Prabhakar Andrew Resnick, Ph. D. Instructor Department of Physiology and Biophysics Case Western Reserve University 216-368-6899 (V) 216-368-4223 (F) |
|
|
Mark Cannell |
Re: Deep penetration in tissue with two photon
|
|
In reply to this post by Edrun Andrea Schnell
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Depth penetration also depends on the nature of the sample -do not forget that a RI mismatch (the cause of the scattering) also robs power at the focal point and in 2P the emission depends on the square of power. With our homebuilt system focussing into a terrible sample (cheddar cheese) 2P non-descanned detection approximately doubled depth penetration to ~70um see: Soeller & Cannell: Two-Photon Microscopy: Imaging in Scattering Samples and Three-Dimensionally Resolved Flash Photolysis MICROSCOPY RESEARCH AND TECHNIQUE 47:182–195 (1999) Cheers Mark On 7/12/2007, at 11:46 PM, Edrun Andrea Schnell wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear list, > > it is a known fact that a two photon laser will penetrate deeper into > tissue, and we have done several experiments to show this, but without > success. We have used spheroids labeled with several dyes, and imaged > with our Zeiss LSM510 with 1) visible laser and descanned detector, > 2) two > photon laser and descanned detector and 3) two photon laser and > non-descanned detector. We have only found a difference of about > 10-20 um > from exp. 1) to exp. 3), whereas it should be around 100 um. > > So I'm wondering if anyone else have done this kind of experiment > and have > any tips as to how to image this increase in penetration depth? > Type of > sample and dye etc.? > > Thanks a lot! > > Regards, > Edrun A. Schnell > > -- > > Edrun Andrea Schnell > Divisional engineer, > Dept. of Physics, NTNU > Hogskoleringen 5, 7491 Trondheim > Norway |
|
|
Craig Brideau |
Re: Deep penetration in tissue with two photon
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal One way I've seen to get around the focal point aberration caused by the sample is to use adaptive optics to compensate. This is not trivial to implement, but there are some good algorithms out there for running the deformable mirror necessary to adjust the focus and wavefront of your system. Such a setup will allow significantly more penetration than an uncorrected system. Craig On Dec 10, 2007 1:36 PM, Mark Cannell <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Depth penetration also depends on the nature of the sample -do not > forget that a RI mismatch (the cause of the scattering) also robs > power at the focal point and in 2P the emission depends on the > square of power. With our homebuilt system focussing into a terrible > sample (cheddar cheese) 2P non-descanned detection approximately > doubled depth penetration to ~70um see: > > Soeller & Cannell: Two-Photon Microscopy: Imaging in Scattering Samples > and Three-Dimensionally Resolved Flash Photolysis MICROSCOPY RESEARCH > AND TECHNIQUE 47:182–195 (1999) > > Cheers Mark > > > On 7/12/2007, at 11:46 PM, Edrun Andrea Schnell wrote: > > > Search the CONFOCAL archive at > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > > Dear list, > > > > it is a known fact that a two photon laser will penetrate deeper into > > tissue, and we have done several experiments to show this, but without > > success. We have used spheroids labeled with several dyes, and imaged > > with our Zeiss LSM510 with 1) visible laser and descanned detector, > > 2) two > > photon laser and descanned detector and 3) two photon laser and > > non-descanned detector. We have only found a difference of about > > 10-20 um > > from exp. 1) to exp. 3), whereas it should be around 100 um. > > > > So I'm wondering if anyone else have done this kind of experiment > > and have > > any tips as to how to image this increase in penetration depth? > > Type of > > sample and dye etc.? > > > > Thanks a lot! > > > > Regards, > > Edrun A. Schnell > > > > -- > > > > Edrun Andrea Schnell > > Divisional engineer, > > Dept. of Physics, NTNU > > Hogskoleringen 5, 7491 Trondheim > > Norway > |
|
|
In reply to this post by Csucs Gabor
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Csucs Gabor <[hidden email]> writes: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear Edrun, > > Although Zeiss officially doesn't support it, but I know that with the > ConfoCor 3 system you can do also 2photon FCS. Actually it should be > possible also with the Leica setup (given that you buy the necessary > filter sets..). You can buy a 2-photon FCS system from ISS (http://www.iss.com/products/albafcs/index.html). I have used their single photon system which works fine but still has a few rough edges. Ian |
|
|
Alberto Diaspro |
Re: Deep penetration in tissue with two photon sorry for possible duplicates - alby
|
|
In reply to this post by Edrun Andrea Schnell
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Sorry for possible duplicates, Steve Paddock fixed a problem I had (it was my fault, thank you Steve) but i did not know about this reply (see below) Ciao ALby Hi following Mark, even if we eat Cheddar cheese, we found the problem related to penetration depth really intricate. There was a very intersting paper by Denk group on JOSA (1) that reported about halos on the superficial layers when focusing deep... we were not able to reproduce them experimentally probbly due to the comparatively small penetration depth experienced - 200/400 micron - even if increasing scatterers in our gel, already used for understanding photobleaching profiles (2), we started findindìg something. As well, we found theorethical results (3) in partial agreement with a Denk paper's figure that unfrtunately is only a sort of viugnette.. I think hard job has to be done for understanding all the factors inluencing the performances... 1)P. Theer and W. Denk, "On the fundamental imaging-depth limit in two- photon microscopy," J. Opt. Soc. Am. A 23, 3139-3149 (2006) 2) Mazza D., Cella, F., Vicidomini G., Krol S., Diaspro A. (2007) Role of three dimensional bleach distribution in confocal and two-photon FRAP experiments. Appl Opt, 46(30):7401-11 3)Ying et al APPLIED OPTICS Vol. 38, No. 1, 1 January 1999 Ciao ALby Il giorno 10/dic/07, alle ore 10:43, Edrun Andrea Schnell ha scritto: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi Maria, > > we are trying to find out how deep it is possible to go, and > especially > the difference between the visible lasers and the 2P. Just to know > what > kind of experiments we can do... In my latest experiments I could > image > around 30-40 um with the argon laser, 45-50 um with 2P, and 60-70 > with 2P > + NDD. I think that both the depth and the difference are a bit to > small > compared to what I have seen elsewhere. > > Thanks for all your answers everyone! We will discuss them in my > group, > and I might have some more comments/questions later. > > Regards, > Edrun > > -- > > Edrun Andrea Schnell > Divisional engineer, > Dept. of Physics, NTNU > Hogskoleringen 5, 7491 Trondheim > Tel.: +47 73 59 36 69 > > On Fri, 7 Dec 2007, Maria Smedh wrote: > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hi Edrun, >> >> How deep are you trying to go? At least our old C-Apochromat 40x/ >> 1.2 W >> objectives only have a working distance of ~0.3 mm (which have been >> debated >> here on the list whether it is counted from the top lens element or >> from the >> cover glass)... >> >> /Maria >> >> >> >> >> >> ~~~~~~~~~~~~~~~~~~ >> >> Maria Smedh, Ph.D. >> >> >> >> Centre for Cellular Imaging >> The Sahlgrenska Academy, Göteborg University >> Visiting address: Medicinareg. 7A, 413 90 Göteborg >> Postal address: Box 435, 40530 Göteborg >> Delivery address: Medicinaregatan 1G, 413 90 Göteborg, Sweden >> >> Phone: + 46 (0)31 786 3704 >> Mobile: + 46 (0)70 298 3424 >> E-mail: [hidden email] >> >> Webpage: http://www.cf.gu.se/ >> >> >> -----Original Message----- >> From: Confocal Microscopy List >> [mailto:[hidden email]] On >> Behalf Of Edrun Andrea Schnell >> Sent: den 7 december 2007 12:20 >> To: [hidden email] >> Subject: Re: Deep penetration in tissue with two photon >> >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> We use the C-Apochromat 40x/1.2 W objective, which is supposed to >> be the >> best for two photon, and yes, we do have an AOM. We don't use any >> mounting >> medium since the spheroids are in solution (PBS, RPMI etc) in glass >> wells. >> >> -Edrun >> >> -- >> >> Edrun Andrea Schnell >> Divisional engineer, >> Dept. of Physics, NTNU >> Hogskoleringen 5, 7491 Trondheim >> Norway >> >> >> On Fri, 7 Dec 2007, Nuno Moreno wrote: >> >>> Search the CONFOCAL archive at >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>> Witch objective and mounting medium are you using? Is the laser >>> passing >>> through an AOM? >>> >>> Edrun Andrea Schnell wrote: >>>> Search the CONFOCAL archive at >>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>>> >>>> Dear list, >>>> >>>> it is a known fact that a two photon laser will penetrate deeper >>>> into >>>> tissue, and we have done several experiments to show this, but >>>> without >>>> success. We have used spheroids labeled with several dyes, and >>>> imaged >>>> with our Zeiss LSM510 with 1) visible laser and descanned >>>> detector, 2) >> two >>>> photon laser and descanned detector and 3) two photon laser and >>>> non-descanned detector. We have only found a difference of about >>>> 10-20 >> um >>>> from exp. 1) to exp. 3), whereas it should be around 100 um. >>>> >>>> So I'm wondering if anyone else have done this kind of experiment >>>> and >> have >>>> any tips as to how to image this increase in penetration depth? >>>> Type of >>>> sample and dye etc.? >>>> >>>> Thanks a lot! >>>> >>>> Regards, >>>> Edrun A. Schnell >>>> >>>> -- >>>> >>>> Edrun Andrea Schnell >>>> Divisional engineer, >>>> Dept. of Physics, NTNU >>>> Hogskoleringen 5, 7491 Trondheim >>>> Norway >>>> >>> >>> -- >>> Nuno Moreno >>> Cell Imaging Unit >>> Instituto Gulbenkian de Ciência >>> http://uic.igc.gulbekian.pt >>> http://www.igc.gulbekian.pt >>> phone +351 214464606 >>> fax +351 214407970 >>> >> > ---------------------------------------------------- "Follow knowledge wherever it leads us." (reading Galileo Galilei) ----------------------------------------------------- Alberto Diaspro, EBSA President-Elect, MicroScoBIO LAMBS-IFOM, Department of Physics, University of Genoa, Via Dodecaneso 33, 16146 Genoa, Italy - fax +39-010314218 - tel +39 0103536426/309; URLs: www.lambs.it ; EBSA is Biophysics in Europe - European Biophysical Societies' Association www.ebsa.org ---------------------------------------------- |
|
|
James Pawley |
Re: Deep penetration in tissue with two photon
|
|
In reply to this post by Craig Brideau
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >One way I've seen to get around the focal point aberration caused by >the sample is to use adaptive optics to compensate. This is not >trivial to implement, but there are some good algorithms out there for >running the deformable mirror necessary to adjust the focus and >wavefront of your system. Such a setup will allow significantly more >penetration than an uncorrected system. > >Craig Hi Craig, I am always very interested in ways to correct SA fast. But it seems to me that, to use the deformable mirror properly, the optical axis must approach it perpendicular. And any way I can think of to do this seems to involve the beam going through a 50/50 beamsplitter, twice. As this will reduce the signal by at least 75%, it seems a high p[rice to pay, Can one use these things at a slight angle? Cheers, Jim P. -- **************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-262-9083 250 N. Mills St., Madison, WI, 53706 [hidden email] "A scientist is not one who can answer questions but one who can question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39 |
|
|
John Girkin |
Re: Deep penetration in tissue with two photon
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal The way that we have used deformable mirrors for improved MP imaging is to use them both at shallow angles, when you effectively loose optical power, but also using a polarising beam splitter and waveplate. The details can be found in the following references and those in the papers themselves. "Practical implementation of adaptive optics in multiphoton microscopy" P N Marsh, D Burns, J M Girkin, Optics Express 11 1123-1130 2003 "Optimisation Algorithms for the Implementation of Adaptive Optics in Confocal and Multiphoton Microscopy" A. J. Wright, D. Burns, B. A. Patterson, S. Poland, G. Valentine, J. M. Girkin, Microscopy Research and Technique, 67, 36 2005 "A Dynamic closed loop system for focus tracking using a spatial light modulator and a deformable membrane mirror" A J Wright, B A Patterson, S Poland, J M Girkin, G Gibson, M J Padgett Optics Express, 14, 222, 2006 Best regards John ________________________________ From: Confocal Microscopy List on behalf of James Pawley Sent: Tue 12/11/2007 21:07 To: [hidden email] Subject: Re: Deep penetration in tissue with two photon Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >One way I've seen to get around the focal point aberration caused by >the sample is to use adaptive optics to compensate. This is not >trivial to implement, but there are some good algorithms out there for >running the deformable mirror necessary to adjust the focus and >wavefront of your system. Such a setup will allow significantly more >penetration than an uncorrected system. > >Craig Hi Craig, I am always very interested in ways to correct SA fast. But it seems to me that, to use the deformable mirror properly, the optical axis must approach it perpendicular. And any way I can think of to do this seems to involve the beam going through a 50/50 beamsplitter, twice. As this will reduce the signal by at least 75%, it seems a high p[rice to pay, Can one use these things at a slight angle? Cheers, Jim P. -- **************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-262-9083 250 N. Mills St., Madison, WI, 53706 [hidden email] "A scientist is not one who can answer questions but one who can question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39 |
|
|
Craig Brideau |
Re: Deep penetration in tissue with two photon
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Yes, these work well enough at even slight angles. The key specs to look for in a deformable mirror are the speed of response (how quickly it can change shape) and the piston/piezo stroke (the amount it can deform the mirror). The most common technology is piezo, which uses little piezo actuators to push-pull the mirror membrane. They respond quickly (depending on the high-voltage drivers) but their stroke is typically measured on the order of a few micrometers. There are magnetic push-pull ones that have strokes up to 50um, which allows for a tremendous range of compensation, but this may be at the expense of speed, depending on the design. If you are using an iterative algorithm to optimize your aberration correction, response speed is important as the algorithm will need to 'try' multiple configurations of the mirror. There's probably newer stuff out there these days that are pushing the speed/stroke numbers I mentioned above; it's a fast moving field. Craig On Dec 11, 2007 2:36 PM, John Girkin <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > The way that we have used deformable mirrors for improved MP imaging is to use them both at shallow angles, when you effectively loose optical power, but also using a polarising beam splitter and waveplate. The details can be found in the following references and those in the papers themselves. > > "Practical implementation of adaptive optics in multiphoton microscopy" P N Marsh, D Burns, J M Girkin, Optics Express 11 1123-1130 2003 > > "Optimisation Algorithms for the Implementation of Adaptive Optics in Confocal and Multiphoton Microscopy" A. J. Wright, D. Burns, B. A. Patterson, S. Poland, G. Valentine, J. M. Girkin, Microscopy Research and Technique, 67, 36 2005 > > > > "A Dynamic closed loop system for focus tracking using a spatial light modulator and a deformable membrane mirror" A J Wright, B A Patterson, S Poland, J M Girkin, G Gibson, M J Padgett Optics Express, 14, 222, 2006 > > > > Best regards > > > > John > > > ________________________________ > > From: Confocal Microscopy List on behalf of James Pawley > Sent: Tue 12/11/2007 21:07 > To: [hidden email] > Subject: Re: Deep penetration in tissue with two photon > > > > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > >Search the CONFOCAL archive at > >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > >One way I've seen to get around the focal point aberration caused by > >the sample is to use adaptive optics to compensate. This is not > >trivial to implement, but there are some good algorithms out there for > >running the deformable mirror necessary to adjust the focus and > >wavefront of your system. Such a setup will allow significantly more > >penetration than an uncorrected system. > > > >Craig > > Hi Craig, > > I am always very interested in ways to correct SA fast. But it seems > to me that, to use the deformable mirror properly, the optical axis > must approach it perpendicular. And any way I can think of to do this > seems to involve the beam going through a 50/50 beamsplitter, twice. > As this will reduce the signal by at least 75%, it seems a high > p[rice to pay, Can one use these things at a slight angle? > > Cheers, > > Jim P. > -- > **************************************** > Prof. James B. Pawley, Ph. 608-263-3147 > Room 223, Zoology Research Building, FAX 608-262-9083 > 250 N. Mills St., Madison, WI, 53706 [hidden email] > "A scientist is not one who can answer questions but one who can > question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39 > |
|
|
Stephen Cody |
Re: Microscope enclosure chambers
|
|
In reply to this post by Andrew Resnick
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear Prabhakar, May I suggest you contact Allan Anderson (Ex. Bio-Rad Engineer) at Clear State Solutions [hidden email] . He has just launched a new microscope enclosure after consulting many of the scientists around Australia. It has some great features, and it is very competitively priced. Cheers Steve Microscopy Manager Central Resource for Advanced Microscopy PO Box 2008 Royal Melbourne Hospital Victoria, 3050 Australia Tel: 61 3 9341 3155 Fax: 61 3 9341 3104 email: [hidden email] www.ludwig.edu.au/labs/confocal.html Tip: Learn how to receive reminders about you microscope booking: www.ludwig.edu.au/confocal/Local/Booking_Hint.htm
-----Original Message----- Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Ours was made by Solent Scientific, for an upright Leica DM 6000. It's designed specifically for what you are talking about (which is also, coincidentally, the type of studies we perform). Andy At 04:59 PM 12/6/2007, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hello, > We are looking for a microscope enclosure chambers that > will control temperature, humidity and gas with feedback for long term studies. >I am looking for enclosures that will also allow ports for tubing to >come out of the chamber. The tubing is connected to a syringe pump >outside the enclosure. > >Any suggestions with feedback on companies to checkout will be great. > >Thanks, > >-Prabhakar Andrew Resnick, Ph. D. Instructor Department of Physiology and Biophysics Case Western Reserve University 216-368-6899 (V) 216-368-4223 (F) This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error. The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research. |
|
|
Stephen Cody |
Re: Deep penetration in tissue with two photon
|
|
In reply to this post by "José A. Feijó"
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear List,
Great discussion……
With regards to the previous comments by Jose on lenses I just stumbled across what sounds like a fantastic lens for multiphoton applications.
It is a 25x dipping lens with a correction collar so that it can be used with a coverslip as well and presumably can be tweaked in between for Spherical Abberations caused by the specimen. Olympus claim that when used on their FV1000-MPE the exit pupil is completely filled. So the full 1.05 NA of the objective should deliver high resolution images.
With this lens I presume we should be able to work at low power, and then optically zoom in for high power images without changing objectives. See the following link for details.
http://www.olympusamerica.com/cpg_section/cpg_headlineDetails.asp?pressNo=562
No commercial affiliation with Olympus, other than I’m waiting delivery of a FV1000-MPE system. I think I know what lens to ask for…. Stephen H. Cody Tip: Learn how to receive reminders about you microscope
booking: -----Original Message-----
we published some of our own evaluations of Nikon
lenses in Protoplasma, 223:132, 2004 Search the CONFOCAL archive athttp://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Our own lab has found that modifying confocal microscopes for 2-photonis a tricky business. As mentioned before, an AOM will cause problemswith the laser. If there is any way to bypass the AOM in your system,or simply remove it, this will help.Another issue is optics and objectives. Most optics and objectivesare designed for the visible range rather than the NIR. From my ownexperience certain medium-high-end Nikon and Olympus lenses tend tohave the best NIR performance, and most others are terrible; absorbing90% or more of the light. A crude test is to use a laser power meterto measure the power after your objective. Place the detector headsome distance away where the light is diverging but still all of ithits the detector surface; the focal point can damage your detector.Compare this to the power before the microscope to get your systemlosses. Don't be afraid to ask your manufacturer for the NIRtransmission spectrum for your lenses. Craig On Dec 7, 2007 5:06 AM, "José A. Feijó" [hidden email] wrote:
-- **********************************************************Jose' A. Feijo', Prof. ---------------------------------------------------------- Dep. Biologia Vegetal, Fac.Ciencias, Universidade LisboaPT-1749-016 Lisboa, PORTUGAL tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48 and/ e Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70__________________________________________________________e.mail: [hidden email] URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38**********************************************************This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error. The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research. |
|
|
Farid Jalali |
Re: Microscope enclosure chambers
|
|
In reply to this post by Stephen Cody
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Germaine to the topic of this thread, does anyone have experience maintaining the atmosphere within these microscope-enclosing chambers at hypoxic O2 concentrations (0.5-0%O2)? We are a cancer research laboratory; many solid tumours have hypoxic regions, and we would like to image live cells under hypoxic conditions. The responses we are looking at are very sensitive to O2 and I am trying to get a sense of whether an enclosure or stage -top approach would be best. We are poised to test a Pathology Devices stage top incubator but I am curious to know if anyone has tried something similar with an enclosure.
Cheers Farid On Dec 11, 2007 8:00 PM, Stephen Cody <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal -- Farid Jalali MSc Senior Research Technician/ Lab Manager Dr. Robert Bristow Lab Applied Molecular Oncology Princess Margaret Hospital Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Microscopy Suite |
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
To the best of my knowledge most of these chambers can control CO2 and temperature but cannot control oxygen concentrations. However a chamber which controls temperature, O2 and CO2 is available from PeCon, Germany (No commercial interest) In this chamber oxygen can be decreased down to about 1%. Creating low oxygen conditions is a very challenging task. I am myself curious to know whether any other commercial microscopy chambers for oxygen control are available. Good luck for your experiments. Best, Venkat Farid Jalali wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Germaine to the topic of this thread, does anyone have experience maintaining the atmosphere within these microscope-enclosing chambers at hypoxic O2 concentrations (0.5-0%O2)? We are a cancer research laboratory; many solid tumours have hypoxic regions, and we would like to image live cells under hypoxic conditions. The responses we are looking at are very sensitive to O2 and I am trying to get a sense of whether an enclosure or stage -top approach would be best. We are poised to test a Pathology Devices stage top incubator but I am curious to know if anyone has tried something similar with an enclosure. |
|
|
Urs Utzinger |
Re: Deep penetration in tissue with two photon
|
|
In reply to this post by Stephen Cody
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
As I heard in the U.S. this lens goes for $20k and that it is currently
available only for FV MPE customers. From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Stephen Cody Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Search the CONFOCAL
archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear List, Great discussion…… With regards to the previous comments by Jose on lenses I just
stumbled across what sounds like a fantastic lens for multiphoton applications.
It is a 25x dipping lens with a correction collar so that it can be
used with a coverslip as well and presumably can be tweaked in between for
Spherical Abberations caused by the specimen. Olympus claim that when used on
their FV1000-MPE the exit pupil is completely filled. So the full
1.05 NA of the objective should deliver high resolution images. With this lens I presume we should be able to work at low power,
and then optically zoom in for high power images without changing objectives. See the following link for details. http://www.olympusamerica.com/cpg_section/cpg_headlineDetails.asp?pressNo=562
No commercial affiliation with Olympus, other than I’m waiting
delivery of a FV1000-MPE system. I think I know what lens to ask for…. Stephen
H. Cody Tip:
Learn how to receive reminders about you microscope booking: -----Original Message----- we published some of our own evaluations of Nikon lenses in
Protoplasma, 223:132, 2004 Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Our own lab has found that modifying confocal microscopes for 2-photon is a tricky business. As mentioned before, an AOM will cause problems with the laser. If there is any way to bypass the AOM in your system, or simply remove it, this will help. Another issue is optics and objectives. Most optics and objectives are designed for the visible range rather than the NIR. From my own experience certain medium-high-end Nikon and Olympus lenses tend to have the best NIR performance, and most others are terrible; absorbing 90% or more of the light. A crude test is to use a laser power meter to measure the power after your objective. Place the detector head some distance away where the light is diverging but still all of it hits the detector surface; the focal point can damage your detector. Compare this to the power before the microscope to get your system losses. Don't be afraid to ask your manufacturer for the NIR transmission spectrum for your lenses. Craig On Dec 7, 2007 5:06 AM, "José A. Feijó" [hidden email] wrote: Search the CONFOCAL archive athttp://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalthanks George, it's good to hear about good experiences (and definitelyyou raise out some good points). But again 10x and 770nm may not be whatmost people expect out of a 2P. And sure enough, Hoechst and DAPI are"the" perfect 2P dyes!George McNamara escreveu:Search the CONFOCAL archive athttp://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalHi Jose,Thanks for the great post!On City of Hope's multiphoton rig, with a Chameleon split between twoLSM510's (see Brian Armstrong's e-signature), I had no problem imaginghundreds of um into freshly excised mouse brain stained with Hoechstfor 30 minutes (perfusion fixed - handy to flush the blood out). 10xobjective lens. Try 770 nm excitation, 10 ug/mL Hoeschst (do initialdilution from 10 mg/mL bottle into dH2O), then can dilute intowhatever the brain is in. 770 nm is also optimal for several (notall!) Alexa dyes according to a JBO article by Mary Dickinson - soAlexa Fluor 488 tomato lectin could be used to label blood vesselsimmediately before sacrificing the mouse.Edrun - This was with fairly low power, but don't be afraid to crankup the power! Also, try more than one objective lens, and testdifferent wavelengths.If working with fixed tissue: If you need to make a fixed specimentransparent, there are several recipes for clearing tissue - seeRobert Zucker's papers using BABB for example. An interesting mountingmedium is 2,2'-thiodiethanol, published by Staudt et al in January.best wishes,GeorgeAt 06:28 AM 12/7/2007, you wrote:Search the CONFOCAL archive athttp://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalthe problem I believe is not 2P physics, but the Zeiss 510. We'vebeen trying to keep a low profile about this, but it's certainlyfrustrating that we can't really get much out of 510 under 2p, andit's painful that we still get much better results with aside-by-side Bio-Rad 1024MP, despite the 10yrs+ difference in termsof design and technology (do mind, sharing the same laser and opticalpath, we just diverge form one to the other with a simmplereflector!!). During the purchase process I made a number ofinquiries about a number of physical parameters affecting thenegative GVD performance of the system, namely the AOM and theexternal detectors path, and NEVER got any satisfactory, even lessquantitative answer from anyone from Zeiss. The AOM sucks, itdestroys the power and the pulsewidth, one good reason for you to seesuch small difference, penetration is pretty much dependent on bothof these. We've tried a simple hand operated ND filter, butunfortunately the fly-back of the beam in the 510 turned out to benasty for most samples. The external PMT's optical design sucks^2,and does not help either.In our hands, and after 2 years of fighting the beast and trying tohave some answers from Zeiss, we are changing our initial plan ofdowngrading the Bio-Rad 1024 to confocal, and focus on the Zeiss for2P to make use of all the nice software features and (oh insanenaivety!) eventually the META detector, and now we find ourselvesbuying video amplifiers from China to repair the Bio-Rad amplifiersand try to keep it running, and eventually downgrade the Zeiss tobare confocal (eventually we will have tow external for sale, in caseanyone's interested...).I voted against the merge Zeiss/BioRad, but was kind of hoping thatZeiss people could eventually learn from their 2P design investment,and their user database experience. That is surely not the case withthe 510. In short I fail to meet anyone with a different experiencethan ours, if there is someone out there on the list that actuallymanaged to bend a 510 to fulfill the kind of expectations we've gotused to with other machines, and have been reported in many papersout there, we would be very interested in learning how. By now Ihave problems seeing it coming from Zeiss, they are probably moreconcerned on developing the forthcoming 610 or whatever (and thenperhaps propose costly upgrades "true that one didn't work, but thisone will..."), but the sad reality is that I feel that Zeiss has NOTcome of age in matters of 2P. Not even with the monopoly of the patent.I would be very happy to retract myself to the list if I'm provenwrong in this regard...JoseEdrun Andrea Schnell escreveu:Search the CONFOCAL archive athttp://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalDear list,it is a known fact that a two photon laser will penetrate deeper intotissue, and we have done several experiments to show this, but withoutsuccess. We have used spheroids labeled with several dyes, and imagedwith our Zeiss LSM510 with 1) visible laser and descanned detector,2) twophoton laser and descanned detector and 3) two photon laser andnon-descanned detector. We have only found a difference of about10-20 umfrom exp. 1) to exp. 3), whereas it should be around 100 um.So I'm wondering if anyone else have done this kind of experimentand haveany tips as to how to image this increase in penetration depth? Type ofsample and dye etc.?Thanks a lot!Regards,Edrun A. Schnell--Edrun Andrea SchnellDivisional engineer,Dept. of Physics, NTNUHogskoleringen 5, 7491 TrondheimNorway--**********************************************************Jose' A. Feijo', Prof.----------------------------------------------------------Dep. Biologia Vegetal, Fac.Ciencias, Universidade LisboaPT-1749-016 Lisboa, PORTUGALtel. +351.21.750.00.47/00/24, fax +351.21.750.00.48and/ eInst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGALtel. +351.21.440.79.41/00/19, fax +351.21.440.79.70__________________________________________________________e.mail: [hidden email]URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38**********************************************************George McNamara, Ph.D.University of Miami, Miller School of MedicineImage CoreMiami, FL 33010[hidden email][hidden email]305-243-8436 officehttp://home.earthlink.net/~pubspectra/http://home.earthlink.net/~geomcnamara/http://www.sylvester.org/health_pro/shared_resources/index.asp (seeAnalytical Imaging Core Facility)--**********************************************************Jose' A. Feijo', Prof.----------------------------------------------------------Dep. Biologia Vegetal, Fac.Ciencias, Universidade LisboaPT-1749-016 Lisboa, PORTUGALtel. +351.21.750.00.47/00/24, fax +351.21.750.00.48and/ eInst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGALtel. +351.21.440.79.41/00/19, fax +351.21.440.79.70__________________________________________________________e.mail: [hidden email]URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38********************************************************** -- ********************************************************** Jose' A. Feijo', Prof. ---------------------------------------------------------- Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa PT-1749-016 Lisboa, PORTUGAL tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48 and/ e Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70 __________________________________________________________ e.mail: [hidden email] URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38 ********************************************************** This communication is
intended only for the named recipient and may contain information that is
confidential, legally privileged or subject to copyright; the Ludwig Institute
for Cancer Research does not waiver any rights if you have received this communication
in error. |
|
|
Christian Soeller |
Re: Deep penetration in tissue with two photon
|
|
In reply to this post by Craig Brideau
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Chirping compensation is also important with even moderate GVD if you are trying to use very short pulses (much less than 120fs). To my knowledge there is no very good reason to use extremely short pulses. In fact, photobleaching and toxicity could be much worse for equal 2P excitation. Christian On 8/12/2007, at 4:21 AM, Craig Brideau wrote:
-- Christian Soeller PhD Dept. of Physiology +64 9 3737599 x82770 University of Auckland Auckland, New Zealand fax +64 9 3737499 |
|
|
Craig Brideau |
Re: Deep penetration in tissue with two photon
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal If you have good GVD compensation a short pulse is better since it will be more effective at the focus within the sample. Since the sample eats most of your power that short pulse width can make a difference, assuming you have the gear to pre-chirp it accordingly. Craig On Dec 11, 2007 11:00 PM, Christian Soeller <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Chirping compensation is also important with even moderate GVD if you are > trying to use very short pulses (much less than 120fs). To my knowledge > there is no very good reason to use extremely short pulses. In fact, > photobleaching and toxicity could be much worse for equal 2P excitation. > > Christian > > > On 8/12/2007, at 4:21 AM, Craig Brideau wrote: > > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Pre-chirp only makes a huge difference when the GVD induced by your > system is quite large. This is probably true when something like an > AOM is present. We built our own compressors with prisms and > optomechanics, but this is not an exercise for the faint-of-heart. > From what I've seen the DeepSee is probably the simplest GVD control > method from a user perspective and apparently can provide quite a high > level of GVD compensation. > > Craig > > > On Dec 7, 2007 7:32 AM, Nuno Moreno <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi, > > In the Zeiss the problem of removing the AOM is that it has a mechanical > shutter that opens before the galvos start to scan. This means that you > will get a burned spot at the center of the FOV (I tried that one). You > can build some electronics to synchronize the slow motor/bar based > shutter with the AOM signals but I would believe it will be an pain...or > maybe not, I have to check that! > > Talking about pre-chirping do you know someone using something similar > to maitai deepsee in a Coherent Mira laser? > > Regards, > NM > > > Craig Brideau wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Our own lab has found that modifying confocal microscopes for 2-photon > is a tricky business. As mentioned before, an AOM will cause problems > with the laser. If there is any way to bypass the AOM in your system, > or simply remove it, this will help. > Another issue is optics and objectives. Most optics and objectives > are designed for the visible range rather than the NIR. From my own > experience certain medium-high-end Nikon and Olympus lenses tend to > have the best NIR performance, and most others are terrible; absorbing > 90% or more of the light. A crude test is to use a laser power meter > to measure the power after your objective. Place the detector head > some distance away where the light is diverging but still all of it > hits the detector surface; the focal point can damage your detector. > Compare this to the power before the microscope to get your system > losses. Don't be afraid to ask your manufacturer for the NIR > transmission spectrum for your lenses. > > Craig > > > On Dec 7, 2007 5:06 AM, "José A. Feijó" <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > thanks George, it's good to hear about good experiences (and definitely > you raise out some good points). But again 10x and 770nm may not be what > most people expect out of a 2P. And sure enough, Hoechst and DAPI are > "the" perfect 2P dyes! > > George McNamara escreveu: > > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi Jose, > > Thanks for the great post! > > On City of Hope's multiphoton rig, with a Chameleon split between two > LSM510's (see Brian Armstrong's e-signature), I had no problem imaging > hundreds of um into freshly excised mouse brain stained with Hoechst > for 30 minutes (perfusion fixed - handy to flush the blood out). 10x > objective lens. Try 770 nm excitation, 10 ug/mL Hoeschst (do initial > dilution from 10 mg/mL bottle into dH2O), then can dilute into > whatever the brain is in. 770 nm is also optimal for several (not > all!) Alexa dyes according to a JBO article by Mary Dickinson - so > Alexa Fluor 488 tomato lectin could be used to label blood vessels > immediately before sacrificing the mouse. > > Edrun - This was with fairly low power, but don't be afraid to crank > up the power! Also, try more than one objective lens, and test > different wavelengths. > > If working with fixed tissue: If you need to make a fixed specimen > transparent, there are several recipes for clearing tissue - see > Robert Zucker's papers using BABB for example. An interesting mounting > medium is 2,2'-thiodiethanol, published by Staudt et al in January. > > best wishes, > > > George > > > > At 06:28 AM 12/7/2007, you wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > the problem I believe is not 2P physics, but the Zeiss 510. We've > been trying to keep a low profile about this, but it's certainly > frustrating that we can't really get much out of 510 under 2p, and > it's painful that we still get much better results with a > side-by-side Bio-Rad 1024MP, despite the 10yrs+ difference in terms > of design and technology (do mind, sharing the same laser and optical > path, we just diverge form one to the other with a simmple > reflector!!). During the purchase process I made a number of > inquiries about a number of physical parameters affecting the > negative GVD performance of the system, namely the AOM and the > external detectors path, and NEVER got any satisfactory, even less > quantitative answer from anyone from Zeiss. The AOM sucks, it > destroys the power and the pulsewidth, one good reason for you to see > such small difference, penetration is pretty much dependent on both > of these. We've tried a simple hand operated ND filter, but > unfortunately the fly-back of the beam in the 510 turned out to be > nasty for most samples. The external PMT's optical design sucks^2, > and does not help either. > > In our hands, and after 2 years of fighting the beast and trying to > have some answers from Zeiss, we are changing our initial plan of > downgrading the Bio-Rad 1024 to confocal, and focus on the Zeiss for > 2P to make use of all the nice software features and (oh insane > naivety!) eventually the META detector, and now we find ourselves > buying video amplifiers from China to repair the Bio-Rad amplifiers > and try to keep it running, and eventually downgrade the Zeiss to > bare confocal (eventually we will have tow external for sale, in case > anyone's interested...). > > I voted against the merge Zeiss/BioRad, but was kind of hoping that > Zeiss people could eventually learn from their 2P design investment, > and their user database experience. That is surely not the case with > the 510. In short I fail to meet anyone with a different experience > than ours, if there is someone out there on the list that actually > managed to bend a 510 to fulfill the kind of expectations we've got > used to with other machines, and have been reported in many papers > out there, we would be very interested in learning how. By now I > have problems seeing it coming from Zeiss, they are probably more > concerned on developing the forthcoming 610 or whatever (and then > perhaps propose costly upgrades "true that one didn't work, but this > one will..."), but the sad reality is that I feel that Zeiss has NOT > come of age in matters of 2P. Not even with the monopoly of the patent. > > I would be very happy to retract myself to the list if I'm proven > wrong in this regard... > > Jose > > Edrun Andrea Schnell escreveu: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear list, > > it is a known fact that a two photon laser will penetrate deeper into > tissue, and we have done several experiments to show this, but without > success. We have used spheroids labeled with several dyes, and imaged > with our Zeiss LSM510 with 1) visible laser and descanned detector, > 2) two > photon laser and descanned detector and 3) two photon laser and > non-descanned detector. We have only found a difference of about > 10-20 um > from exp. 1) to exp. 3), whereas it should be around 100 um. > > So I'm wondering if anyone else have done this kind of experiment > and have > any tips as to how to image this increase in penetration depth? Type of > sample and dye etc.? > > Thanks a lot! > > Regards, > Edrun A. Schnell > > -- > > Edrun Andrea Schnell > Divisional engineer, > Dept. of Physics, NTNU > Hogskoleringen 5, 7491 Trondheim > Norway > > > > -- > > > ********************************************************** > Jose' A. Feijo', Prof. > ---------------------------------------------------------- > Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa > PT-1749-016 Lisboa, PORTUGAL > > tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48 > > and/ e > > Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL > > tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70 > __________________________________________________________ > e.mail: [hidden email] > URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38 > ********************************************************** > > > > > > George McNamara, Ph.D. > University of Miami, Miller School of Medicine > Image Core > Miami, FL 33010 > [hidden email] > [hidden email] > 305-243-8436 office > http://home.earthlink.net/~pubspectra/ > http://home.earthlink.net/~geomcnamara/ > http://www.sylvester.org/health_pro/shared_resources/index.asp (see > Analytical Imaging Core Facility) > > > -- > > > > ********************************************************** > Jose' A. Feijo', Prof. > ---------------------------------------------------------- > Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa > PT-1749-016 Lisboa, PORTUGAL > > tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48 > > and/ e > > Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL > > tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70 > __________________________________________________________ > e.mail: [hidden email] > URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38 > ********************************************************** > > > > > > -- > > Nuno Moreno > Cell Imaging Unit > Instituto Gulbenkian de Ciência > http://uic.igc.gulbekian.pt > http://www.igc.gulbekian.pt > phone +351 214464606 > fax +351 214407970 > > > > > > > -- > Christian Soeller PhD Dept. of Physiology +64 9 3737599 x82770 > University of Auckland Auckland, New Zealand fax +64 9 3737499 > > |
|
|
Alberto Diaspro |
Re: Deep penetration in tissue with two photon
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal ... well, however not pulses, let say, too short lets try indicating a number for short pulses... 140 fs at the back focal plane of the obejective Alby Il giorno 12/dic/07, alle ore 08:25, Craig Brideau ha scritto: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > If you have good GVD compensation a short pulse is better since it > will be more effective at the focus within the sample. Since the > sample eats most of your power that short pulse width can make a > difference, assuming you have the gear to pre-chirp it accordingly. > > Craig > > On Dec 11, 2007 11:00 PM, Christian Soeller > <[hidden email]> wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> Chirping compensation is also important with even moderate GVD if >> you are >> trying to use very short pulses (much less than 120fs). To my >> knowledge >> there is no very good reason to use extremely short pulses. In fact, >> photobleaching and toxicity could be much worse for equal 2P >> excitation. >> >> Christian >> >> >> On 8/12/2007, at 4:21 AM, Craig Brideau wrote: >> >> >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Pre-chirp only makes a huge difference when the GVD induced by your >> system is quite large. This is probably true when something like an >> AOM is present. We built our own compressors with prisms and >> optomechanics, but this is not an exercise for the faint-of-heart. >> From what I've seen the DeepSee is probably the simplest GVD control >> method from a user perspective and apparently can provide quite a >> high >> level of GVD compensation. >> >> Craig >> >> >> On Dec 7, 2007 7:32 AM, Nuno Moreno <[hidden email]> wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hi, >> >> In the Zeiss the problem of removing the AOM is that it has a >> mechanical >> shutter that opens before the galvos start to scan. This means that >> you >> will get a burned spot at the center of the FOV (I tried that one). >> You >> can build some electronics to synchronize the slow motor/bar based >> shutter with the AOM signals but I would believe it will be an >> pain...or >> maybe not, I have to check that! >> >> Talking about pre-chirping do you know someone using something >> similar >> to maitai deepsee in a Coherent Mira laser? >> >> Regards, >> NM >> >> >> Craig Brideau wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Our own lab has found that modifying confocal microscopes for 2- >> photon >> is a tricky business. As mentioned before, an AOM will cause >> problems >> with the laser. If there is any way to bypass the AOM in your >> system, >> or simply remove it, this will help. >> Another issue is optics and objectives. Most optics and objectives >> are designed for the visible range rather than the NIR. From my own >> experience certain medium-high-end Nikon and Olympus lenses tend to >> have the best NIR performance, and most others are terrible; >> absorbing >> 90% or more of the light. A crude test is to use a laser power meter >> to measure the power after your objective. Place the detector head >> some distance away where the light is diverging but still all of it >> hits the detector surface; the focal point can damage your detector. >> Compare this to the power before the microscope to get your system >> losses. Don't be afraid to ask your manufacturer for the NIR >> transmission spectrum for your lenses. >> >> Craig >> >> >> On Dec 7, 2007 5:06 AM, "José A. Feijó" <[hidden email]> wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> thanks George, it's good to hear about good experiences (and >> definitely >> you raise out some good points). But again 10x and 770nm may not be >> what >> most people expect out of a 2P. And sure enough, Hoechst and DAPI are >> "the" perfect 2P dyes! >> >> George McNamara escreveu: >> >> >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hi Jose, >> >> Thanks for the great post! >> >> On City of Hope's multiphoton rig, with a Chameleon split between two >> LSM510's (see Brian Armstrong's e-signature), I had no problem >> imaging >> hundreds of um into freshly excised mouse brain stained with Hoechst >> for 30 minutes (perfusion fixed - handy to flush the blood out). 10x >> objective lens. Try 770 nm excitation, 10 ug/mL Hoeschst (do initial >> dilution from 10 mg/mL bottle into dH2O), then can dilute into >> whatever the brain is in. 770 nm is also optimal for several (not >> all!) Alexa dyes according to a JBO article by Mary Dickinson - so >> Alexa Fluor 488 tomato lectin could be used to label blood vessels >> immediately before sacrificing the mouse. >> >> Edrun - This was with fairly low power, but don't be afraid to crank >> up the power! Also, try more than one objective lens, and test >> different wavelengths. >> >> If working with fixed tissue: If you need to make a fixed specimen >> transparent, there are several recipes for clearing tissue - see >> Robert Zucker's papers using BABB for example. An interesting >> mounting >> medium is 2,2'-thiodiethanol, published by Staudt et al in January. >> >> best wishes, >> >> >> George >> >> >> >> At 06:28 AM 12/7/2007, you wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> the problem I believe is not 2P physics, but the Zeiss 510. We've >> been trying to keep a low profile about this, but it's certainly >> frustrating that we can't really get much out of 510 under 2p, and >> it's painful that we still get much better results with a >> side-by-side Bio-Rad 1024MP, despite the 10yrs+ difference in terms >> of design and technology (do mind, sharing the same laser and optical >> path, we just diverge form one to the other with a simmple >> reflector!!). During the purchase process I made a number of >> inquiries about a number of physical parameters affecting the >> negative GVD performance of the system, namely the AOM and the >> external detectors path, and NEVER got any satisfactory, even less >> quantitative answer from anyone from Zeiss. The AOM sucks, it >> destroys the power and the pulsewidth, one good reason for you to see >> such small difference, penetration is pretty much dependent on both >> of these. We've tried a simple hand operated ND filter, but >> unfortunately the fly-back of the beam in the 510 turned out to be >> nasty for most samples. The external PMT's optical design sucks^2, >> and does not help either. >> >> In our hands, and after 2 years of fighting the beast and trying to >> have some answers from Zeiss, we are changing our initial plan of >> downgrading the Bio-Rad 1024 to confocal, and focus on the Zeiss for >> 2P to make use of all the nice software features and (oh insane >> naivety!) eventually the META detector, and now we find ourselves >> buying video amplifiers from China to repair the Bio-Rad amplifiers >> and try to keep it running, and eventually downgrade the Zeiss to >> bare confocal (eventually we will have tow external for sale, in case >> anyone's interested...). >> >> I voted against the merge Zeiss/BioRad, but was kind of hoping that >> Zeiss people could eventually learn from their 2P design investment, >> and their user database experience. That is surely not the case with >> the 510. In short I fail to meet anyone with a different experience >> than ours, if there is someone out there on the list that actually >> managed to bend a 510 to fulfill the kind of expectations we've got >> used to with other machines, and have been reported in many papers >> out there, we would be very interested in learning how. By now I >> have problems seeing it coming from Zeiss, they are probably more >> concerned on developing the forthcoming 610 or whatever (and then >> perhaps propose costly upgrades "true that one didn't work, but this >> one will..."), but the sad reality is that I feel that Zeiss has NOT >> come of age in matters of 2P. Not even with the monopoly of the >> patent. >> >> I would be very happy to retract myself to the list if I'm proven >> wrong in this regard... >> >> Jose >> >> Edrun Andrea Schnell escreveu: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Dear list, >> >> it is a known fact that a two photon laser will penetrate deeper into >> tissue, and we have done several experiments to show this, but >> without >> success. We have used spheroids labeled with several dyes, and imaged >> with our Zeiss LSM510 with 1) visible laser and descanned detector, >> 2) two >> photon laser and descanned detector and 3) two photon laser and >> non-descanned detector. We have only found a difference of about >> 10-20 um >> from exp. 1) to exp. 3), whereas it should be around 100 um. >> >> So I'm wondering if anyone else have done this kind of experiment >> and have >> any tips as to how to image this increase in penetration depth? >> Type of >> sample and dye etc.? >> >> Thanks a lot! >> >> Regards, >> Edrun A. Schnell >> >> -- >> >> Edrun Andrea Schnell >> Divisional engineer, >> Dept. of Physics, NTNU >> Hogskoleringen 5, 7491 Trondheim >> Norway >> >> >> >> -- >> >> >> ********************************************************** >> Jose' A. Feijo', Prof. >> ---------------------------------------------------------- >> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa >> PT-1749-016 Lisboa, PORTUGAL >> >> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48 >> >> and/ e >> >> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL >> >> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70 >> __________________________________________________________ >> e.mail: [hidden email] >> URL: http://www.igc.gulbenkian.pt/code/research.php? >> lang=en&unit_id=38 >> ********************************************************** >> >> >> >> >> >> George McNamara, Ph.D. >> University of Miami, Miller School of Medicine >> Image Core >> Miami, FL 33010 >> [hidden email] >> [hidden email] >> 305-243-8436 office >> http://home.earthlink.net/~pubspectra/ >> http://home.earthlink.net/~geomcnamara/ >> http://www.sylvester.org/health_pro/shared_resources/index.asp (see >> Analytical Imaging Core Facility) >> >> >> -- >> >> >> >> ********************************************************** >> Jose' A. Feijo', Prof. >> ---------------------------------------------------------- >> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa >> PT-1749-016 Lisboa, PORTUGAL >> >> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48 >> >> and/ e >> >> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL >> >> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70 >> __________________________________________________________ >> e.mail: [hidden email] >> URL: http://www.igc.gulbenkian.pt/code/research.php? >> lang=en&unit_id=38 >> ********************************************************** >> >> >> >> >> >> -- >> >> Nuno Moreno >> Cell Imaging Unit >> Instituto Gulbenkian de Ciência >> http://uic.igc.gulbekian.pt >> http://www.igc.gulbekian.pt >> phone +351 214464606 >> fax +351 214407970 >> >> >> >> >> >> >> -- >> Christian Soeller PhD Dept. of Physiology +64 9 3737599 x82770 >> University of Auckland Auckland, New Zealand fax +64 9 3737499 >> >> ---------------------------------------------------- "Follow knowledge wherever it leads us." (reading Galileo Galilei) ----------------------------------------------------- Alberto Diaspro, EBSA President-Elect, MicroScoBIO LAMBS-IFOM, Department of Physics, University of Genoa, Via Dodecaneso 33, 16146 Genoa, Italy - fax +39-010314218 - tel +39 0103536426/309; URLs: www.lambs.it ; EBSA is Biophysics in Europe - European Biophysical Societies' Association www.ebsa.org ---------------------------------------------- |
|
|
Dale Callaham |
Re: Recent two-photon thread regarding "photon depth"
|
|
In reply to this post by Edrun Andrea Schnell
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I just thought that the listers might want to check their "Junk" mail folders. I just realized that I have been missing a whole thread with a subject line including the words "Deep Penetration" that Seamonkey (a Mozilla product) has been automatically flagging and moving to the JUNK folder for understandable reasons. Maybe a different subject line would be better? Edrun Andrea Schnell wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear list, > > it is a known fact that a two photon laser will penetrate deeper into > tissue, and we have done several experiments to show this, but without > success. We have used spheroids labeled with several dyes, and imaged > with our Zeiss LSM510 with 1) visible laser and descanned detector, 2) two > photon laser and descanned detector and 3) two photon laser and > non-descanned detector. We have only found a difference of about 10-20 um > from exp. 1) to exp. 3), whereas it should be around 100 um. > > So I'm wondering if anyone else have done this kind of experiment and have > any tips as to how to image this increase in penetration depth? Type of > sample and dye etc.? > > Thanks a lot! > > Regards, > Edrun A. Schnell > > -- > > Edrun Andrea Schnell > Divisional engineer, > Dept. of Physics, NTNU > Hogskoleringen 5, 7491 Trondheim > Norway |
|
|
Craig Brideau |
Re: Recent two-photon thread regarding "photon depth"
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Actually, if you have any control over your junk filter tell it to never spamify messages from the confocal listserv address. You do raise a point though, as occasionally you might receive an email directly from a lister rather than the list itself. Craig On Dec 13, 2007 6:50 AM, Dale Callaham <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > I just thought that the listers might want to check their "Junk" mail > folders. I just realized that I have been missing a whole thread with a > subject line including the words "Deep Penetration" that Seamonkey (a > Mozilla product) has been automatically flagging and moving to the JUNK > folder for understandable reasons. Maybe a different subject line would > be better? > > Edrun Andrea Schnell wrote: > > Search the CONFOCAL archive at > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > > Dear list, > > > > it is a known fact that a two photon laser will penetrate deeper into > > tissue, and we have done several experiments to show this, but without > > success. We have used spheroids labeled with several dyes, and imaged > > with our Zeiss LSM510 with 1) visible laser and descanned detector, 2) two > > photon laser and descanned detector and 3) two photon laser and > > non-descanned detector. We have only found a difference of about 10-20 um > > from exp. 1) to exp. 3), whereas it should be around 100 um. > > > > So I'm wondering if anyone else have done this kind of experiment and have > > any tips as to how to image this increase in penetration depth? Type of > > sample and dye etc.? > > > > Thanks a lot! > > > > Regards, > > Edrun A. Schnell > > > > -- > > > > Edrun Andrea Schnell > > Divisional engineer, > > Dept. of Physics, NTNU > > Hogskoleringen 5, 7491 Trondheim > > Norway > |
«
Return to Confocal Microscopy List
|
1 view|%1 views
| Free forum by Nabble | Edit this page |