http://confocal-microscopy-list.275.s1.nabble.com/High-speed-spinning-disc-confocal-with-EMCCD-camera-tp7583142p7583225.html
Michael, I typed out a longer reply, but I think I can boil it down. Which
built a 1p SPIM. The goal was C. elegans development timelapses, gentler
than 1p spinning disk. Turned out the worms HATED 2p (bleached/died much
1p spinning disk). I used temporal focus for photoactivation in another
project, but it left me curious. Why did the worms hate 2p so much?
suspect all three, but still don't know. I expected the two systems would
parallel. We tried different exposure times, power levels, wavelengths, but
> *****
> 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 Andrew,
>
> As you point out, the 1p absorption cross section in the NIR is very low as
> compared to visible, but I'm not sure you appreciate just how much lower.
> Going from 400 to 800 nm for instance, you reduce the absorption in whole
> human tissue by roughly 3 orders of magnitude. So 1 mW of 800nm light has
> the same 1p absorption as 1 microwatt of 400 nm. Often, damage in
> ultrafast systems is almost entirely through multiphoton effects, which is
> a pretty good place to operate.
>
> Regarding laser repetition rates, its rare to be limited by laser rep rate
> with an 80MHz system (that would be a very fast scanner), but if you are,
> you can easily double or quadruple the pulse rate of a ti:sapphire laser
> using beam splitters. However, its usually advantageous to stay below
> 80MHz, as above that you run into the FM radio and then cellular bands
> which are very noisy and require quite a lot more effort to work in.
>
> I don't think there is a difference in bleaching between 1 and 2p
> absorption in general. Usually though bleaching is lower with 2 photon
> because the area of excitation is more tightly confined (a plane is thinner
> for a given NA).
>
> Regarding the more general question of how to image faster, I think it
> depends on what you want to do. Confocal is at the least disadvantage when
> operated on single layer samples like monolayers because there is
> negligible scattering and no need for depth selection. The relative
> simplicity of it then allows for very highly parallel systems. Likewise
> multispot multiphoton will work best for less scattering samples. If the
> sample is thicker or more scattering, single pixel multiphoton has a large
> advantage in that the light collection is not descanned and so much more
> total signal can be collected (for a given, lower illumination power) while
> the low 1p absorption minimizes out of plane photobleaching. Unfortunately
> though, very fast scanning is hard, which limits the speed of single spot
> systems somewhat.
>
> Mike
>
> On Sun, Jan 4, 2015 at 1:14 PM, Andrew York <
>
[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.
> > *****
> >
> > 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:
> >
> > > *****
> > > 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.
> > > *****
> > >
> > > 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_JSBGnaEcBqKN1nFKNq> > > >>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:
> > > >>>
>
http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFKN> > > >>>qOL1
> > >
> >>>Rax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfo
> > > >>>calm
> > > >>>icroscopy
> > > >>>Post images on
> > > >>>
>
http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFKN> > > >>>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_JSBGnaEcBqKN1nFK> > > >>>>NqOL
> > >
> >>>>1Rax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dcon
> > > >>>>foca
> > > >>>>lmicroscopy
> > > >>>> Post images on
> > > >>>>
>
http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFK> > > >>>>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]
> > > >>>>
> > >
> > >
> > > --
> > > ****************************************
> > > James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt,
> BC,
> > > Canada, V0N3A0, Phone 604-885-0840, email <
[hidden email]> NEW!
> NEW!
> > > AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
> > >
> >
>