http://confocal-microscopy-list.275.s1.nabble.com/High-speed-spinning-disc-confocal-with-EMCCD-camera-tp7583142p7583240.html
laser for a given level of 2p excitation. Furthermore, if you're doing
more photo damage). Intuitively at least (and I could be wrong), I would
> *****
> 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.
> *****
>
> Michael, I typed out a longer reply, but I think I can boil it down. Which
> has lower bleaching/toxicity/heating, 1p SPIM or parallel point-scanning
> 2p? Why?
>
> My anecdotal experience: My first postdoc project was to build a temporal
> focus system (extremely fast parallel 2p scanning), while another postdoc
> 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
> faster than 1p spinning disk), but loved 1p SPIM (30x gentler/faster than
> 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?
> Heating? Nonlinear damage mechanisms? Inherently lower efficiency? I
> suspect all three, but still don't know. I expected the two systems would
> perform about the same; neither bleaches out-of-plane, both are highly
> parallel. We tried different exposure times, power levels, wavelengths, but
> there was no combination that left us anywhere near the gentleness and
> signal levels of the 1p SPIM.
>
> On Tue, Jan 6, 2015 at 3:16 PM, Michael Giacomelli <
[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 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
> > > >
> > >
> >
>