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spinning disc X1 or W1

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SUBSCRIBE CONFOCALMICROSCOPY Virginie Lecaudey

spinning disc X1 or W1

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Hello,

my lab is planning on purchasing a spinning disc system to perform live imaging on zebrafish
embryos. We have experience with the Zeiss setup (CSU-X1 and Zen Blue software) with a
simple axiocam camera and older experience with different version of the PerkinElmer
spinning discs with Hamamatso EM-CCD camera.
Working with zebrafish, we are quite tempting to switch to the CSU-W1 proposed by
Nikon/Andor but would be really happy to get opinion of experts on:

1) How do the CSU-W1 compare to the X1 in terms of required illumintation? Do we need
much longer exposure time (and thus much more light on the sample...)?

2) Is it really a problem to image fast processes with the W1

3) in terms of camera, how does the Zyla 4.2 sCMOS compare to the iXon EMCCD camera?

4) Does anybody have experience with the Borealis system from Andor? How much does it
really improve homogeneity of illumination?

Many thanks in advance for any advice and comments!

Virginie

George McNamara

Re: spinning disc X1 or W1

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Hi Virginie,

There are another disk option for you to consider:

X-Light V2
http://www.crestopt.com/html/x-light_v2_tp.html

vt-iSIM
http://www.visitech.co.uk/vt-isim.html
if you were in the US, you could go through
http://www.biovis.com/vtisim.html

whatever system you choose, I encourage putting Microvolution GPU
deconvolution downstream
http://www.microvolution.com/
(yes, that's an image from my lab on their home page - I do not have a
financial stake in the company). Apparently SVI now has GPU
deconvolution in Huygens as a possible alternative.

The sCMOs cameras (PCO, Andor, Hamamatsu, and even Photometrics is now a
believer) have certain advantages over EMCCD. The key question is: what
camera is best for your application in the context of your entire system
(and budget)?

best wishes,

George

On 3/4/2016 1:39 PM, SUBSCRIBE CONFOCALMICROSCOPY Virginie Lecaudey 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.
> *****
>
> Hello,
>
> my lab is planning on purchasing a spinning disc system to perform live imaging on zebrafish
> embryos. We have experience with the Zeiss setup (CSU-X1 and Zen Blue software) with a
> simple axiocam camera and older experience with different version of the PerkinElmer
> spinning discs with Hamamatso EM-CCD camera.
> Working with zebrafish, we are quite tempting to switch to the CSU-W1 proposed by
> Nikon/Andor but would be really happy to get opinion of experts on:
>
> 1) How do the CSU-W1 compare to the X1 in terms of required illumintation? Do we need
> much longer exposure time (and thus much more light on the sample...)?
>
> 2) Is it really a problem to image fast processes with the W1
>
> 3) in terms of camera, how does the Zyla 4.2 sCMOS compare to the iXon EMCCD camera?
>
> 4) Does anybody have experience with the Borealis system from Andor? How much does it
> really improve homogeneity of illumination?
>
> Many thanks in advance for any advice and comments!
>
> Virginie
>

--

George McNamara, Ph.D.
Single Cells Analyst, T-Cell Therapy Lab (Cooper Lab)
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42
http://works.bepress.com/gmcnamara/75
https://www.linkedin.com/in/georgemcnamara

Lutz Schaefer

Re: spinning disc X1 or W1

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*****

Dear Virginie,
unfortunately I can't give you much advice on how the different Yokogawa
attachments compare. Perhaps you may want to consider ApoTome instead?
Regarding deconvolution processing it is a good idea to stick with ZEN Blue
as there will be a blazingly fast GPU based deconvolution become available
with the next release.

Regards
Lutz

__________________________________
L u t z S c h a e f e r
Research Scientist
Mathematical modeling / Computational microscopy
Advanced Imaging Methodology Consultation
16-715 Doon Village Rd.
Kitchener, ON, N2P 2A2, Canada
Phone/Fax: +1 519 894 8870
Email: [hidden email]
___________________________________

On 3/4/2016 1:39 PM, SUBSCRIBE CONFOCALMICROSCOPY Virginie Lecaudey 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.
> *****
>
> Hello,
>
> my lab is planning on purchasing a spinning disc system to perform live
> imaging on zebrafish
> embryos. We have experience with the Zeiss setup (CSU-X1 and Zen Blue
> software) with a
> simple axiocam camera and older experience with different version of the
> PerkinElmer
> spinning discs with Hamamatso EM-CCD camera.
> Working with zebrafish, we are quite tempting to switch to the CSU-W1
> proposed by
> Nikon/Andor but would be really happy to get opinion of experts on:
>
> 1) How do the CSU-W1 compare to the X1 in terms of required illumintation?
> Do we need
> much longer exposure time (and thus much more light on the sample...)?
>
> 2) Is it really a problem to image fast processes with the W1
>
> 3) in terms of camera, how does the Zyla 4.2 sCMOS compare to the iXon
> EMCCD camera?
>
> 4) Does anybody have experience with the Borealis system from Andor? How
> much does it
> really improve homogeneity of illumination?
>
> Many thanks in advance for any advice and comments!
>
> Virginie
>

phil laissue-2

Re: spinning disc X1 or W1

In reply to this post by SUBSCRIBE CONFOCALMICROSCOPY Virginie Lecaudey

*****
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,
Another consideration could be phototoxicity. Parthasarathy's group showed
nicely how it affects e.g. development of the opercle with spinning disk,
but not lightsheet:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3843985/
Kind regards,
Philippe

_________________________________________
Philippe Laissue, PhD
Royal Society Industry Fellow
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
Lightsheets in Sheffield: www.lsfm2016.org

On 4 March 2016 at 19:39, SUBSCRIBE CONFOCALMICROSCOPY Virginie Lecaudey <
[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.
> *****
>
> Hello,
>
> my lab is planning on purchasing a spinning disc system to perform live
> imaging on zebrafish
> embryos. We have experience with the Zeiss setup (CSU-X1 and Zen Blue
> software) with a
> simple axiocam camera and older experience with different version of the
> PerkinElmer
> spinning discs with Hamamatso EM-CCD camera.
> Working with zebrafish, we are quite tempting to switch to the CSU-W1
> proposed by
> Nikon/Andor but would be really happy to get opinion of experts on:
>
> 1) How do the CSU-W1 compare to the X1 in terms of required illumintation?
> Do we need
> much longer exposure time (and thus much more light on the sample...)?
>
> 2) Is it really a problem to image fast processes with the W1
>
> 3) in terms of camera, how does the Zyla 4.2 sCMOS compare to the iXon
> EMCCD camera?
>
> 4) Does anybody have experience with the Borealis system from Andor? How
> much does it
> really improve homogeneity of illumination?
>
> Many thanks in advance for any advice and comments!
>
> Virginie
>

Kurt Thorn

Re: spinning disc X1 or W1

In reply to this post by SUBSCRIBE CONFOCALMICROSCOPY Virginie Lecaudey

We have just set up a Borealis CSU-W1. We haven't done a direct
comparison with a CSU-X1, but I can say that the Borealis makes the
overall efficiency of the excitation pathway quite high. We are mostly
using 100 mW lasers for excitation and get more than enough power at the
sample (in fact, we typically run at much less than full power to avoid
photobleaching and phototoxicity). In principle, the CSU-W1 runs at 200
FPS, though we haven't run it that fast.

The Zyla 4.2 sCMOS is definitely less sensitive than an EMCCD. We added
a iXon 888 onto the 2nd port of our W1 because several users imaging dim
samples couldn't see them on the sCMOS camera. The new 4.2 plus with
82% QE should be better, but I suspect it will still perform less well
than an EMCCD camera, though I haven't tried one yet.

The Borealis system does improve illumination homogeneity. I haven't
compared it to a non-Borealis system, but I believe you should be able
to get 10-15% uniformity across the field of view.

Best,
Kurt

>
> Many thanks in advance for any advice and comments!
>
> Virginie
>

--
Kurt Thorn
Associate Professor
Director, Nikon Imaging Center
http://thornlab.ucsf.edu/
http://nic.ucsf.edu/blog/

mcammer

Re: spinning disc X1 or W1

In reply to this post by phil laissue-2

To second Philippe's comment, our first impressions of the Zeiss lightsheet (20X water lens) with both zebrafish and c elegans are that it is less phototoxic than confocals, but we haven't had it long enough to know for sure.
=========================================================================
Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
Cell: 914-309-3270 ** Office: Skirball 2nd Floor main office, back right **
http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of phil laissue
Sent: Monday, March 07, 2016 6:39 AM
To: [hidden email]
Subject: Re: spinning disc X1 or W1

Hi there,
Another consideration could be phototoxicity. Parthasarathy's group showed nicely how it affects e.g. development of the opercle with spinning disk, but not lightsheet:
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ncbi.nlm.nih.gov_pmc_articles_PMC3843985_&d=CwIBaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=6GLAN78YnmZcHCIkUHLwHPWr3sE6kW1HWidhqlmUf8w&s=Fgi_wzcZBqw_BARyjismOBo5czVCdYRqrJvK4YA3Eko&e=
Kind regards,
Philippe

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jerie

Re: spinning disc X1 or W1

In reply to this post by SUBSCRIBE CONFOCALMICROSCOPY Virginie Lecaudey

*****
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.
*****

Dear Virginie,

1) How do the CSU-W1 compare to the X1 in terms of required illumintation?
Do we need much longer exposure time (and thus much more light on the
sample...)?
W1 has an about 2x larger center distance of pinholes (5µm W1, 2,53µm X1
when back projected into the sample plane [1]) resulting in an about 4x
lower light throughput (using the 50µm pinhole disc); the W1 field of view
is 3.88 times larger than X1. [2] together resulting in far (~15x) less
light per area/time when illuminating with W1 than with X1 using the same
laser power.
So you will indeed either need longer exposure or higher illumination
power, however the light will be distributed over a larger area, and so,
phototoxicity will be similar for similar contrast images in both systems.

2) Is it really a problem to image fast processes with the W1
The recommended minimum integration time for the W1 is 5ms, for the X1 high
end version its 0,5ms (ie. max frame rate is 200 frames per sec resp. 2000
fps) [2]. both IXON888 and Zyla(4,2 or 5,5) offer lower full frame rates
(of 56 and 100 fps), so you might be limited by the camera frame rate or
have to use binning or subregions before the W1 speed is becoming limiting.
Would your measurements require mor than 200 fps?

3) in terms of camera, how does the Zyla 4.2 sCMOS compare to the iXon
EMCCD camera?
Below 34 photons/pixel, the SNR of Ixon888 images is slightly better than
Zyla4.2 at binning 2. (binning 2 is required to compare the same active
area, because pixels of the Ixon888 are factor 4 larger than Zyla 4.2) [3].
For high NA, high spatial resolution imaging, the smaller pixel size of
Zyla will allow you to sample at a higher rate, though both camera variants
will not allow Nyquist rate sampling at 1,3NA 100x magnification using W1.
Zyla will allow Nyquist rate sampling with the X1 at 1,3NA 100x.

4) Does anybody have experience with the Borealis system from Andor? How
much does it really improve homogeneity of illumination?
I have no experience with the Borealis system personally, but colleagues
report the improvement in field flatness and illumination intensity to be
about a factor of 3, also in case adaptive optics systems of other
providers are used.

No commercial interest in any of the above mentioned products or companies.

[1] https://svi.nl/YokogawaDisk
[2] http://www.yokogawa.com/scanner/product/products_csu1_e.htm
[3] http://www.magersci.com/brochures/imager/Zyla4.2.pdf

Abraços,
Jens

Dr. Jens Rietdorf, visiting scientist @ center for technological
development in health CDTS, Rio de Janeiro, Brasil.