Yokogawa CSU-W1 spinning disk engineering with SoRa and w/o the light looser invention

[Vitaly Boyko-2]

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 Dear List,
It looks like Yokogawa Marketing staff is "scared" to release data on axial resolution of the relatively new CSU-W1 unit?1. with 50 um disk and NA 1.4 objective?2. with 25 um disk and NA 1.4 lens?3. with SoRa (50 um disk) and NA 1.4 before and after non-linear deconvolution? Is there a linear deconvolution algorithm for the spinning disk confocal available?4. w/o SoRa "invention" and NA 1.4 glass?
I would greatly appreciate professional response rather from Yokogawa engineers than from Yokogawa Marketing.
Also, how come CSU-W1 unit cannot be designed with both removable SoRa and inter-exchangeable 25/50 um disks?
Good morning Japan! 
Look forward to hearing solid pro and against arguments?
Many thanks in advance.
Best regards,
Vitaly

[Zdenek Svindrych-2]

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Hi Vitaly,
the long answer is: there are different "measures" of resolution, like FWHM
of the PSF, the OTF cutoff, and many more. They all behave well for lateral
resolution, meaning they give you comparable results for many microscopy
modalities.
It's more difficult with axial resolution, and on top of that, there is
another important measure "sectioning ability" that can be vaguely defined
as ability to cope with thick densely labeled samples. And those two are
much harder to compare among different microscopy (superresolution)
techniques.
I personally think in terms of OTF cutoff. It's a well defined, "hard"
resolution limit (at least for widefield, confocal, Airyscan, SIM, MSIM,
SMI, ISM, SoRa, Opra, instant SIM, you name it) that cannot be "improved"
by deconvolution alone. Then SoRa brings about no axial resolution
improvement, as was shown by Sheppard and Enderlein. This does not apply to
Airyscan, as Zeiss does do a bit more math than just "summing the shifted
PSFs".
Then there is the practical/experimental point of view, where I think the
most important quantity that affects the image quality is SNR.
To better compare with other techniques, bear in mind that in SoRa the
effective pinhole size is doubled by the action of the microlenses, and
scaled down by a factor 2.8 or 3 by the magnification changer between the
scope and the disk. So they come out a bit smaller than 50 um (for the
purpose of calculating axial resolution), but the disk throughput (and
pinhole crosstalk) is equivalent to 100 um pinhole disk (approx). This
sounds like a clever tradeoff to me, but no doubt one of the easiest from
the design point of view.
Back to your questions, I don't have the numbers, I'm not Yokogawa
engineer. But remember the resolution will depend on the lens magnification
as well! Also, I don't think (but I may very well be wrong) Yokogawa offers
deconvolution software for their W1.
Finally, SoRa is not "removable", it's a special disk; and you can only
switch between two disks (if I understood your question correctly).
Btw, as for the short answer, I don't think there is any :-).
Best, zdenek


On Mon, Aug 5, 2019 at 10:05 PM Vitaly Boyko <
[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.
> *****
>
>  Dear List,
> It looks like Yokogawa Marketing staff is "scared" to release data on
> axial resolution of the relatively new CSU-W1 unit?1. with 50 um disk and
> NA 1.4 objective?2. with 25 um disk and NA 1.4 lens?3. with SoRa (50 um
> disk) and NA 1.4 before and after non-linear deconvolution? Is there a
> linear deconvolution algorithm for the spinning disk confocal available?4.
> w/o SoRa "invention" and NA 1.4 glass?
> I would greatly appreciate professional response rather from Yokogawa
> engineers than from Yokogawa Marketing.
> Also, how come CSU-W1 unit cannot be designed with both removable SoRa and
> inter-exchangeable 25/50 um disks?
> Good morning Japan!
> Look forward to hearing solid pro and against arguments?
> Many thanks in advance.
> Best regards,
> Vitaly
>

--
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

[Vitaly Boyko-2]

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 Hi Zdenek,
thank you very much for your reply. For the axial resolution, FWHM would be good enough for the beginning, a kind of obvious, with 63x NA 1.4 lens w/o SoRa and with N.A. 1.49 lens with SoRa.  
I have done cross-comparison between CSU-W1 with and w/o SoRa, and got better results both laterally and axially with 0.6 AU pinhole for the Alexa 488 (and the same pinhole size for Alexa 594, which would be ca. 0.54 AU) on Leica SP8. Airyscan alike SoRa is struggling to collect enough photons per voxel to achieve low photo toxicity for long-term imaging of live specimen (0.5x or 1x sampling along 50-80 um in z). So far, Lattice SIM seems to outperform all of the above for thin samples, but again, Zeiss have been stubborn (and rarely listen) with the choice of lasers, and as "stubbornly" expected Zeiss offers only four very "old-fashioned" laser lines (Zeiss still has to learn that exciting with 496 nm argon line gives better SNR for tissues or immune cells (macrophages) with higher auto-fluorescence). Are their high end confocals finally come with a tunable white light laser??? Zeiss should respond promptly?
Has someone done a very simple experiment combining 100 nm and 200 nm multi-color TetraSpec beads, and calculated ratio of, let say, Alexa-488 mean intensity for 200 nm bead and 100 nm bead before and after deconvolution (Airyscan, SoRa, Lattice SIM, iSIM etc.)??? Training set of ca. 5000 beads should be good enough for the beginning. The same could be done when beads are placed in matrigel or any other 3D media 100 um, 200 um, or 500 um away from the coveglass?  Another question to all vendors with positively evolved Broad Vision: Leica (Leica has WLL, not much to complain here except for their mediocre z-galvo, 63x NA 1.4 oil "non-holding" objectives", all others are equally guilty of cheap thermally remarkably expandable aluminum), Zeiss, Nikon, Olympus, Andor and any other new broadly experienced, 100% customer oriented vendors. Sorry, the question is just below finally:
Which vendor (WLL-plus vendors are excluded on a positive note) can offer 8-10 laser lines fixture (50 mW minimum) such as:
1. 375 nm +/- 5 nm,2. 440 nm +/- 3 nm,3. 486 nm +/- 3 nm,4. 495 nm +/- 5 nm,5. 510 nm +/- 3 nm,6. 550 nm +/- 5 nm,7. 572 nm +/- 5 nm,8. 585 nm +/- 5 nm,   9. 648 nm +/- 5 nm,10. 750 nm +/- 5 nm
Cheers,
Vitaly        On Tuesday, August 6, 2019, 11:03:55 AM EDT, Zdenek Svindrych <[hidden email]> wrote:  
 
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Hi Vitaly,
the long answer is: there are different "measures" of resolution, like FWHM
of the PSF, the OTF cutoff, and many more. They all behave well for lateral
resolution, meaning they give you comparable results for many microscopy
modalities.
It's more difficult with axial resolution, and on top of that, there is
another important measure "sectioning ability" that can be vaguely defined
as ability to cope with thick densely labeled samples. And those two are
much harder to compare among different microscopy (superresolution)
techniques.
I personally think in terms of OTF cutoff. It's a well defined, "hard"
resolution limit (at least for widefield, confocal, Airyscan, SIM, MSIM,
SMI, ISM, SoRa, Opra, instant SIM, you name it) that cannot be "improved"
by deconvolution alone. Then SoRa brings about no axial resolution
improvement, as was shown by Sheppard and Enderlein. This does not apply to
Airyscan, as Zeiss does do a bit more math than just "summing the shifted
PSFs".
Then there is the practical/experimental point of view, where I think the
most important quantity that affects the image quality is SNR.
To better compare with other techniques, bear in mind that in SoRa the
effective pinhole size is doubled by the action of the microlenses, and
scaled down by a factor 2.8 or 3 by the magnification changer between the
scope and the disk. So they come out a bit smaller than 50 um (for the
purpose of calculating axial resolution), but the disk throughput (and
pinhole crosstalk) is equivalent to 100 um pinhole disk (approx). This
sounds like a clever tradeoff to me, but no doubt one of the easiest from
the design point of view.
Back to your questions, I don't have the numbers, I'm not Yokogawa
engineer. But remember the resolution will depend on the lens magnification
as well! Also, I don't think (but I may very well be wrong) Yokogawa offers
deconvolution software for their W1.
Finally, SoRa is not "removable", it's a special disk; and you can only
switch between two disks (if I understood your question correctly).
Btw, as for the short answer, I don't think there is any :-).
Best, zdenek


On Mon, Aug 5, 2019 at 10:05 PM Vitaly Boyko <
[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.
> *****
>
>  Dear List,
> It looks like Yokogawa Marketing staff is "scared" to release data on
> axial resolution of the relatively new CSU-W1 unit?1. with 50 um disk and
> NA 1.4 objective?2. with 25 um disk and NA 1.4 lens?3. with SoRa (50 um
> disk) and NA 1.4 before and after non-linear deconvolution? Is there a
> linear deconvolution algorithm for the spinning disk confocal available?4.
> w/o SoRa "invention" and NA 1.4 glass?
> I would greatly appreciate professional response rather from Yokogawa
> engineers than from Yokogawa Marketing.
> Also, how come CSU-W1 unit cannot be designed with both removable SoRa and
> inter-exchangeable 25/50 um disks?
> Good morning Japan!
> Look forward to hearing solid pro and against arguments?
> Many thanks in advance.
> Best regards,
> Vitaly
>

--
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth
 

[Alison J. North]

*****
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 Vitaly,

While I too am a huge fan of the Leica WLL, and I would likewise enjoy a
greater range of lasers to select from on many/most of our systems, I am
not sure that airing public criticisms of specific companies is very
helpful, particularly at a time when they have been showing enormous
willingness and enthusiasm to work alongside those of us in core
facilities.   As you may know, Zeiss recently held their first "Core
Companies Convergence" meeting up at Thornwood, where they encouraged
many of us from core facilities to share our wish lists and make
requests concerning future developments and service - and they listened
carefully to what we said.  Likewise, a group from ELMI (Roland Nitschke
and others) have been working extensively with the companies towards
better quality control on microscopes, and I believe the open responses
from the companies are posted and available for you to read.  I find
these developments extremely encouraging, as well as all of the
interactions between the various national and international microscopy
networks (including our new BINA, on this side of the pond) and many
microscope companies.

So could you please try to put a more positive spin on your requests, in
support of this era of constructive interactions? I'm sure you don't
want to risk the tone of your e-mails being compared to a certain
person's tweets... :-)

As to imaging 5,000 beads on each of multiple systems, that is a great
idea, and something we would all like to try ... if we could find the
time!  But if you can do it, and share the results with us, that would
be just fabulous!  In the meantime, I must go back to re-making the OTFs
on my OMX, so that my users can get on with their research...

All the best,

Alison


On 8/7/2019 2:04 PM, Vitaly Boyko wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=fgQUQlkPxEpA9ceUEfAy-G7VPcvwLIDwc0_0q3YUGhY&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=T-WKys_2h3pyoSUp_YlPJTwxs4QXhx1fqYgt0YepCBQ&e=  and include the link in your posting.
> *****
>
>   Hi Zdenek,
> thank you very much for your reply. For the axial resolution, FWHM would be good enough for the beginning, a kind of obvious, with 63x NA 1.4 lens w/o SoRa and with N.A. 1.49 lens with SoRa.
> I have done cross-comparison between CSU-W1 with and w/o SoRa, and got better results both laterally and axially with 0.6 AU pinhole for the Alexa 488 (and the same pinhole size for Alexa 594, which would be ca. 0.54 AU) on Leica SP8. Airyscan alike SoRa is struggling to collect enough photons per voxel to achieve low photo toxicity for long-term imaging of live specimen (0.5x or 1x sampling along 50-80 um in z). So far, Lattice SIM seems to outperform all of the above for thin samples, but again, Zeiss have been stubborn (and rarely listen) with the choice of lasers, and as "stubbornly" expected Zeiss offers only four very "old-fashioned" laser lines (Zeiss still has to learn that exciting with 496 nm argon line gives better SNR for tissues or immune cells (macrophages) with higher auto-fluorescence). Are their high end confocals finally come with a tunable white light laser??? Zeiss should respond promptly?
> Has someone done a very simple experiment combining 100 nm and 200 nm multi-color TetraSpec beads, and calculated ratio of, let say, Alexa-488 mean intensity for 200 nm bead and 100 nm bead before and after deconvolution (Airyscan, SoRa, Lattice SIM, iSIM etc.)??? Training set of ca. 5000 beads should be good enough for the beginning. The same could be done when beads are placed in matrigel or any other 3D media 100 um, 200 um, or 500 um away from the coveglass?  Another question to all vendors with positively evolved Broad Vision: Leica (Leica has WLL, not much to complain here except for their mediocre z-galvo, 63x NA 1.4 oil "non-holding" objectives", all others are equally guilty of cheap thermally remarkably expandable aluminum), Zeiss, Nikon, Olympus, Andor and any other new broadly experienced, 100% customer oriented vendors. Sorry, the question is just below finally:
> Which vendor (WLL-plus vendors are excluded on a positive note) can offer 8-10 laser lines fixture (50 mW minimum) such as:
> 1. 375 nm +/- 5 nm,2. 440 nm +/- 3 nm,3. 486 nm +/- 3 nm,4. 495 nm +/- 5 nm,5. 510 nm +/- 3 nm,6. 550 nm +/- 5 nm,7. 572 nm +/- 5 nm,8. 585 nm +/- 5 nm,   9. 648 nm +/- 5 nm,10. 750 nm +/- 5 nm
> Cheers,
> Vitaly        On Tuesday, August 6, 2019, 11:03:55 AM EDT, Zdenek Svindrych <[hidden email]> wrote:
>  
>   *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=fgQUQlkPxEpA9ceUEfAy-G7VPcvwLIDwc0_0q3YUGhY&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=T-WKys_2h3pyoSUp_YlPJTwxs4QXhx1fqYgt0YepCBQ&e=  and include the link in your posting.
> *****
>
> Hi Vitaly,
> the long answer is: there are different "measures" of resolution, like FWHM
> of the PSF, the OTF cutoff, and many more. They all behave well for lateral
> resolution, meaning they give you comparable results for many microscopy
> modalities.
> It's more difficult with axial resolution, and on top of that, there is
> another important measure "sectioning ability" that can be vaguely defined
> as ability to cope with thick densely labeled samples. And those two are
> much harder to compare among different microscopy (superresolution)
> techniques.
> I personally think in terms of OTF cutoff. It's a well defined, "hard"
> resolution limit (at least for widefield, confocal, Airyscan, SIM, MSIM,
> SMI, ISM, SoRa, Opra, instant SIM, you name it) that cannot be "improved"
> by deconvolution alone. Then SoRa brings about no axial resolution
> improvement, as was shown by Sheppard and Enderlein. This does not apply to
> Airyscan, as Zeiss does do a bit more math than just "summing the shifted
> PSFs".
> Then there is the practical/experimental point of view, where I think the
> most important quantity that affects the image quality is SNR.
> To better compare with other techniques, bear in mind that in SoRa the
> effective pinhole size is doubled by the action of the microlenses, and
> scaled down by a factor 2.8 or 3 by the magnification changer between the
> scope and the disk. So they come out a bit smaller than 50 um (for the
> purpose of calculating axial resolution), but the disk throughput (and
> pinhole crosstalk) is equivalent to 100 um pinhole disk (approx). This
> sounds like a clever tradeoff to me, but no doubt one of the easiest from
> the design point of view.
> Back to your questions, I don't have the numbers, I'm not Yokogawa
> engineer. But remember the resolution will depend on the lens magnification
> as well! Also, I don't think (but I may very well be wrong) Yokogawa offers
> deconvolution software for their W1.
> Finally, SoRa is not "removable", it's a special disk; and you can only
> switch between two disks (if I understood your question correctly).
> Btw, as for the short answer, I don't think there is any :-).
> Best, zdenek
>
>
> On Mon, Aug 5, 2019 at 10:05 PM Vitaly Boyko <
> [hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=fgQUQlkPxEpA9ceUEfAy-G7VPcvwLIDwc0_0q3YUGhY&e=
>> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=T-WKys_2h3pyoSUp_YlPJTwxs4QXhx1fqYgt0YepCBQ&e=  and include the link in your posting.
>> *****
>>
>>    Dear List,
>> It looks like Yokogawa Marketing staff is "scared" to release data on
>> axial resolution of the relatively new CSU-W1 unit?1. with 50 um disk and
>> NA 1.4 objective?2. with 25 um disk and NA 1.4 lens?3. with SoRa (50 um
>> disk) and NA 1.4 before and after non-linear deconvolution? Is there a
>> linear deconvolution algorithm for the spinning disk confocal available?4.
>> w/o SoRa "invention" and NA 1.4 glass?
>> I would greatly appreciate professional response rather from Yokogawa
>> engineers than from Yokogawa Marketing.
>> Also, how come CSU-W1 unit cannot be designed with both removable SoRa and
>> inter-exchangeable 25/50 um disks?
>> Good morning Japan!
>> Look forward to hearing solid pro and against arguments?
>> Many thanks in advance.
>> Best regards,
>> Vitaly
>>
>

--
Alison J. North, Ph.D.,
Research Associate Professor and
Senior Director of the Bio-Imaging Resource Center,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office    ++ 212 327 7488
Tel: lab        ++ 212 327 7486
Fax:            ++ 212 327 7489

[Vitaly Boyko-2]

*****
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*****

 Alison,
I would agree with you, but I have been talking to Zeiss, Leica and others since 2004, already 15 years, and I think they have had enough time to learn what flexibility and tunability stand for.Quantitative Biology is still mostly in the air, than a daily routine. Measure&quantify (aka measure seven times and cut once, not other way around), not "emotional cherry-picking".
Cheers,
Vitaly
  
    On Wednesday, August 7, 2019, 03:29:06 PM EDT, Alison J. North <[hidden email]> wrote:  
 
 *****
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*****

Dear Vitaly,

While I too am a huge fan of the Leica WLL, and I would likewise enjoy a
greater range of lasers to select from on many/most of our systems, I am
not sure that airing public criticisms of specific companies is very
helpful, particularly at a time when they have been showing enormous
willingness and enthusiasm to work alongside those of us in core
facilities.   As you may know, Zeiss recently held their first "Core
Companies Convergence" meeting up at Thornwood, where they encouraged
many of us from core facilities to share our wish lists and make
requests concerning future developments and service - and they listened
carefully to what we said.  Likewise, a group from ELMI (Roland Nitschke
and others) have been working extensively with the companies towards
better quality control on microscopes, and I believe the open responses
from the companies are posted and available for you to read.  I find
these developments extremely encouraging, as well as all of the
interactions between the various national and international microscopy
networks (including our new BINA, on this side of the pond) and many
microscope companies.

So could you please try to put a more positive spin on your requests, in
support of this era of constructive interactions? I'm sure you don't
want to risk the tone of your e-mails being compared to a certain
person's tweets... :-)

As to imaging 5,000 beads on each of multiple systems, that is a great
idea, and something we would all like to try ... if we could find the
time!  But if you can do it, and share the results with us, that would
be just fabulous!  In the meantime, I must go back to re-making the OTFs
on my OMX, so that my users can get on with their research...

All the best,

Alison


On 8/7/2019 2:04 PM, Vitaly Boyko wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=fgQUQlkPxEpA9ceUEfAy-G7VPcvwLIDwc0_0q3YUGhY&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=T-WKys_2h3pyoSUp_YlPJTwxs4QXhx1fqYgt0YepCBQ&e=  and include the link in your posting.
> *****
>
>  Hi Zdenek,
> thank you very much for your reply. For the axial resolution, FWHM would be good enough for the beginning, a kind of obvious, with 63x NA 1.4 lens w/o SoRa and with N.A. 1.49 lens with SoRa.
> I have done cross-comparison between CSU-W1 with and w/o SoRa, and got better results both laterally and axially with 0.6 AU pinhole for the Alexa 488 (and the same pinhole size for Alexa 594, which would be ca. 0.54 AU) on Leica SP8. Airyscan alike SoRa is struggling to collect enough photons per voxel to achieve low photo toxicity for long-term imaging of live specimen (0.5x or 1x sampling along 50-80 um in z). So far, Lattice SIM seems to outperform all of the above for thin samples, but again, Zeiss have been stubborn (and rarely listen) with the choice of lasers, and as "stubbornly" expected Zeiss offers only four very "old-fashioned" laser lines (Zeiss still has to learn that exciting with 496 nm argon line gives better SNR for tissues or immune cells (macrophages) with higher auto-fluorescence). Are their high end confocals finally come with a tunable white light laser??? Zeiss should respond promptly?
> Has someone done a very simple experiment combining 100 nm and 200 nm multi-color TetraSpec beads, and calculated ratio of, let say, Alexa-488 mean intensity for 200 nm bead and 100 nm bead before and after deconvolution (Airyscan, SoRa, Lattice SIM, iSIM etc.)??? Training set of ca. 5000 beads should be good enough for the beginning. The same could be done when beads are placed in matrigel or any other 3D media 100 um, 200 um, or 500 um away from the coveglass?  Another question to all vendors with positively evolved Broad Vision: Leica (Leica has WLL, not much to complain here except for their mediocre z-galvo, 63x NA 1.4 oil "non-holding" objectives", all others are equally guilty of cheap thermally remarkably expandable aluminum), Zeiss, Nikon, Olympus, Andor and any other new broadly experienced, 100% customer oriented vendors. Sorry, the question is just below finally:
> Which vendor (WLL-plus vendors are excluded on a positive note) can offer 8-10 laser lines fixture (50 mW minimum) such as:
> 1. 375 nm +/- 5 nm,2. 440 nm +/- 3 nm,3. 486 nm +/- 3 nm,4. 495 nm +/- 5 nm,5. 510 nm +/- 3 nm,6. 550 nm +/- 5 nm,7. 572 nm +/- 5 nm,8. 585 nm +/- 5 nm,   9. 648 nm +/- 5 nm,10. 750 nm +/- 5 nm
> Cheers,
> Vitaly        On Tuesday, August 6, 2019, 11:03:55 AM EDT, Zdenek Svindrych <[hidden email]> wrote:
> 
>  *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=fgQUQlkPxEpA9ceUEfAy-G7VPcvwLIDwc0_0q3YUGhY&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=T-WKys_2h3pyoSUp_YlPJTwxs4QXhx1fqYgt0YepCBQ&e=  and include the link in your posting.
> *****
>
> Hi Vitaly,
> the long answer is: there are different "measures" of resolution, like FWHM
> of the PSF, the OTF cutoff, and many more. They all behave well for lateral
> resolution, meaning they give you comparable results for many microscopy
> modalities.
> It's more difficult with axial resolution, and on top of that, there is
> another important measure "sectioning ability" that can be vaguely defined
> as ability to cope with thick densely labeled samples. And those two are
> much harder to compare among different microscopy (superresolution)
> techniques.
> I personally think in terms of OTF cutoff. It's a well defined, "hard"
> resolution limit (at least for widefield, confocal, Airyscan, SIM, MSIM,
> SMI, ISM, SoRa, Opra, instant SIM, you name it) that cannot be "improved"
> by deconvolution alone. Then SoRa brings about no axial resolution
> improvement, as was shown by Sheppard and Enderlein. This does not apply to
> Airyscan, as Zeiss does do a bit more math than just "summing the shifted
> PSFs".
> Then there is the practical/experimental point of view, where I think the
> most important quantity that affects the image quality is SNR.
> To better compare with other techniques, bear in mind that in SoRa the
> effective pinhole size is doubled by the action of the microlenses, and
> scaled down by a factor 2.8 or 3 by the magnification changer between the
> scope and the disk. So they come out a bit smaller than 50 um (for the
> purpose of calculating axial resolution), but the disk throughput (and
> pinhole crosstalk) is equivalent to 100 um pinhole disk (approx). This
> sounds like a clever tradeoff to me, but no doubt one of the easiest from
> the design point of view.
> Back to your questions, I don't have the numbers, I'm not Yokogawa
> engineer. But remember the resolution will depend on the lens magnification
> as well! Also, I don't think (but I may very well be wrong) Yokogawa offers
> deconvolution software for their W1.
> Finally, SoRa is not "removable", it's a special disk; and you can only
> switch between two disks (if I understood your question correctly).
> Btw, as for the short answer, I don't think there is any :-).
> Best, zdenek
>
>
> On Mon, Aug 5, 2019 at 10:05 PM Vitaly Boyko <
> [hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=fgQUQlkPxEpA9ceUEfAy-G7VPcvwLIDwc0_0q3YUGhY&e=
>> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=T-WKys_2h3pyoSUp_YlPJTwxs4QXhx1fqYgt0YepCBQ&e=  and include the link in your posting.
>> *****
>>
>>    Dear List,
>> It looks like Yokogawa Marketing staff is "scared" to release data on
>> axial resolution of the relatively new CSU-W1 unit?1. with 50 um disk and
>> NA 1.4 objective?2. with 25 um disk and NA 1.4 lens?3. with SoRa (50 um
>> disk) and NA 1.4 before and after non-linear deconvolution? Is there a
>> linear deconvolution algorithm for the spinning disk confocal available?4.
>> w/o SoRa "invention" and NA 1.4 glass?
>> I would greatly appreciate professional response rather from Yokogawa
>> engineers than from Yokogawa Marketing.
>> Also, how come CSU-W1 unit cannot be designed with both removable SoRa and
>> inter-exchangeable 25/50 um disks?
>> Good morning Japan!
>> Look forward to hearing solid pro and against arguments?
>> Many thanks in advance.
>> Best regards,
>> Vitaly
>>
>

--
Alison J. North, Ph.D.,
Research Associate Professor and
Senior Director of the Bio-Imaging Resource Center,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office    ++ 212 327 7488
Tel: lab        ++ 212 327 7486
Fax:            ++ 212 327 7489

 

[Rosemary White]

*****
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.
*****

Just to add a further (probably unnecessary) 2c worth...

I remember a time when the implementation of multiline lasers was patented, and unless companies bought the (expensive) license, they had to install separate lasers for each laser line. I imagine there are patents on most/all of the new technological innovations in confocal and other types of imaging, from lasers (the multiphoton picosecond vs. femtosecond patent issue springs to mind) to optical fibres to detectors. Patent negotiations can be long and expensive, and I can well imagine that these types of negotiations are going on in the background, and may be one reason why the latest technologies are not implemented immediately in all confocals.

cheers,
Rosemary

Dr Rosemary White
CSIRO Black Mountain
GPO Box 1700
ACT 2601, Australia

M: 61-0468966713
E: [hidden email]
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Vitaly Boyko [[hidden email]]
Sent: Thursday, 8 August 2019 6:13 AM
To: [hidden email]
Subject: Re: Yokogawa CSU-W1 spinning disk engineering with SoRa and w/o the light looser invention

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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*****

 Alison,
I would agree with you, but I have been talking to Zeiss, Leica and others since 2004, already 15 years, and I think they have had enough time to learn what flexibility and tunability stand for.Quantitative Biology is still mostly in the air, than a daily routine. Measure&quantify (aka measure seven times and cut once, not other way around), not "emotional cherry-picking".
Cheers,
Vitaly

    On Wednesday, August 7, 2019, 03:29:06 PM EDT, Alison J. North <[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.
*****

Dear Vitaly,

While I too am a huge fan of the Leica WLL, and I would likewise enjoy a
greater range of lasers to select from on many/most of our systems, I am
not sure that airing public criticisms of specific companies is very
helpful, particularly at a time when they have been showing enormous
willingness and enthusiasm to work alongside those of us in core
facilities.   As you may know, Zeiss recently held their first "Core
Companies Convergence" meeting up at Thornwood, where they encouraged
many of us from core facilities to share our wish lists and make
requests concerning future developments and service - and they listened
carefully to what we said.  Likewise, a group from ELMI (Roland Nitschke
and others) have been working extensively with the companies towards
better quality control on microscopes, and I believe the open responses
from the companies are posted and available for you to read.  I find
these developments extremely encouraging, as well as all of the
interactions between the various national and international microscopy
networks (including our new BINA, on this side of the pond) and many
microscope companies.

So could you please try to put a more positive spin on your requests, in
support of this era of constructive interactions? I'm sure you don't
want to risk the tone of your e-mails being compared to a certain
person's tweets... :-)

As to imaging 5,000 beads on each of multiple systems, that is a great
idea, and something we would all like to try ... if we could find the
time!  But if you can do it, and share the results with us, that would
be just fabulous!  In the meantime, I must go back to re-making the OTFs
on my OMX, so that my users can get on with their research...

All the best,

Alison


On 8/7/2019 2:04 PM, Vitaly Boyko wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=fgQUQlkPxEpA9ceUEfAy-G7VPcvwLIDwc0_0q3YUGhY&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=T-WKys_2h3pyoSUp_YlPJTwxs4QXhx1fqYgt0YepCBQ&e=  and include the link in your posting.
> *****
>
>  Hi Zdenek,
> thank you very much for your reply. For the axial resolution, FWHM would be good enough for the beginning, a kind of obvious, with 63x NA 1.4 lens w/o SoRa and with N.A. 1.49 lens with SoRa.
> I have done cross-comparison between CSU-W1 with and w/o SoRa, and got better results both laterally and axially with 0.6 AU pinhole for the Alexa 488 (and the same pinhole size for Alexa 594, which would be ca. 0.54 AU) on Leica SP8. Airyscan alike SoRa is struggling to collect enough photons per voxel to achieve low photo toxicity for long-term imaging of live specimen (0.5x or 1x sampling along 50-80 um in z). So far, Lattice SIM seems to outperform all of the above for thin samples, but again, Zeiss have been stubborn (and rarely listen) with the choice of lasers, and as "stubbornly" expected Zeiss offers only four very "old-fashioned" laser lines (Zeiss still has to learn that exciting with 496 nm argon line gives better SNR for tissues or immune cells (macrophages) with higher auto-fluorescence). Are their high end confocals finally come with a tunable white light laser??? Zeiss should respond promptly?
> Has someone done a very simple experiment combining 100 nm and 200 nm multi-color TetraSpec beads, and calculated ratio of, let say, Alexa-488 mean intensity for 200 nm bead and 100 nm bead before and after deconvolution (Airyscan, SoRa, Lattice SIM, iSIM etc.)??? Training set of ca. 5000 beads should be good enough for the beginning. The same could be done when beads are placed in matrigel or any other 3D media 100 um, 200 um, or 500 um away from the coveglass?  Another question to all vendors with positively evolved Broad Vision: Leica (Leica has WLL, not much to complain here except for their mediocre z-galvo, 63x NA 1.4 oil "non-holding" objectives", all others are equally guilty of cheap thermally remarkably expandable aluminum), Zeiss, Nikon, Olympus, Andor and any other new broadly experienced, 100% customer oriented vendors. Sorry, the question is just below finally:
> Which vendor (WLL-plus vendors are excluded on a positive note) can offer 8-10 laser lines fixture (50 mW minimum) such as:
> 1. 375 nm +/- 5 nm,2. 440 nm +/- 3 nm,3. 486 nm +/- 3 nm,4. 495 nm +/- 5 nm,5. 510 nm +/- 3 nm,6. 550 nm +/- 5 nm,7. 572 nm +/- 5 nm,8. 585 nm +/- 5 nm,   9. 648 nm +/- 5 nm,10. 750 nm +/- 5 nm
> Cheers,
> Vitaly        On Tuesday, August 6, 2019, 11:03:55 AM EDT, Zdenek Svindrych <[hidden email]> wrote:
> 
>  *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=fgQUQlkPxEpA9ceUEfAy-G7VPcvwLIDwc0_0q3YUGhY&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=T-WKys_2h3pyoSUp_YlPJTwxs4QXhx1fqYgt0YepCBQ&e=  and include the link in your posting.
> *****
>
> Hi Vitaly,
> the long answer is: there are different "measures" of resolution, like FWHM
> of the PSF, the OTF cutoff, and many more. They all behave well for lateral
> resolution, meaning they give you comparable results for many microscopy
> modalities.
> It's more difficult with axial resolution, and on top of that, there is
> another important measure "sectioning ability" that can be vaguely defined
> as ability to cope with thick densely labeled samples. And those two are
> much harder to compare among different microscopy (superresolution)
> techniques.
> I personally think in terms of OTF cutoff. It's a well defined, "hard"
> resolution limit (at least for widefield, confocal, Airyscan, SIM, MSIM,
> SMI, ISM, SoRa, Opra, instant SIM, you name it) that cannot be "improved"
> by deconvolution alone. Then SoRa brings about no axial resolution
> improvement, as was shown by Sheppard and Enderlein. This does not apply to
> Airyscan, as Zeiss does do a bit more math than just "summing the shifted
> PSFs".
> Then there is the practical/experimental point of view, where I think the
> most important quantity that affects the image quality is SNR.
> To better compare with other techniques, bear in mind that in SoRa the
> effective pinhole size is doubled by the action of the microlenses, and
> scaled down by a factor 2.8 or 3 by the magnification changer between the
> scope and the disk. So they come out a bit smaller than 50 um (for the
> purpose of calculating axial resolution), but the disk throughput (and
> pinhole crosstalk) is equivalent to 100 um pinhole disk (approx). This
> sounds like a clever tradeoff to me, but no doubt one of the easiest from
> the design point of view.
> Back to your questions, I don't have the numbers, I'm not Yokogawa
> engineer. But remember the resolution will depend on the lens magnification
> as well! Also, I don't think (but I may very well be wrong) Yokogawa offers
> deconvolution software for their W1.
> Finally, SoRa is not "removable", it's a special disk; and you can only
> switch between two disks (if I understood your question correctly).
> Btw, as for the short answer, I don't think there is any :-).
> Best, zdenek
>
>
> On Mon, Aug 5, 2019 at 10:05 PM Vitaly Boyko <
> [hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=fgQUQlkPxEpA9ceUEfAy-G7VPcvwLIDwc0_0q3YUGhY&e=
>> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=T-WKys_2h3pyoSUp_YlPJTwxs4QXhx1fqYgt0YepCBQ&e=  and include the link in your posting.
>> *****
>>
>>    Dear List,
>> It looks like Yokogawa Marketing staff is "scared" to release data on
>> axial resolution of the relatively new CSU-W1 unit?1. with 50 um disk and
>> NA 1.4 objective?2. with 25 um disk and NA 1.4 lens?3. with SoRa (50 um
>> disk) and NA 1.4 before and after non-linear deconvolution? Is there a
>> linear deconvolution algorithm for the spinning disk confocal available?4.
>> w/o SoRa "invention" and NA 1.4 glass?
>> I would greatly appreciate professional response rather from Yokogawa
>> engineers than from Yokogawa Marketing.
>> Also, how come CSU-W1 unit cannot be designed with both removable SoRa and
>> inter-exchangeable 25/50 um disks?
>> Good morning Japan!
>> Look forward to hearing solid pro and against arguments?
>> Many thanks in advance.
>> Best regards,
>> Vitaly
>>
>

--
Alison J. North, Ph.D.,
Research Associate Professor and
Senior Director of the Bio-Imaging Resource Center,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office    ++ 212 327 7488
Tel: lab        ++ 212 327 7486
Fax:            ++ 212 327 7489

[Vitaly Boyko-2]

*****
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 Rosemary,likely, as these days folks patent everything including obvious things. Plus we have excess of costly lawyers with limited understanding of advanced imaging, not mentioning quantitative data analysis.Though in many cases they are "cuddled" by patent biologists who rarely study photo chemistry and/or optics, thus often ending up with numerous examples of a "cul-de-sac" finale. Best, Vitaly
   
    On Wednesday, August 7, 2019, 06:48:29 PM EDT, Rosemary White <[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.
*****

Just to add a further (probably unnecessary) 2c worth...

I remember a time when the implementation of multiline lasers was patented, and unless companies bought the (expensive) license, they had to install separate lasers for each laser line. I imagine there are patents on most/all of the new technological innovations in confocal and other types of imaging, from lasers (the multiphoton picosecond vs. femtosecond patent issue springs to mind) to optical fibres to detectors. Patent negotiations can be long and expensive, and I can well imagine that these types of negotiations are going on in the background, and may be one reason why the latest technologies are not implemented immediately in all confocals.

cheers,
Rosemary

Dr Rosemary White
CSIRO Black Mountain
GPO Box 1700
ACT 2601, Australia

M: 61-0468966713
E: [hidden email]
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Vitaly Boyko [[hidden email]]
Sent: Thursday, 8 August 2019 6:13 AM
To: [hidden email]
Subject: Re: Yokogawa CSU-W1 spinning disk engineering with SoRa and w/o the light looser invention

*****
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.
*****

 Alison,
I would agree with you, but I have been talking to Zeiss, Leica and others since 2004, already 15 years, and I think they have had enough time to learn what flexibility and tunability stand for.Quantitative Biology is still mostly in the air, than a daily routine. Measure&quantify (aka measure seven times and cut once, not other way around), not "emotional cherry-picking".
Cheers,
Vitaly

    On Wednesday, August 7, 2019, 03:29:06 PM EDT, Alison J. North <[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.
*****

Dear Vitaly,

While I too am a huge fan of the Leica WLL, and I would likewise enjoy a
greater range of lasers to select from on many/most of our systems, I am
not sure that airing public criticisms of specific companies is very
helpful, particularly at a time when they have been showing enormous
willingness and enthusiasm to work alongside those of us in core
facilities.  As you may know, Zeiss recently held their first "Core
Companies Convergence" meeting up at Thornwood, where they encouraged
many of us from core facilities to share our wish lists and make
requests concerning future developments and service - and they listened
carefully to what we said.  Likewise, a group from ELMI (Roland Nitschke
and others) have been working extensively with the companies towards
better quality control on microscopes, and I believe the open responses
from the companies are posted and available for you to read.  I find
these developments extremely encouraging, as well as all of the
interactions between the various national and international microscopy
networks (including our new BINA, on this side of the pond) and many
microscope companies.

So could you please try to put a more positive spin on your requests, in
support of this era of constructive interactions? I'm sure you don't
want to risk the tone of your e-mails being compared to a certain
person's tweets... :-)

As to imaging 5,000 beads on each of multiple systems, that is a great
idea, and something we would all like to try ... if we could find the
time!  But if you can do it, and share the results with us, that would
be just fabulous!  In the meantime, I must go back to re-making the OTFs
on my OMX, so that my users can get on with their research...

All the best,

Alison


On 8/7/2019 2:04 PM, Vitaly Boyko wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=fgQUQlkPxEpA9ceUEfAy-G7VPcvwLIDwc0_0q3YUGhY&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=T-WKys_2h3pyoSUp_YlPJTwxs4QXhx1fqYgt0YepCBQ&e=  and include the link in your posting.
> *****
>
>  Hi Zdenek,
> thank you very much for your reply. For the axial resolution, FWHM would be good enough for the beginning, a kind of obvious, with 63x NA 1.4 lens w/o SoRa and with N.A. 1.49 lens with SoRa.
> I have done cross-comparison between CSU-W1 with and w/o SoRa, and got better results both laterally and axially with 0.6 AU pinhole for the Alexa 488 (and the same pinhole size for Alexa 594, which would be ca. 0.54 AU) on Leica SP8. Airyscan alike SoRa is struggling to collect enough photons per voxel to achieve low photo toxicity for long-term imaging of live specimen (0.5x or 1x sampling along 50-80 um in z). So far, Lattice SIM seems to outperform all of the above for thin samples, but again, Zeiss have been stubborn (and rarely listen) with the choice of lasers, and as "stubbornly" expected Zeiss offers only four very "old-fashioned" laser lines (Zeiss still has to learn that exciting with 496 nm argon line gives better SNR for tissues or immune cells (macrophages) with higher auto-fluorescence). Are their high end confocals finally come with a tunable white light laser??? Zeiss should respond promptly?
> Has someone done a very simple experiment combining 100 nm and 200 nm multi-color TetraSpec beads, and calculated ratio of, let say, Alexa-488 mean intensity for 200 nm bead and 100 nm bead before and after deconvolution (Airyscan, SoRa, Lattice SIM, iSIM etc.)??? Training set of ca. 5000 beads should be good enough for the beginning. The same could be done when beads are placed in matrigel or any other 3D media 100 um, 200 um, or 500 um away from the coveglass?  Another question to all vendors with positively evolved Broad Vision: Leica (Leica has WLL, not much to complain here except for their mediocre z-galvo, 63x NA 1.4 oil "non-holding" objectives", all others are equally guilty of cheap thermally remarkably expandable aluminum), Zeiss, Nikon, Olympus, Andor and any other new broadly experienced, 100% customer oriented vendors. Sorry, the question is just below finally:
> Which vendor (WLL-plus vendors are excluded on a positive note) can offer 8-10 laser lines fixture (50 mW minimum) such as:
> 1. 375 nm +/- 5 nm,2. 440 nm +/- 3 nm,3. 486 nm +/- 3 nm,4. 495 nm +/- 5 nm,5. 510 nm +/- 3 nm,6. 550 nm +/- 5 nm,7. 572 nm +/- 5 nm,8. 585 nm +/- 5 nm,  9. 648 nm +/- 5 nm,10. 750 nm +/- 5 nm
> Cheers,
> Vitaly        On Tuesday, August 6, 2019, 11:03:55 AM EDT, Zdenek Svindrych <[hidden email]> wrote:
> 
>  *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=fgQUQlkPxEpA9ceUEfAy-G7VPcvwLIDwc0_0q3YUGhY&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=T-WKys_2h3pyoSUp_YlPJTwxs4QXhx1fqYgt0YepCBQ&e=  and include the link in your posting.
> *****
>
> Hi Vitaly,
> the long answer is: there are different "measures" of resolution, like FWHM
> of the PSF, the OTF cutoff, and many more. They all behave well for lateral
> resolution, meaning they give you comparable results for many microscopy
> modalities.
> It's more difficult with axial resolution, and on top of that, there is
> another important measure "sectioning ability" that can be vaguely defined
> as ability to cope with thick densely labeled samples. And those two are
> much harder to compare among different microscopy (superresolution)
> techniques.
> I personally think in terms of OTF cutoff. It's a well defined, "hard"
> resolution limit (at least for widefield, confocal, Airyscan, SIM, MSIM,
> SMI, ISM, SoRa, Opra, instant SIM, you name it) that cannot be "improved"
> by deconvolution alone. Then SoRa brings about no axial resolution
> improvement, as was shown by Sheppard and Enderlein. This does not apply to
> Airyscan, as Zeiss does do a bit more math than just "summing the shifted
> PSFs".
> Then there is the practical/experimental point of view, where I think the
> most important quantity that affects the image quality is SNR.
> To better compare with other techniques, bear in mind that in SoRa the
> effective pinhole size is doubled by the action of the microlenses, and
> scaled down by a factor 2.8 or 3 by the magnification changer between the
> scope and the disk. So they come out a bit smaller than 50 um (for the
> purpose of calculating axial resolution), but the disk throughput (and
> pinhole crosstalk) is equivalent to 100 um pinhole disk (approx). This
> sounds like a clever tradeoff to me, but no doubt one of the easiest from
> the design point of view.
> Back to your questions, I don't have the numbers, I'm not Yokogawa
> engineer. But remember the resolution will depend on the lens magnification
> as well! Also, I don't think (but I may very well be wrong) Yokogawa offers
> deconvolution software for their W1.
> Finally, SoRa is not "removable", it's a special disk; and you can only
> switch between two disks (if I understood your question correctly).
> Btw, as for the short answer, I don't think there is any :-).
> Best, zdenek
>
>
> On Mon, Aug 5, 2019 at 10:05 PM Vitaly Boyko <
> [hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=fgQUQlkPxEpA9ceUEfAy-G7VPcvwLIDwc0_0q3YUGhY&e=
>> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=uDCkj_ueA2XO08p1EtmCfuxCf7zNwnG-vbpvVbkDtW4&s=T-WKys_2h3pyoSUp_YlPJTwxs4QXhx1fqYgt0YepCBQ&e=  and include the link in your posting.
>> *****
>>
>>   Dear List,
>> It looks like Yokogawa Marketing staff is "scared" to release data on
>> axial resolution of the relatively new CSU-W1 unit?1. with 50 um disk and
>> NA 1.4 objective?2. with 25 um disk and NA 1.4 lens?3. with SoRa (50 um
>> disk) and NA 1.4 before and after non-linear deconvolution? Is there a
>> linear deconvolution algorithm for the spinning disk confocal available?4.
>> w/o SoRa "invention" and NA 1.4 glass?
>> I would greatly appreciate professional response rather from Yokogawa
>> engineers than from Yokogawa Marketing.
>> Also, how come CSU-W1 unit cannot be designed with both removable SoRa and
>> inter-exchangeable 25/50 um disks?
>> Good morning Japan!
>> Look forward to hearing solid pro and against arguments?
>> Many thanks in advance.
>> Best regards,
>> Vitaly
>>
>

--
Alison J. North, Ph.D.,
Research Associate Professor and
Senior Director of the Bio-Imaging Resource Center,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office    ++ 212 327 7488
Tel: lab        ++ 212 327 7486
Fax:            ++ 212 327 7489  

[Sylvie Le Guyader]

*****
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 Vitali

Interesting comparison at the start of your email. However in my opinion, a few more parameters will come into play when I have to choose between the systems you mention: how deep you can go in the sample and the speed of imaging.

In that respect only a spinning disk with a sensitive camera can fulfil my expectations. At our facility imaging live organoids in gels has literally exploded. That is not something our users want to do with a single point confocal with a 0.6 AU pinhole.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7C,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
Follow our microscopy blog!

On 7 Aug 2019 20:04, Vitaly Boyko <[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 Zdenek,
thank you very much for your reply. For the axial resolution, FWHM would be good enough for the beginning, a kind of obvious, with 63x NA 1.4 lens w/o SoRa and with N.A. 1.49 lens with SoRa.
I have done cross-comparison between CSU-W1 with and w/o SoRa, and got better results both laterally and axially with 0.6 AU pinhole for the Alexa 488 (and the same pinhole size for Alexa 594, which would be ca. 0.54 AU) on Leica SP8. Airyscan alike SoRa is struggling to collect enough photons per voxel to achieve low photo toxicity for long-term imaging of live specimen (0.5x or 1x sampling along 50-80 um in z). So far, Lattice SIM seems to outperform all of the above for thin samples, but again, Zeiss have been stubborn (and rarely listen) with the choice of lasers, and as "stubbornly" expected Zeiss offers only four very "old-fashioned" laser lines (Zeiss still has to learn that exciting with 496 nm argon line gives better SNR for tissues or immune cells (macrophages) with higher auto-fluorescence). Are their high end confocals finally come with a tunable white light laser??? Zeiss should respond promptly?
Has someone done a very simple experiment combining 100 nm and 200 nm multi-color TetraSpec beads, and calculated ratio of, let say, Alexa-488 mean intensity for 200 nm bead and 100 nm bead before and after deconvolution (Airyscan, SoRa, Lattice SIM, iSIM etc.)??? Training set of ca. 5000 beads should be good enough for the beginning. The same could be done when beads are placed in matrigel or any other 3D media 100 um, 200 um, or 500 um away from the coveglass?  Another question to all vendors with positively evolved Broad Vision: Leica (Leica has WLL, not much to complain here except for their mediocre z-galvo, 63x NA 1.4 oil "non-holding" objectives", all others are equally guilty of cheap thermally remarkably expandable aluminum), Zeiss, Nikon, Olympus, Andor and any other new broadly experienced, 100% customer oriented vendors. Sorry, the question is just below finally:
Which vendor (WLL-plus vendors are excluded on a positive note) can offer 8-10 laser lines fixture (50 mW minimum) such as:
1. 375 nm +/- 5 nm,2. 440 nm +/- 3 nm,3. 486 nm +/- 3 nm,4. 495 nm +/- 5 nm,5. 510 nm +/- 3 nm,6. 550 nm +/- 5 nm,7. 572 nm +/- 5 nm,8. 585 nm +/- 5 nm,   9. 648 nm +/- 5 nm,10. 750 nm +/- 5 nm
Cheers,
Vitaly        On Tuesday, August 6, 2019, 11:03:55 AM EDT, Zdenek Svindrych <[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 Vitaly,
the long answer is: there are different "measures" of resolution, like FWHM
of the PSF, the OTF cutoff, and many more. They all behave well for lateral
resolution, meaning they give you comparable results for many microscopy
modalities.
It's more difficult with axial resolution, and on top of that, there is
another important measure "sectioning ability" that can be vaguely defined
as ability to cope with thick densely labeled samples. And those two are
much harder to compare among different microscopy (superresolution)
techniques.
I personally think in terms of OTF cutoff. It's a well defined, "hard"
resolution limit (at least for widefield, confocal, Airyscan, SIM, MSIM,
SMI, ISM, SoRa, Opra, instant SIM, you name it) that cannot be "improved"
by deconvolution alone. Then SoRa brings about no axial resolution
improvement, as was shown by Sheppard and Enderlein. This does not apply to
Airyscan, as Zeiss does do a bit more math than just "summing the shifted
PSFs".
Then there is the practical/experimental point of view, where I think the
most important quantity that affects the image quality is SNR.
To better compare with other techniques, bear in mind that in SoRa the
effective pinhole size is doubled by the action of the microlenses, and
scaled down by a factor 2.8 or 3 by the magnification changer between the
scope and the disk. So they come out a bit smaller than 50 um (for the
purpose of calculating axial resolution), but the disk throughput (and
pinhole crosstalk) is equivalent to 100 um pinhole disk (approx). This
sounds like a clever tradeoff to me, but no doubt one of the easiest from
the design point of view.
Back to your questions, I don't have the numbers, I'm not Yokogawa
engineer. But remember the resolution will depend on the lens magnification
as well! Also, I don't think (but I may very well be wrong) Yokogawa offers
deconvolution software for their W1.
Finally, SoRa is not "removable", it's a special disk; and you can only
switch between two disks (if I understood your question correctly).
Btw, as for the short answer, I don't think there is any :-).
Best, zdenek


On Mon, Aug 5, 2019 at 10:05 PM Vitaly Boyko <
[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.
> *****
>
>  Dear List,
> It looks like Yokogawa Marketing staff is "scared" to release data on
> axial resolution of the relatively new CSU-W1 unit?1. with 50 um disk and
> NA 1.4 objective?2. with 25 um disk and NA 1.4 lens?3. with SoRa (50 um
> disk) and NA 1.4 before and after non-linear deconvolution? Is there a
> linear deconvolution algorithm for the spinning disk confocal available?4.
> w/o SoRa "invention" and NA 1.4 glass?
> I would greatly appreciate professional response rather from Yokogawa
> engineers than from Yokogawa Marketing.
> Also, how come CSU-W1 unit cannot be designed with both removable SoRa and
> inter-exchangeable 25/50 um disks?
> Good morning Japan!
> Look forward to hearing solid pro and against arguments?
> Many thanks in advance.
> Best regards,
> Vitaly
>

--
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth





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[George McNamara]

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Sylvie,

Single point confocal microscopy using a Resonant scanner (Olympus
FV3000RS, 20x/0.75NA, 1 Airy unit pinhole, sometimes 3 for getting most
of the ~10 um cells thickness) is working great here imaging 2D gut
epithelial monolayers ... yes, we (that is, my colleagues in the
research labs) routinely grow the cultures in 3D, then we plate onto
(more or less transparent) transwells. Most imaging is done transferring
one transwell into a 35 mm imaging dish (i.e. 28 mm diameter glass
bottom). We typically use 16 frame averaging (~1 fps) with GCaMP6s for
Ca++ ion imaging (epub: https://www.ncbi.nlm.nih.gov/pubmed/31365292 ,
though not that much Ca++ imaging there). When Nathan Shaner's new
AausFP1 fluorescent protein (4.8x brighter than EGFP, and narrow
excitation and emission spectra ... this, YFP version, and similarly
narrow ex spectra fluorophores will make having WLL laser with AOBS or
'old school' AOBS, extremely useful ... WLL's are pulsed, so also
FastFLIM friendly): Lambert ... Shaner,/Aequorea victoria’s/secrets
https://www.biorxiv.org/content/10.1101/677344v2 ... if AausFP1CaMP# is
~5x brighter than "6s", implies could image about 5 fps with same SNR
(our cells probably do not need to image that fast, so could go for
lower laser light level, less risk of photobleaching or phototoxicity).

Vitaly - you wanted 8 laser lines (or WLL) ... our FV3000RS has 7 laser
lines, 4 GaAsP PMTs (internal decks), 2 GaAs PMTs (external ... I would
like more, someday, with 'fast photon counting' from each ... maybe
someday fast FLIM if enough users & applications enable finding the
money), described at http://confocal.jhu.edu/current-equipment/fv3000/ 
(NIH S10 grant summary near the bottom -- thanks as always to NIH, study
section, and especially U.S. taxpayers, for enabling this grant).

sincerely,

George

p.s. with respect to spinning disk confocal microscopes: we just
designated our Andor Revolution X1 / Olympus inverted microscope as
"legacy" = available to fully trained users (who mostly moved to the
FV3000RS), not actively encouraging use. I hope we do not buy another
spinning disk ... an identical twin to our FV3000RS would be much more
useful to our user base ... as would a fully tricked out Leica SP8 DMi8,
i.e. 4 SMD HyD's internal (GaAsP faceplate), 4 SMD HyD's on X1 external
port (say 2 GaAsP, 2 GaAs), sure a white light laser (especially one
reaching ~350 nm excitation for Brilliant Ultraviolets and perhaps next
gen quantum dots). Our newest instrument - thanks again to NIH,
reviewers, U.S. taxpayers, for NIH NIDDK P30 grant supplement money and
Prof. Mark Donowitz for matching funds ... we moved a 60x oil immersion
objective lens and our Olympus Cellsens GPU deconvolution license from
FV3000RS to here, so thanks again for S10 grant money!) -- is our
FISHscope,

http://confocal.jhu.edu/current-equipment/fishscope/

major application is locally produced (at much lower reagent cost than
commercial) single molecule RNA fluorescence in situ hybridization
(smFISH) ... I am also anticipating future "21plex" immunofluorescence
(and/or combined with smFISH), using Brilliants, SuperBrights, maybe
next gen quantum dots, etc. I am looking forward to replacing current
(under 1 teraflop - budget friendly PC being used) GPU card with NVidia
RTX Titan 2080 Ti (<2% cost of the microscope), 15 teraflops GPU.


A couple of the G.I. labs I work with adopted

On 8/8/2019 2:56 AM, Sylvie Le Guyader 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 Vitali
>
> Interesting comparison at the start of your email. However in my opinion, a few more parameters will come into play when I have to choose between the systems you mention: how deep you can go in the sample and the speed of imaging.
>
> In that respect only a spinning disk with a sensitive camera can fulfil my expectations. At our facility imaging live organoids in gels has literally exploded. That is not something our users want to do with a single point confocal with a 0.6 AU pinhole.
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7C,
> Room 7362 (lab)/7840 (office)
> 14157 Huddinge, Sweden
> mobile: [phone]
> LCI website
> Follow our microscopy blog!
>
> On 7 Aug 2019 20:04, Vitaly Boyko <[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 Zdenek,
> thank you very much for your reply. For the axial resolution, FWHM would be good enough for the beginning, a kind of obvious, with 63x NA 1.4 lens w/o SoRa and with N.A. 1.49 lens with SoRa.
> I have done cross-comparison between CSU-W1 with and w/o SoRa, and got better results both laterally and axially with 0.6 AU pinhole for the Alexa 488 (and the same pinhole size for Alexa 594, which would be ca. 0.54 AU) on Leica SP8. Airyscan alike SoRa is struggling to collect enough photons per voxel to achieve low photo toxicity for long-term imaging of live specimen (0.5x or 1x sampling along 50-80 um in z). So far, Lattice SIM seems to outperform all of the above for thin samples, but again, Zeiss have been stubborn (and rarely listen) with the choice of lasers, and as "stubbornly" expected Zeiss offers only four very "old-fashioned" laser lines (Zeiss still has to learn that exciting with 496 nm argon line gives better SNR for tissues or immune cells (macrophages) with higher auto-fluorescence). Are their high end confocals finally come with a tunable white light laser??? Zeiss should respond promptly?
> Has someone done a very simple experiment combining 100 nm and 200 nm multi-color TetraSpec beads, and calculated ratio of, let say, Alexa-488 mean intensity for 200 nm bead and 100 nm bead before and after deconvolution (Airyscan, SoRa, Lattice SIM, iSIM etc.)??? Training set of ca. 5000 beads should be good enough for the beginning. The same could be done when beads are placed in matrigel or any other 3D media 100 um, 200 um, or 500 um away from the coveglass?  Another question to all vendors with positively evolved Broad Vision: Leica (Leica has WLL, not much to complain here except for their mediocre z-galvo, 63x NA 1.4 oil "non-holding" objectives", all others are equally guilty of cheap thermally remarkably expandable aluminum), Zeiss, Nikon, Olympus, Andor and any other new broadly experienced, 100% customer oriented vendors. Sorry, the question is just below finally:
> Which vendor (WLL-plus vendors are excluded on a positive note) can offer 8-10 laser lines fixture (50 mW minimum) such as:
> 1. 375 nm +/- 5 nm,2. 440 nm +/- 3 nm,3. 486 nm +/- 3 nm,4. 495 nm +/- 5 nm,5. 510 nm +/- 3 nm,6. 550 nm +/- 5 nm,7. 572 nm +/- 5 nm,8. 585 nm +/- 5 nm,   9. 648 nm +/- 5 nm,10. 750 nm +/- 5 nm
> Cheers,
> Vitaly        On Tuesday, August 6, 2019, 11:03:55 AM EDT, Zdenek Svindrych <[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 Vitaly,
> the long answer is: there are different "measures" of resolution, like FWHM
> of the PSF, the OTF cutoff, and many more. They all behave well for lateral
> resolution, meaning they give you comparable results for many microscopy
> modalities.
> It's more difficult with axial resolution, and on top of that, there is
> another important measure "sectioning ability" that can be vaguely defined
> as ability to cope with thick densely labeled samples. And those two are
> much harder to compare among different microscopy (superresolution)
> techniques.
> I personally think in terms of OTF cutoff. It's a well defined, "hard"
> resolution limit (at least for widefield, confocal, Airyscan, SIM, MSIM,
> SMI, ISM, SoRa, Opra, instant SIM, you name it) that cannot be "improved"
> by deconvolution alone. Then SoRa brings about no axial resolution
> improvement, as was shown by Sheppard and Enderlein. This does not apply to
> Airyscan, as Zeiss does do a bit more math than just "summing the shifted
> PSFs".
> Then there is the practical/experimental point of view, where I think the
> most important quantity that affects the image quality is SNR.
> To better compare with other techniques, bear in mind that in SoRa the
> effective pinhole size is doubled by the action of the microlenses, and
> scaled down by a factor 2.8 or 3 by the magnification changer between the
> scope and the disk. So they come out a bit smaller than 50 um (for the
> purpose of calculating axial resolution), but the disk throughput (and
> pinhole crosstalk) is equivalent to 100 um pinhole disk (approx). This
> sounds like a clever tradeoff to me, but no doubt one of the easiest from
> the design point of view.
> Back to your questions, I don't have the numbers, I'm not Yokogawa
> engineer. But remember the resolution will depend on the lens magnification
> as well! Also, I don't think (but I may very well be wrong) Yokogawa offers
> deconvolution software for their W1.
> Finally, SoRa is not "removable", it's a special disk; and you can only
> switch between two disks (if I understood your question correctly).
> Btw, as for the short answer, I don't think there is any :-).
> Best, zdenek
>
>
> On Mon, Aug 5, 2019 at 10:05 PM Vitaly Boyko <
> [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.
>> *****
>>
>>   Dear List,
>> It looks like Yokogawa Marketing staff is "scared" to release data on
>> axial resolution of the relatively new CSU-W1 unit?1. with 50 um disk and
>> NA 1.4 objective?2. with 25 um disk and NA 1.4 lens?3. with SoRa (50 um
>> disk) and NA 1.4 before and after non-linear deconvolution? Is there a
>> linear deconvolution algorithm for the spinning disk confocal available?4.
>> w/o SoRa "invention" and NA 1.4 glass?
>> I would greatly appreciate professional response rather from Yokogawa
>> engineers than from Yokogawa Marketing.
>> Also, how come CSU-W1 unit cannot be designed with both removable SoRa and
>> inter-exchangeable 25/50 um disks?
>> Good morning Japan!
>> Look forward to hearing solid pro and against arguments?
>> Many thanks in advance.
>> Best regards,
>> Vitaly
>>
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> --
> Zdenek Svindrych, Ph.D.
> Research Associate - Imaging Specialist
> Department of Biochemistry and Cell Biology
> Geisel School of Medicine at Dartmouth
>
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