http://confocal-microscopy-list.275.s1.nabble.com/High-speed-spinning-disc-confocal-with-EMCCD-camera-tp7583142p7583215.html
Look, I'm not trying to sell any solution, I'm just contributing to the discussion. Of course the 2P cross-section is smaller - 2P would not be depth-selective and (moderately) super-resolution if it were not. But IR is MUCH less damaging to living tissue than UV/blue. As to photo-bleaching, I think you are wrong BUT you can be right! Years ago, at a conference in Taiwan, a speaker presented data that FITC bleached much more rapidly in 2P then in 1P. The problem was he hadn't considered the photochemistry. Fluorescein is a symmetrical molecule, and as such 2P excitation cannot occur at half the 1P frequency. The excitation curves had been published, but he hadn't looked at them. If he'd excited at the (published) 2P maximum he would not have found this. Exciting ANY fluorochrome to above the S1 level will naturally increase bleaching.
Fluorescence lifetimes of common fluorochromes range from 1.1ns (eosin) to 3.41ns (Texas Red). So your have a point, but it is not tenfold.
Not being able to saturate excitation is a good thing, not a bad thing! I am perpetually telling people to reduce their excitation to 50% and check that their excitation decreases by the same amount - if not they are saturating (and destroying) their fluorochrome. Nobody ever follows my advice - they just want a bright (and usually saturated and therefore uninformative) image.
Subject: Re: High speed spinning disc confocal with EMCCD camera - commercial response
Good point about two-photon, the confinement of bleaching and reduced crosstalk is quite nice. Devils advocate arguments against going fast with 2p, compared to 1p spinning disk:
1. 2p cross sections are very very low compared to 1p; it takes a lot of power to saturate each 2p spot (~mWs each), which can add up to impractical levels pretty fast (>1 W average power). Even though IR light is absorbed less than visible, low cross section combined with high parallelization can mean non-negligible heating. Getting the same degree of parallelization as a spinning disk isn't likely, so your instantaneously glowing volume will be a lot smaller and ultimate speed limit will be a lot slower.
2. Typical pulse rates for 2p (>10 ns) are long compared to fluorescent lifetimes (~1 ns?), so your molecules spend a lot of time not glowing, and the speed-limiting signal per second takes another 5-10x hit compared to CW visible excitation.
3. I'm pretty sure you get fewer signal photons per bleaching event with 2p compared to 1p, when imaging a single plane. Can anyone confirm/deny? I know bleaching rates blow up past a certain 2p intensity, but I'm not sure they ever get as low as with 1p, for the same amount of signal produced.
(of course, this is offset by the absence of out-of-plane bleaching Guy mentioned, so for a thick enough sample where you're imaging the entire volume, you're clearly better off with 2p)
4. I'm not even sure you can saturate excitation with 2p, compared to 1p.
Has anyone studied this? Which comes first, saturation of excitation, or 2p photobleaching rates greatly exceeding 1p rates?
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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> *****
>
> Multi-beam multiphoton (eg LaVision Biotec) also limits bleaching to
> the focal plane and has the advantage over spinning disk confocal that
> there is no cross-talk. No commercial association, but I do know a
> very satisfied user.
>
> Guy
>
> Guy Cox, Honorary Associate Professor
> School of Medical Sciences
>
> Australian Centre for Microscopy and Microanalysis, Madsen, F09,
> University of Sydney, NSW 2006
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:
[hidden email]]
> On Behalf Of James Pawley
> Sent: Sunday, 4 January 2015 11:47 AM
> To:
[hidden email]
> Subject: Re: High speed spinning disc confocal with EMCCD camera -
> commercial response
>
> *****
> 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.
> *****
>
> >*****
> >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.
> >*****
>
>
>
> Details aside, data rate will always be proportional to how much light
> is detected/second. More beams will produce more data/second.
> Single beam instruments really can't compete because they intensity in
> a focused confocal spot is already close to singlet-state saturation.
> But the quality of the data will vary between techniques.
> What do you "need to see"?.
>
> I would bet on light sheet/SPIM. Damage only in the illuminated plane
> and simple optics to a (effective) high-QE EM-CCD or sCMOS camera.
>
> JP
>
> >Hi all,
> >
> >Does anyone think it would be possible to tabulate a 'speed limit'
> >for the various options discussed? I know it sounds near impossible
> >to come up with a standard basis for comparison, but let's say
> >something approximating a 512x512 acquisition either fixed or or a
> >volume that includes 10 z steps (e.g., using a piezo stage when
> >relevant). It would be great to have an order of magnitude idea how
> >to compare technologies like a resonant scanner, Optera-type swept
> >field scanner, spinning disc, VCS super-spinning disc or light sheet
> >instrument when FPS is a major priority and excitation light is not
> >limiting. Maybe we could crowdsource it from what users actually get in practice.
> >
> >All the best,
> >
> >
> >Tim
> >
> >Timothy Feinstein, Ph.D. | Manager, Core for Confocal Microscopy and
> >Quantitative Imaging
> >333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
> >Phone: 616-234-5819 | Email:
[hidden email]
> >
> >
> >
> >
> >
> >
> >
> >On 12/30/14, 2:36 AM, "Andrea Latini" <
[hidden email]> wrote:
> >
> >>*****
> >>To join, leave or search the confocal microscopy listserv, go to:
> >>
http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFK> >>Nq
> >>OL1R
> >>ax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfo
> >>ca
> >>lmic
> >>roscopy
> >>Post images on
> >>
http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFK> >>Nq OLwF bleD9dw&u=http%3a%2f%2fwww%2eimgur%2ecom and include the
> >>link in your posting.
> >>*****
> >>
> >>Dear Andrew,
> >>the VCS (Video Confocal Super Resolution), module is an X-Light
> >>Spinning disk system add-on.
> >>the disk is out of the optical path when in VCS mode (i.e. 'bypass'
> mode).
> >>basically, it's a new implementation of structured illumination
> >>technology aimed to fast image acquisition with no resolution
> >>limitations that are spinning disk related.
> >>
> >>I'll be pleased to discuss more, please get in touch.
> >>
> >>Regards.
> >>
> >>Andrea
> >>
[hidden email]
> >>
> >>
> >>On Mon, 29 Dec 2014 16:58:15 -0500, Andrew York
> >><
[hidden email]> wrote:
> >>
> >>>*****
> >>>To join, leave or search the confocal microscopy listserv, go to:
> >>>
http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nF> >>>KN
> >>>qOL1
> >>>Rax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dcon
> >>>fo
> >>>calm
> >>>icroscopy
> >>>Post images on
> >>>
http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nF> >>>KN qOLw FbleD9dw&u=http%3a%2f%2fwww%2eimgur%2ecom and include the
> >>>link in your posting.
> >>>*****
> >>>
> >>> Is there information available about this product? Is this an
> >>>implementation of Enderlein's spinning disk paper? Also, 80 nm seems...
> >>>optimistic? Is this with very short wavelength light, or just a
> >>>slightly different definition of resolution than I'm used to?
> >>>
> >>>On Mon, Dec 29, 2014 at 4:10 PM, Andrea Latini
> >>><
[hidden email]>
> >>>wrote:
> >>>
> >>>> *****
> >>>> To join, leave or search the confocal microscopy listserv, go to:
> >>>>
> >>>>
http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1n> >>>>FK
> >>>>NqOL
> >>>>1Rax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dc
> >>>>on
> >>>>foca
> >>>>lmicroscopy
> >>>> Post images on
> >>>>
http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1n> >>>>FK NqOL wFbleD9dw&u=http%3a%2f%2fwww%2eimgur%2ecom and include the
> >>>>link in your
> > >>>posting.
> >>>> *****
> >>>>
> >>>> - commercial response
> >>>>
> >>>> thanks for reporting your experience with our Confocals Marco.
> >>>>
> >>>> the new Video Super Resolution module for XLight allows for 50ms
> >>>>exposure
> >>>> time and <1
> >>>> second, 80nm spatial resolution; this is possible with large
> >>>>Cuda
> >>>> programming we've been
> >>>> developing during past months and introduced @SfN 2014 as a product.
> >>>>
> >>>> soon on our website and in your Lab, hopefully!
> >>>>
> >>>> Cheers.
> >>>>
> >>>> Andrea
> >>>>
> >>>> CrestOptics
> >>>>
[hidden email]
> >>>>
>
>
> --
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> BC, Canada, V0N3A0, Phone 604-885-0840, email <
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