http://confocal-microscopy-list.275.s1.nabble.com/High-speed-spinning-disc-confocal-with-EMCCD-camera-tp7583142p7583249.html
Good question about 1p vs 2p light sheet. I don't know, but that ought to
distinguish between heating vs. poor signal per bleaching event. Fluorphore
> *****
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>
> Andrew, that's an interesting account. I reckon there are only a few
> people in the world who have been able to make (an almost) direct
> comparison like this so far. What do you think the result would have been
> if 1p scanned light sheet were compared to 2p scanned light sheet (assuming
> the 2p wavelength is chosen to reside at the fluorophore 2p max absorption)?
>
> When you did your tests with C.elegans, what was the fluorochrome?
>
> John Oreopoulos
>
>
> On 2015-01-06, at 5:05 PM, Andrew York wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
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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
> >>>>
> >>>
> >>
>