Fast & sensitive LSM versus Spinning Disk

[offterdi1]

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Hi
There had been a discussion on that topic already 10 years ago...
I was wondering if it is really still valid to say that a spinning disk
microscope with an EM-CCD is always better for fast live imaging than a
latest generation fast scanning LSM equipped with sensitive detectors- we
have a Leica SP5 with RS and HyDs. 10 years ago people argued that with the
PMTs you will loose a lot of light compared to the SD system. But now we do
have much better detectors on the LSM such as GaSPs and HyDs!  Is it really
still a big advantage to use a spinning disk system compared to a confocal.
Does anyone know a systematic side by side comparison in terms of
signal-to-noise, speed, bleaching properties...?

In other words- is it still better to go for spinning disk, when you need a
fast live cell imaging or can a fast & sensitive LSM truly compete?


Best
Martin
***********************
Martin Offterdinger, PhD
Medical University Innsbruck
CCB
Division of Neurobiochemistry
Biooptics
Innrain 80-82, 1st floor, room 01.370
A-6020 Innsbruck
Austria
***********************
phone number: [phone]
http://www.i-med.ac.at/neurobiochemistry/neurobiochemistry/Biooptics/Main.html

[Gary Laevsky]

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Hi Martin,

I think it really depends on HOW fast you want to go, HOW sensitive you
need to be (intensity of fluor), and what kind of resolution do you need.

Our users are regularly told the instrument to be used is going to be
application specific.

Do you need 100 fps or 5 fps?  Bright fluor or single molecule?  Resolution
or morphology?

John Murray et al published a great paper in 2007 (Evaluating performance
in three-dimensional fluorescence microscopy) comparing the modalities.

While the comparison technique holds, using the newer technologies will be
a moving target.

Besides comparing CCD/EM-CCDs to PMTs, you would have to add sCMOS to the
mix.  This would be a pretty big undertaking!

Gary

On Wed, Apr 22, 2015 at 5:10 AM, Martin Offterdinger <
[hidden email]> wrote:

> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi
> There had been a discussion on that topic already 10 years ago...
> I was wondering if it is really still valid to say that a spinning disk
> microscope with an EM-CCD is always better for fast live imaging than a
> latest generation fast scanning LSM equipped with sensitive detectors- we
> have a Leica SP5 with RS and HyDs. 10 years ago people argued that with the
> PMTs you will loose a lot of light compared to the SD system. But now we do
> have much better detectors on the LSM such as GaSPs and HyDs!  Is it really
> still a big advantage to use a spinning disk system compared to a confocal.
> Does anyone know a systematic side by side comparison in terms of
> signal-to-noise, speed, bleaching properties...?
>
> In other words- is it still better to go for spinning disk, when you need a
> fast live cell imaging or can a fast & sensitive LSM truly compete?
>
>
> Best
> Martin
> ***********************
> Martin Offterdinger, PhD
> Medical University Innsbruck
> CCB
> Division of Neurobiochemistry
> Biooptics
> Innrain 80-82, 1st floor, room 01.370
> A-6020 Innsbruck
> Austria
> ***********************
> phone number: [phone]
>
> http://www.i-med.ac.at/neurobiochemistry/neurobiochemistry/Biooptics/Main.html
>

--
Best,

Gary Laevsky, Ph.D.
Confocal Imaging Facility Manager
Dept. of Molecular Biology
Washington Rd.
Princeton University
Princeton, New Jersey, 08544-1014
(O) 609 258 5432
(C) 508 507 1310

[phil laissue-2]

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It's not just about detection though. Spinning disk will have several
orders of magnitude lower power density compared to CLSM. Still, both
options illuminate the whole sample for every frame, so lightsheet options
might be worth looking at.

_________________________________________
Philippe Laissue, PhD, Director of Bioimaging Unit
School of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / (0044) 07842 676 456
[hidden email]
privatewww.essex.ac.uk/~plaissue

On 22 April 2015 at 10:10, Martin Offterdinger <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi
> There had been a discussion on that topic already 10 years ago...
> I was wondering if it is really still valid to say that a spinning disk
> microscope with an EM-CCD is always better for fast live imaging than a
> latest generation fast scanning LSM equipped with sensitive detectors- we
> have a Leica SP5 with RS and HyDs. 10 years ago people argued that with the
> PMTs you will loose a lot of light compared to the SD system. But now we do
> have much better detectors on the LSM such as GaSPs and HyDs!  Is it really
> still a big advantage to use a spinning disk system compared to a confocal.
> Does anyone know a systematic side by side comparison in terms of
> signal-to-noise, speed, bleaching properties...?
>
> In other words- is it still better to go for spinning disk, when you need a
> fast live cell imaging or can a fast & sensitive LSM truly compete?
>
>
> Best
> Martin
> ***********************
> Martin Offterdinger, PhD
> Medical University Innsbruck
> CCB
> Division of Neurobiochemistry
> Biooptics
> Innrain 80-82, 1st floor, room 01.370
> A-6020 Innsbruck
> Austria
> ***********************
> phone number: [phone]
>
> http://www.i-med.ac.at/neurobiochemistry/neurobiochemistry/Biooptics/Main.html
>

[Csúcs Gábor-3]

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Dear Martin,

Unfortunately, we do not have time to make a really quantitative
comparison but our day to day experience says that with a spinning disk
(irrespective whether EM-CCD or sCMOS) we have significantly better
results on thin, weak, dynamic samples (like visualizing microtubules in
yeast) then with a high-end confocal (Leica SP8 with HyD-s and 8kHZ
resonant scanner). With our sCMOS based system we can get a 4MP (so
relatively large FOV) image with 10-100 ms camera integration time for a
"standard" (of course this is ill defined) GFP labeled sample without
significant bleaching. This is (to my knowledge) impossible with any point
scanning system.

Greetings    Gabor


On 4/22/15 11:10 AM, "Martin Offterdinger"
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>Hi
>There had been a discussion on that topic already 10 years ago...
>I was wondering if it is really still valid to say that a spinning disk
>microscope with an EM-CCD is always better for fast live imaging than a
>latest generation fast scanning LSM equipped with sensitive detectors- we
>have a Leica SP5 with RS and HyDs. 10 years ago people argued that with
>the
>PMTs you will loose a lot of light compared to the SD system. But now we
>do
>have much better detectors on the LSM such as GaSPs and HyDs!  Is it
>really
>still a big advantage to use a spinning disk system compared to a
>confocal.
>Does anyone know a systematic side by side comparison in terms of
>signal-to-noise, speed, bleaching properties...?
>
>In other words- is it still better to go for spinning disk, when you need
>a
>fast live cell imaging or can a fast & sensitive LSM truly compete?
>
>
>Best
>Martin
>***********************
>Martin Offterdinger, PhD
>Medical University Innsbruck
>CCB
>Division of Neurobiochemistry
>Biooptics
>Innrain 80-82, 1st floor, room 01.370
>A-6020 Innsbruck
>Austria
>***********************
>phone number: [phone]
>http://www.i-med.ac.at/neurobiochemistry/neurobiochemistry/Biooptics/Main.
>html

[Jens]

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Hi Martin,

I tried for quite some to image and track virus particles (about 600 FP/particle) in the nucleus. Even state of the art LSMs were not able to give sufficient signal to image and track in the 20-30 fps range. The spinning disks I tried performed better but the best results were obtained after switching to oblique illumination (Hilo, "poor man’s lightsheet") on a TIRF rig with EM-CCD camera. With that setup it was possible to image and automatically track at up to 80fps. Since then I built an oblique setup with ring-illumination at the BFP and this seems to be the ultimate solution right now with nice and even illumination of the FOV. This however works only for pretty flat cells in the 5 - 10 um max range. I am curious how the newer light sheet techniques like diSPIM will perform in comparison.

Best,

Jens

--------------------------------------------------------------
Jens B. Bosse Ph.D.
Enquist Lab
Department of Molecular Biology
and
Princeton Neuroscience Institute
Princeton University
301 Schultz Lab
Washington Rd
08544 Princeton, NJ, USA

Phone: [phone]
Email: [hidden email]
Web: http://molbio.princeton.edu/labs/enquist/

This electronic communication, including any attached documents, may contain confidential and/or legally privileged information that is intended only for use by the recipient(s) named above.  If you have received this communication in error, please notify the sender immediately and delete the communication and any attachments.  

> On Apr 22, 2015, at 5:10 AM, Martin Offterdinger <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi
> There had been a discussion on that topic already 10 years ago...
> I was wondering if it is really still valid to say that a spinning disk
> microscope with an EM-CCD is always better for fast live imaging than a
> latest generation fast scanning LSM equipped with sensitive detectors- we
> have a Leica SP5 with RS and HyDs. 10 years ago people argued that with the
> PMTs you will loose a lot of light compared to the SD system. But now we do
> have much better detectors on the LSM such as GaSPs and HyDs!  Is it really
> still a big advantage to use a spinning disk system compared to a confocal.
> Does anyone know a systematic side by side comparison in terms of
> signal-to-noise, speed, bleaching properties...?
>
> In other words- is it still better to go for spinning disk, when you need a
> fast live cell imaging or can a fast & sensitive LSM truly compete?
>
>
> Best
> Martin
> ***********************
> Martin Offterdinger, PhD
> Medical University Innsbruck
> CCB
> Division of Neurobiochemistry
> Biooptics
> Innrain 80-82, 1st floor, room 01.370
> A-6020 Innsbruck
> Austria
> ***********************
> phone number: [phone]
> http://www.i-med.ac.at/neurobiochemistry/neurobiochemistry/Biooptics/Main.html

[Jason Kirk]

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The main difference to remember between camera based imaging systems and
point scanning detection systems is dwell time.  For a given frame rate the
camera based solutions (SD, TIRF, LightSheet) will always give you a
significantly longer dwell time than a point scanner ever could due to the
nature of the raster scan vs. parallel imaging approach.

While point scanning detection technology has improved greatly over the past
few years - it has not improved THAT much.

As always in these types of cases it depends on how fast you need to go.  IN
my experience for experiments requiring <10 fps then the LSMs can be used
quite effectively.  For >30 fps the SD and LightSheet systems will be better
solutions.

On Wed, 22 Apr 2015 04:10:41 -0500, Martin Offterdinger
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>Hi
>There had been a discussion on that topic already 10 years ago...
>I was wondering if it is really still valid to say that a spinning disk
>microscope with an EM-CCD is always better for fast live imaging than a
>latest generation fast scanning LSM equipped with sensitive detectors- we
>have a Leica SP5 with RS and HyDs. 10 years ago people argued that with the
>PMTs you will loose a lot of light compared to the SD system. But now we do
>have much better detectors on the LSM such as GaSPs and HyDs!  Is it really
>still a big advantage to use a spinning disk system compared to a confocal.
>Does anyone know a systematic side by side comparison in terms of
>signal-to-noise, speed, bleaching properties...?
>
>In other words- is it still better to go for spinning disk, when you need a
>fast live cell imaging or can a fast & sensitive LSM truly compete?
>
>
>Best
>Martin
>***********************
>Martin Offterdinger, PhD
>Medical University Innsbruck
>CCB
>Division of Neurobiochemistry
>Biooptics
>Innrain 80-82, 1st floor, room 01.370
>A-6020 Innsbruck
>Austria
>***********************
>phone number: [phone]
>http://www.i-med.ac.at/neurobiochemistry/neurobiochemistry/Biooptics/Main.html