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Dear Benedikt and others,
We have a Leica SP5 tandem scanner and a PerkinElmer Ultraview ERS
(6 laserlines, Hamamatsu EM CCD with 8µm pixel size).
Regarding your questions:
- It is easier to setup the fast time-lapse experiment on a spining disk
for fast time-lapse imaging (I can only judge for the PerkinElmer). So
the spinning disks are frequently used for fast time-lapse imaging in
our facility.
- But you can get quite comparable time-lapse sequences with a bit
more 'fine-tuning' on the SP5 (at least for some cases):
a) use accumulation (unfortunately only frame accumulation
available yet) to compensate for lower brightness due to
shorter pixel dwell time instead of increasing the laser power
=> this is similar to increasing the exposure time on a
spinning disk without saturating the fluorophores
b) use less lines (rectangle instead of square format) if possible
c) you can acquire up to 3 channels simultaneous or line-by-line
sequential leading to the same timing for up to 3 channels
compared to frame-by-frame sequential (on the Ultraview - there
are also spinning disks available with 2 cameras and simultaneous
acquisition)
d) often you can open the pinhole at least a bit in time-lapse
imaging
leading to brighter images (not possible on the Yokogawa spinning
disks)
e) you can activate the bidirectional (and optimize the phase with
the
transmission channel as this always has enough brightness and
suitable
structures)
f) you are more flexible adjusting the desired detection range
(zoom!
Illuminating only the detected region)
g) the spinning disk tends to have more problems with thicker
samples
(crosstalk between the pinholes)
- The SP5 (in my opinion) is in a comparable range regarding photobleaching/
phototoxicity as long as one doesn´t use too much laser power (using
rather
accumulations instead of increasing laser power - see 'a)' above).
No commercial interest in both systems.
Hope this helps,
Regards,
Stefan
> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List
> [mailto:[log in to unmask]] Im Auftrag von James Pawley
> Gesendet: Dienstag, 13. November 2007 19:29
> An: [log in to unmask]
> Betreff: Re: Leica resonant scanner-live for cell imaging
>
> 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
> >
> >Hi Benedikt,
> >
> >we acquired a Leica resonant scanner based confocal about 6
> months ago.
> >Before buying this system we tested different types of microscopes
> >including three spinning disc based systems (Andor, PE and Visitec
> >QLC100), two point-scan based systems (Leica and Visitec
> >Eye) and the new Zeiss LSM5 Live system. We brought our own samples
> >(transgenic plant material) and used these to test the different
> >systems in terms of speed, photobleaching and overall image quality.
> >
> >We found the spinning disc based systems to be slightly
> faster than the
> >Leica system especially when acquiring square images (e.g.
> >512x512 pixels). However the speed of the Leica system increases
> >significantly when you reduce the number of lines in the Y
> direction.
> >You should have no problem acquiring 10 fps with a resolution of
> >512x512 pixels resolution and about 15-20 fps with a resolution of
> >512x128 pixels.
> >
> >In our experience the level of photobleaching is quite low
> when using
> >the Leica system. I would say it is comparable to the spinning disc
> >systems we tested and significantly lower than the Visitec
> Eye system
> >that bleached my sample (transgenic GFP5-ER plants) within seconds
> >(this system did however work nicely for calcium imaging in
> dendrites
> >so the level of photobleaching depends on the properties of the
> >fluorechrome).
> >
> >In the end we chose the Leica system because it was
> sufficiently fast,
> >had an adjustable and round pinhole making it fully
> confocal, had low
> >levels of photobleaching, had a very flexible bandpass
> filter solution
> >and could easily and relatively cheaply be upgraded to a
> tandem scanner
> >system (both resonant and conventional scanner in the same system).
> >
> >
> >Best regards
> >Martin Seem
> >-------------------------------------------------------------
> ------------
> >
> >Martin Seem
> >Graduate student
> >Group for Cell and Molecular Biology
> >Norwegian University of Science and Technology
>
> Thank you for this Martin,
>
> Could you please tell us about the type of CCD cameras used with the
> various disk-scanners? EM-CCD or normal CCD?
>
> Thanks,
>
> 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 [log in to unmask]
> "A scientist is not one who can answer questions but one who can
> question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39
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