http://confocal-microscopy-list.275.s1.nabble.com/High-speed-spinning-disc-confocal-with-EMCCD-camera-tp7583142p7583229.html
According to my photochemist colleague Scott Kable the difference is ~ 10 orders of magnitude.
Subject: Re: High speed spinning disc confocal with EMCCD camera - commercial response
being working on R2PI technique (Resonant Two Photons Inonization), for long time, I'm wondering on how much the single phon absorption is taking place compared to the two photons one.
in other words, do you have any estimate for the 1phothon/2photons cross section ratio?
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>
>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:
>
>> *****
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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:
>>
>> > *****
>> > 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_JSBGnaEcBqKN1>> > >>nFKNq
>> > >>OL1R
>> > >>ax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-