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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 <[log in to unmask]> 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 <
[log in to unmask]> wrote:
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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, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth
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