Re: Leica resonant scanner-live for cell imaging

Posted by Rietdorf, Jens on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Leica-resonant-scanner-live-for-cell-imaging-tp590925p590936.html

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Dear Benedikt,

 

in my opinion, the major advantage of a parallel compared to single beam scanner is a result of the low saturation level of the fluorophores (the laser power is split up to many beamlets or a line or other pattern) and therefore reducing the population of molecules in dark states, while keeping a high overall photon flux. In resonant scanners, this beneficial effect (also) results from avoiding dark states,  its descibed in this article:

 

Donnert, G., C. Eggeling and S. W. Hell: Major signal increase in fluorescence microscopy through dark-state relaxation. Nature Methods 4 81-86 (2007)

 

Among the parallel scanning devices, the spinning disk is (again in my opinion) the most efficient device, because it avoids turning times of scanners, which are dead times for the acquisition, because beams will have to be blanked during the turnaround. Parallel scanners with CCD detectors typically outperform PMTs in terms of QE over a broad wavelength range.

 

Point scanners on the other hand are more flexible, ie you can scan lines or AOIs, rotate scanfields etc., also confocality is not compromised under any circumstance or in any direction. For deep-in tissue imaging (above 50microm), single beam scanners typically outperform parallel scanners.

 

For full frame confocal of living specimen of moderate thickness (up to 25microm), my favorite scanner is still the Yokogawa CSU-22 (no commercial interest) in combinaton with a EMCCD camera and powerful lasers >25mW.

regards, jens

 

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Dr. Jens Rietdorf[hidden email]
Head Microscopy
Novartis Research Foundation
Friedrich-Miescher-Institute, wro1066.2.32
Maulbeerstr.66, CH-4058 Basel, Switzerland
phone [phone] mobil [phone]
Email:rietdorf(at)fmi.ch

 

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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kost, Benedikt
Sent: Montag, 12. November 2007 14:03
To: [hidden email]
Subject: Leica resonant scanner-live for cell imaging

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Can anyone compare the performance of the Leica SP5 resonant scanner with
that of a spinning disk (yokagawa) confocal system in terms of
- image quality
- photobleaching/phototxicity
- general "usefulness" for live cell confocal imaging

The resonant scanner was recently demonstrated to us and I was quite
impressed. I guess it is not as fast as spinning disk confocals can be, but
you seem to be able to image at a rate of at least 10 fps at 512x512, which
is all we need. The images we got were very nice, but  I did not have the chance to do extensive tests.

 

I would appreciate very much comments from anyone who has experience using the resonant scanner for live cell confocal imaging.

Many thanks,

Benedikt Kost
____________________________________________________

Benedikt Kost, PhD
Warwick HRI
Wellesbourne
Warwickshire, CV35 9EF
UK

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