SP5 resonant scanner
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Hello,
I'm looking for information about practical experiences with the SP5 resonant scanner.
Leica is claiming: resonant scanners have a much better signal/noise when compared with normal scanning devices, but still offer true optical sectioning at the diffraction limit.
Do you have any practical evidence on this?
Thanks for your help.
Dimitri
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All resonant scanners really do is go faster. They do have smoother motion because they are operating at a mechanical resonance point, but they are not some sort of magic bullet. They just sweep the laser faster, which if you have a weak sample will require you to turn up your laser as the sample will have a shorter effective exposure time.
Craig On Mon, Apr 7, 2008 at 7:03 AM, Gurrie Dimitri <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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In reply to this post by Gurrie Dimitri
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal forget the better do signal/noise ratio. Actually because pixel dwell time is short, the pmts have less time to make a more statistically accurate measurement. What leica claims is that due to faster scanning, the number of molecules in the exited state and triples which get one more photon is decreased, consequently decreasing bleaching. As far as I remember, the resonant scanner operates at 8000 lps which makes a pixel occurring in the range of tens of ns. If you remember a typical relaxation half life time is <10ns, meaning that at some degree this can be true. Apart from the numbers, we do realize that bleaching is in fact less but it is not linear (some time because you have to use more laser power its actually higher). This can be due to the lifetime of the fluophore, due to environment conditions, etc... About speed I remember taking pictures at 3ms frame rate with a 16 pixel height, having the complete confocality. I had never try it but I would say that the sp5x (fiber white laser) can have a laser power limitation when using the resonant head. No commercial interest, NM Gurrie Dimitri wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Hello, > > I'm looking for information about practical experiences with the SP5 > resonant scanner. > > Leica is claiming: resonant scanners have a much better signal/noise > when compared with normal scanning devices, but still offer true optical > sectioning at the diffraction limit. > > Do you have any practical evidence on this? > > Thanks for your help. > > Dimitri > > > ------------------------------------------------------------------------ > Envoyé avec Yahoo! Mail > <http://us.rd.yahoo.com/mailuk/taglines/isp/control/*http://us.rd.yahoo.com/evt=52423/*http://fr.docs.yahoo.com/mail/overview/index.html>. > Une boite mail plus intelligente. -- 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 |
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Search the CONFOCAL archive at
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Nuno Moreno wrote:
"What leica claims is that due to faster scanning,
the number of molecules in the exited state and triples which get one more photon is decreased, consequently decreasing bleaching. As far as I remember, the resonant scanner operates at 8000 lps which makes a pixel occurring in the range of tens of ns. If you remember a typical relaxation half life time is <10ns, meaning that at some degree this can be true." I'm not sure I buy this. If we got down to 1ns dwell time than maybe - but then we wouldn't get an image because the dwell is shorter than the lifetime.
In a conventional 4ms dwell time the likelihood of two photons arriving within
one 10ns period is probably lower than with the fast scanner because we
can use a lower laser intensity. Once the dwell time exceeds the relaxation
time I can't see that this effect could apply. Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Do you think it can have some effect for triplet states? Guy Cox wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Nuno Moreno wrote: > "What leica claims is that due to faster scanning, > the number of molecules in the exited state and triples which get one > more photon is decreased, consequently decreasing bleaching. As far as I > remember, the resonant scanner operates at 8000 lps which makes a pixel > occurring in the range of tens of ns. If you remember a typical > relaxation half life time is <10ns, meaning that at some degree this can > be true." > > I'm not sure I buy this. If we got down to 1ns dwell time than maybe - but > then we wouldn't get an image because the dwell is shorter than the > lifetime. > In a conventional 4ms dwell time the likelihood of two photons arriving > within > one 10ns period is probably lower than with the fast scanner because we > can use a lower laser intensity. Once the dwell time exceeds the > relaxation > time I can't see that this effect could apply. > > Guy > > > Optical Imaging Techniques in Cell Biology > by Guy Cox CRC Press / Taylor & Francis > http://www.guycox.com/optical.htm > ______________________________________________ > Associate Professor Guy Cox, MA, DPhil(Oxon) > Electron Microscope Unit, Madsen Building F09, > University of Sydney, NSW 2006 > ______________________________________________ > Phone +61 2 9351 3176 Fax +61 2 9351 7682 > Mobile 0413 281 861 > ______________________________________________ > http://www.guycox.net > <https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=http://www.guycox.net> > -- 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 |
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Hi all,
Just to muddy the waters a little more...
The extra laser power is needed not only because the frame time
is so fast (i,e., because you still need the same number of detected
photons to make an image of a given quality) but also because only a
small fraction of the a sinusoidal horizontal scan is linear enough to
use effectively (You can try to linearize it by skewing the pixel
clock, but only so much). This fraction is about 30%, even if you scan
in both directions, 15% if you scan in one direction, versus 60-90%
for non-resonant scanners. Assuming that the beam is blanked or masked
when not scanning the area from which the signal is collected, this
still means that the beam power must be 3-6x higher with resonant
scanning even if you collect each image for the same time (i.e., if
you compare a Kalman average of 25-30 video scans or collect a single,
1s scan).
As far as triplet-state effects however, there may be something
to the argument. Triplet states last a lot longer than singlet states:
ms to microseconds vs ns. So the fact that, after the ~30ns
pixel dwell time, any triplet excitation will have a chance to decay
during the frame time before again being assaulted by photons that
might excite it from the triplet state to a state where permanent
damage would occur, might be an advantage.
Of course, we need to remember that it isn't quite this simple.
Assuming Nyquist sampling, each molecule is actually illuminated for a
time equal to 4 pixel-dwell times, and this happens on 4 successive
scan lines. Even so, any molecule excited into the triplet state in
this way would spend relatively less time under illumination with a
resonant, video-rate raster than with a 1 second linear scan (assuming
512x512 raster, 3 microseconds/pixel).
Cheers,
Jim P.
**********************************************
Prof. James B. Pawley, Room 223, Zoology Research Building, 1117 Johnson Ave., Madison, WI, 53706 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada Info:
http://www.3dcourse.ubc.ca/
Do you think it can have some effect for triplet states?
-- |
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In reply to this post by Nuno Moreno
Search the CONFOCAL archive at
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All,
a summary of the effects is given in the publication below.
Martin
MRT Letter: High Speed Scanning Has the Potential to
Increase Fluorescence Yield and to Reduce Photobleaching Rolf T. Borlinghaus
Microsc. Res. Tech. 69: 2006. Wiley-Liss, Inc.
------------------------------------------------------------------------------ Martin Hoppe, Ph.D.
Head of Market Management & Confocal Applications
Life Science Division
Leica Microsystems CMS GmbH
Am Friedensplatz 3 | 68165 Mannheim (Germany)
Phone : +49 621 7028 1100 | Fax : +49 621 7028 1180
Cell: +49 172 623 0409
-----Ursprüngliche Mitteilung----- Von: Nuno Moreno <[hidden email]> An: [hidden email] Verschickt: Di., 8. Apr. 2008, 16:10 Thema: Re: SP5 resonant scanner Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Do you think it can have some effect for triplet states? Guy Cox wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Nuno Moreno wrote: > "What leica claims is that due to faster scanning, > the number of molecules in the exited state and triples which get one > more photon is decreased, consequently decreasing bleaching. As far as I > remember, the resonant scanner operates at 8000 lps which makes a pixel > occurring in the range of tens of ns. If you remember a typical > relaxation half life time is <10ns, meaning that at some degree this can > be true." > > I'm not sure I buy this. If we got down to 1ns dwell time than maybe - but > then we wouldn't get an image because the dwell is shorter than the > lifetime. > In a conventional 4ms dwell time the likelihood of two photons arriving > within > one 10ns period is probably lower than with the fast scanner because we > can use a lower laser intensity. Once the dwell time exceeds the > relaxation > time I can't see that this effect could apply. > > Guy > > > Optical Imaging Techniques in Cell Biology > by Guy Cox CRC Press / Taylor & Francis > http://www.guycox.com/optical.htm > ______________________________________________ > Associate Professor Guy Cox, MA, DPhil(Oxon) > Electron Microscope Unit, Madsen Building F09, > University of Sydney, NSW 2006 > ______________________________________________ > Phone +61 2 9351 3176 Fax +61 2 9351 7682 > Mobile 0413 281 861 > ______________________________________________ > http://www.guycox.net > <https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=http://www.guycox.net> > -- 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 Bei AOL gibt's jetzt kostenlos eMail für alle! Was es sonst noch umsonst bei AOL gibt, finden Sie hier heraus AOL.de. |
 
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Martin Hoppe-2 |
Re: SP5 resonant scanner - complete reference
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In reply to this post by Nuno Moreno
Search the CONFOCAL archive at
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Sorry,
here is the complete reference: Borlinghaus, R.T.: "High speed scanning has the potential to increase fluorescence yield and to reduce photobleaching" Microsc. Res. Tech. 2006 Sep; 69(9): pp 689-692.
------------------------------------------------------------------------------ Martin Hoppe, Ph.D.
Head of Market Management & Confocal Applications
Life Science Division
Leica Microsystems CMS GmbH
Am Friedensplatz 3 | 68165 Mannheim (Germany)
Phone : +49 621 7028 1100 | Fax : +49 621 7028 1180
Cell: +49 172 623 0409
-----Ursprüngliche Mitteilung----- Von: Nuno Moreno <[hidden email]> An: [hidden email] Verschickt: Di., 8. Apr. 2008, 16:10 Thema: Re: SP5 resonant scanner Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Do you think it can have some effect for triplet states? Guy Cox wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Nuno Moreno wrote: > "What leica claims is that due to faster scanning, > the number of molecules in the exited state and triples which get one > more photon is decreased, consequently decreasing bleaching. As far as I > remember, the resonant scanner operates at 8000 lps which makes a pixel > occurring in the range of tens of ns. If you remember a typical > relaxation half life time is <10ns, meaning that at some degree this can > be true." > > I'm not sure I buy this. If we got down to 1ns dwell time than maybe - but > then we wouldn't get an image because the dwell is shorter than the > lifetime. > In a conventional 4ms dwell time the likelihood of two photons arriving > within > one 10ns period is probably lower than with the fast scanner because we > can use a lower laser intensity. Once the dwell time exceeds the > relaxation > time I can't see that this effect could apply. > > Guy > > > Optical Imaging Techniques in Cell Biology > by Guy Cox CRC Press / Taylor & Francis > http://www.guycox.com/optical.htm > ______________________________________________ > Associate Professor Guy Cox, MA, DPhil(Oxon) > Electron Microscope Unit, Madsen Building F09, > University of Sydney, NSW 2006 > ______________________________________________ > Phone +61 2 9351 3176 Fax +61 2 9351 7682 > Mobile 0413 281 861 > ______________________________________________ > http://www.guycox.net > <https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=http://www.guycox.net> > -- 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 Bei AOL gibt's jetzt kostenlos eMail für alle! Was es sonst noch umsonst bei AOL gibt, finden Sie hier heraus AOL.de. |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Please, Mats contact me... Dear folks sorry for this Alby-Mats message ... it seems my e-mails do not reach Mats...thank you for comphrension. All the best from Erice Alby ---------------------------------------------------- "Water slowly flowed under the sky" (Cesare Pavese) ----------------------------------------------------- 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 ---------------------------------------------- |
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