Re: High speed spinning disc confocal with EMCCD camera - commercial response

Posted by Andrew York on
URL: http://confocal-microscopy-list.275.s1.nabble.com/High-speed-spinning-disc-confocal-with-EMCCD-camera-tp7583142p7583211.html

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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?

On Sat, Jan 3, 2015 at 11:09 PM, Guy Cox <[hidden email]> wrote:

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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
>
> *****
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>
>
> 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: [phone] | Email: [hidden email]
> >
> >
> >
> >
> >
> >
> >
> >On 12/30/14, 2:36 AM, "Andrea Latini" <[hidden email]> wrote:
> >
> >>*****
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> >>OL1R
> >>ax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfoca
> >>lmic
> >>roscopy
> >>Post images on
> >>http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFKNq
> >>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:
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> >>>qOL1
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> >>>calm
> >>>icroscopy
> >>>Post images on
> >>>http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFKN
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> >>>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:
> >>>
> >>>>  *****
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> >>>>foca
> >>>>lmicroscopy
> >>>>  Post images on
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> >  >>>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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