Leica resonant scanner-live for cell imaging

[Kost, Benedikt]

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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

Tel:       [phone]    
Fax:      [phone]

e-mail:  b.kost@...
internet: http://www2.warwick.ac.uk/fac/sci/whri/

[Friedrich Johenning]

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Dear Benedikt,
I used a custom-built (not the Leica-Product) resonant scanner and a
spinning-disc confocal with a fast CCD camera for calcium imaging in
dendrites and spines of patched neurons in acute brain slices. Although
we never systematically quantified signal to noise ratio vs.
phototoxicity, from my own experience it seems that the spinning disc
confocal is better when it comes to photodamage. The reason is that
resonant scanners have a very short pixel dwell time, which you have to
compensate for by illumination intensity, whereas the spinning disc
uses a parallel scan which permits for longer exposures repeated
several times during one illumination frame. Wang et al., Journal of
Microscopy, 2005 is a very nice study where the difference between
parallel scanning and single-point scanning is thoroughly analysed.
When dealing with large fluorescent volumes, spinning disc confocals
have the problem of crosstalk between adjacent channels, but this
dependes on the type of preparation you use. A disadvantage of the
spinning disc is that it only has one pinhole size which limits the
objectives you can use. Maybe you should also look into the visitec
infinity 3 scanner if you want parallel scanning with different pinhole
sizes.
Best,
Friedrich


Dr. Friedrich W. Johenning
NWFZ -Campus Mitte / AG Schmitz
Charite der Universitätsmedizin Berlin
Charitéplatz 1
10117 Berlin
Tel.: [phone]
Mobil: 0173 2144701
Fax: [phone]

[Rietdorf, Jens]

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[Martin Seem]

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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

[Ken Bell-2]

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Dear Gentlemen,
My name is Lukasz Kilianek.
I'm a Product Specialist at VisiTech International -manufacturer of VT
Infinity and VT Eye confocal units.

I follow your discussion about system comparison in term of speed and
photobleaching.

Let me point out that original mail was about VT Infinity. The last mail
pertains to VT Eye system.
Let me explain here - VT Infinity is a multiple point scanner, VY Eye is
a single spot scanner.
It makes a huge difference in performance.

With best regards

Lukasz Kilianek
Product Specialist
VisiTech International Ltd
Unit 92, Silverbriar
Sunderland Enterprise Park (East)
Sunderland SR5 2TQ, UK
Tel [phone]
Fax [phone]
email: [hidden email]
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Martin Seem
Sent: 13 November 2007 13:57
To: [hidden email]
Subject: Re: Leica resonant scanner-live for cell imaging

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

[James Pawley]

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  [phone]
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

[Martin Seem]

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

Hi Jim,

we tested the PE Ultraview at to separate occasions. Once with a normal
ORCA CCD-camera at the Molecular Imaging Center (MIC) in Bergen
(Norway) and once with an EM CCD camera at PEs training facility at
Seer Green right outside London. The VisiTech QLC100 was a CSU10 based
model equipped with an ORCA CCD-camera while the Andor system used an
EM CCD camera.

Thanks,
Martin
------------------------------------------------------------------------

Martin Seem
Graduate student
Group for Cell and Molecular Biology
Norwegian University of Science and Technology




Quoting James Pawley <[hidden email]>:

> 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  [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
>

[Stefan Terjung]

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

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:[hidden email]] Im Auftrag von James Pawley
> Gesendet: Dienstag, 13. November 2007 19:29
> An: [hidden email]
> 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  [phone]
> 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

[Michael Weber-4]

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

Just to add that, line accumulation is available since LAS AF v1.8. So
you can finally set it up using "Live" Scan.

How does 2x Accumulation compete compared to 2x more laser power? The
total illumination power added to one pixel is in the end simliar. So
signal-to-noise and bleaching effects should be comparable as well, or not?

cheers,
Michael


Stefan Terjung wrote:
> - 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

[Stefan Terjung]

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

Dear Michael,

Ok, I should have installed 1.8 obviously, good to know.

If you plot fluorescence intensity against excitation intensity you get a
saturation curve. As one typically uses high power for point scanning one
easily gets into this saturation range.

This means that increasing the excitation light even more to compensate for
faster scanning you don´t gain as much fluorescence intensity (there is a
limited number of fluorophores and if (nearly) all are excited you can´t
excite more) and it´s even more likely that you bleach stronger as it´s more
likely that you excite a excited fluorophore (which significantly increases
the chance to bleach).

Regards,

Stefan

> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List
> [mailto:[hidden email]] Im Auftrag von Michael Weber
> Gesendet: Mittwoch, 28. November 2007 13:45
> An: [hidden email]
> Betreff: Re: AW: Leica resonant scanner-live for cell imaging
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Just to add that, line accumulation is available since LAS AF
> v1.8. So you can finally set it up using "Live" Scan.
>
> How does 2x Accumulation compete compared to 2x more laser
> power? The total illumination power added to one pixel is in
> the end simliar. So signal-to-noise and bleaching effects
> should be comparable as well, or not?
>
> cheers,
> Michael
>
>
> Stefan Terjung wrote:
> > - 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