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Hi all,
I think we would have a real problem trying to make a light sheet
bright enough to excite 2-photon fluorescence. In general one needs a
fairly high NA objective to focus a single few-mW beam (or a small
cluster of them) into a spot so small that the intensity is
sufficient to cause useful 2-photon fluorescence.
Trying to do this in the form of a light sheet would have two huge problems:
1) The optics needed to make the sheet would have to be fairly high
NA and as a result the required cylindrical optics would form
something like two wedges of illumination, touching at the focal
plane, i.e., because excitation goes with the square of the
intensity, the effort to make a sheet would actually produce a
"squashed line" of excitation. (There would also be the practical
problem of making a high NA-lens with cylindrical optical components)
2) Were you to succeed in having magically produced a light sheet
with sufficient intensity (perhaps by sticking with the low-NA
cylindrical optics but using a massively more powerful laser) then it
would be hard to imagine a cell not being cooked by the 1-photon
absorption by the water.
In 2 photon-land, the most points anyone has illuminated at one time
and kept the cell alive is 64, not 250,000.
Cheers,
Jim Pawley
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>*****
>
>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 <[log in to unmask]
>> 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 <[log in to unmask]> 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 <
>> >> [log in to unmask]> 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 <[log in to unmask]>
>> 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:
>> >> [log in to unmask]]
>> >>>> On Behalf Of James Pawley
>> >>>> Sent: Sunday, 4 January 2015 11:47 AM
>> >>>> To: [log in to unmask]
>> >>>> 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: [log in to unmask]
>> >>>>>
>> >>>>>
>> >>>>>
>> >>>>>
>> >>>>>
>> >>>>>
>> >>>>>
>> >>>>> On 12/30/14, 2:36 AM, "Andrea Latini" <[log in to unmask]> 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
>> >>>>>> [log in to unmask]
>> >>>>>>
>> >>>>>>
>> >>>>>> On Mon, 29 Dec 2014 16:58:15 -0500, Andrew York
>> >>>>>> <[log in to unmask]> 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 <
>> [log in to unmask]
>> >>>
>> >>>>>>> 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
>> >>>>>>>> [log in to unmask]
>> >>>>>>>>
>> >>>>
>> >>>>
>> >>>> --
>> >>>> ****************************************
>> >>>> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt,
>> >> BC,
>> >>>> Canada, V0N3A0, Phone 604-885-0840, email <[log in to unmask]> NEW!
>> >> NEW!
>> >>>> AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
>> >>>>
>> >>>
>> >>
>>
--
****************************************
James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt,
BC, Canada, V0N3A0,
Phone 604-885-0840, email <[log in to unmask]>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
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