http://confocal-microscopy-list.275.s1.nabble.com/High-speed-spinning-disc-confocal-with-EMCCD-camera-tp7583142p7583250.html
bright enough to excite 2-photon fluorescence. In general one needs a
"squashed line" of excitation. (There would also be the practical
>*****
>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 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
>was GFP.
>
>On Tue, Jan 6, 2015 at 9:24 PM, John Oreopoulos <
[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.
>> *****
>>
>> 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:
>> >
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
>> >>>>
>> >>>
>> >>
>>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146