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High speed spinning disc confocal with EMCCD camera

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Andreas Bruckbauer

Re: High speed spinning disc confocal with EMCCD camera

all these two-photon light sheet
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John, Jim and others

all these two-photon light sheet papers use a beam scanned in the y-direction to produce the light sheet, this helps to increase the intensity to achieve two-photon fluorescence and has the advantage of only cooking the part of the sample which is temporally in the beam...

One should also look at the 2011 Planchon et al. Nature Methods Bessel beam paper where they use 2P and 1P excitation. Interestingly in the 2012 Gao et al. Cell paper they drop the two-photon mode in favor of a multi-beam parallel one-photon mode which was further improved in the recent lattice light sheet paper.

best wishes

Andreas

-----Original Message-----
From: John Oreopoulos <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Fri, 9 Jan 2015 7:25
Subject: Re: High speed spinning disc confocal with EMCCD camera

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But Jim,

2-photon light sheet microscopy has been demonstrated a few times now:

http://www.nature.com/nmeth/journal/v8/n9/full/nmeth.1652.html

http://www.nature.com/nmeth/journal/v11/n6/full/nmeth.2963.html

http://www.nature.com/cr/journal/vaop/ncurrent/full/cr2014124a.html

John Oreopoulos

On 2015-01-08, at 10:41 PM, James Pawley 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 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
>
>> *****
>> 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
>>> >>>>
>>> >>>
>>> >>
>>>
>
>
> --
> ****************************************
> 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

Guy Cox-2

Re: High speed spinning disc confocal with EMCCD camera

*****
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*****

Basically we're seeing a convergence here. It's hard to see why these schemes are going to give you any different image from doing an XZ scan in conventional multiphoton.

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 Andreas Bruckbauer
Sent: Friday, 9 January 2015 8:20 PM
To: [hidden email]
Subject: Re: High speed spinning disc confocal with EMCCD camera

all these two-photon light sheet
*****
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.
*****

John, Jim and others

all these two-photon light sheet papers use a beam scanned in the y-direction to produce the light sheet, this helps to increase the intensity to achieve two-photon fluorescence and has the advantage of only cooking the part of the sample which is temporally in the beam...

One should also look at the 2011 Planchon et al. Nature Methods Bessel beam paper where they use 2P and 1P excitation. Interestingly in the 2012 Gao et al. Cell paper they drop the two-photon mode in favor of a multi-beam parallel one-photon mode which was further improved in the recent lattice light sheet paper.

best wishes

Andreas

-----Original Message-----
From: John Oreopoulos <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Fri, 9 Jan 2015 7:25
Subject: Re: High speed spinning disc confocal with EMCCD camera

*****
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.
*****

But Jim,

2-photon light sheet microscopy has been demonstrated a few times now:

http://www.nature.com/nmeth/journal/v8/n9/full/nmeth.1652.html

http://www.nature.com/nmeth/journal/v11/n6/full/nmeth.2963.html

http://www.nature.com/cr/journal/vaop/ncurrent/full/cr2014124a.html

John Oreopoulos

On 2015-01-08, at 10:41 PM, James Pawley 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 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.

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

James Pawley

Re: High speed spinning disc confocal with EMCCD camera

>*****
>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.
>*****
>
>Basically we're seeing a convergence here. It's
>hard to see why these schemes are going to give
>you any different image from doing an XZ scan in
>conventional multiphoton.
>
> Guy
>
>Guy Cox, Honorary Associate Professor
>School of Medical Sciences
>
>Australian Centre for Microscopy and Microanalysis,
>Madsen, F09, University of Sydney, NSW 2006
>

Well, I hope that we can all agree that there are some small differences.

On the normal 2-photon side, the advantages would
seem to be that you only need to arrange one lens
facing the specimen and you can use non-descanned
detection.

The disadvantage is that you must work closer to
saturation (at east during the pulse) which with
some dyes, will cause much faster bleaching, and
may affect all dies to some extent (in terms of
excitations before death).

On the 2-photon-from-the-side, the disadvantages
might include the fact that the raw (straight
from the CCD) image, z resolution is going to be
quite a lot worse, you cannot correlate scattered
fluorescence photons with their site of origin as
well as with single-beam 2-photon. Finally, you
have to arrange for 2 objectives to approach the
specimen in the correct geometry.

The advantage is that the imaging speed will
potentially be faster and the damage per
collected photon less because

1) At any instant you collect from a fuzzy line
of illumination converging at NA 0.06, rather
than a much smaller blobby circle. So you can
work farther from saturation and still collect
data fast.

2) As this line of excitation is quite wide
(4µm?) and you are collecting the data onto the
CCD using another lens that can work at much
higher NA, the fuzzy line of illumination
actually will cover a volume of the specimen that
will be recorded on many lines of the CCD at any
instant. This also helps to keep the required
dose to any molecule low at any instant (reducing
the change of re-exciting an already excited
molecule) even though the data total rate is
still high.

3) You will collect your data on a CCD a device
that has a much higher effective QE than even the
best PMT (although not much better than an MPPC,
as long as the light signal is spread out to
cover this detector approximately evenly). As
detected signal is the limitation, higher QE
means that again you can again reduce the
excitation intensity. The LaVision Biotec also
uses a CCD but only "illuminates" up to 64 spots,
not 10-20 lines.

The proof here is in the pudding: I have not seen
any images from normal 2P close to the 4D embryo
images made with the side-scan 2P (or even SPIM).
Although these side-scan images are essentially
low resolution images, they show what the
researcher needs and do so using a process that
seems to allow the embryo to survive and grow.

Both 2P systems have the disadvantage of
collecting signal only during a brief period
after each pulse and are somewhat less convenient
than 1P when viewing more than one fluorophor.

Does that sound fair?

Jim Pawley

>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On
>Behalf Of Andreas Bruckbauer
>Sent: Friday, 9 January 2015 8:20 PM
>To: [hidden email]
>Subject: Re: High speed spinning disc confocal with EMCCD camera
>
>all these two-photon light sheet
>*****
>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.
>*****
>
>
> John, Jim and others
>
>all these two-photon light sheet papers use a
>beam scanned in the y-direction to produce the
>light sheet, this helps to increase the
>intensity to achieve two-photon fluorescence and
>has the advantage of only cooking the part of
>the sample which is temporally in the beam...
>
>One should also look at the 2011 Planchon et al.
>Nature Methods Bessel beam paper where they use
>2P and 1P excitation. Interestingly in the 2012
>Gao et al. Cell paper they drop the two-photon
>mode in favor of a multi-beam parallel
>one-photon mode which was further improved in
>the recent lattice light sheet paper.
>
>best wishes
>
>Andreas
>
>
>
>
>
>-----Original Message-----
>From: John Oreopoulos <[hidden email]>
>To: CONFOCALMICROSCOPY <[hidden email]>
>Sent: Fri, 9 Jan 2015 7:25
>Subject: Re: High speed spinning disc confocal with EMCCD camera
>
>
>*****
>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.
>*****
>
>But Jim,
>
>2-photon light sheet microscopy has been demonstrated a few times now:
>
>http://www.nature.com/nmeth/journal/v8/n9/full/nmeth.1652.html
>
>http://www.nature.com/nmeth/journal/v11/n6/full/nmeth.2963.html
>
>http://www.nature.com/cr/journal/vaop/ncurrent/full/cr2014124a.html
>
>John Oreopoulos
>
>
>On 2015-01-08, at 10:41 PM, James Pawley 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 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
>>
>>> *****
>>> 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
>>>> >>>>
>>>> >>>
>>>> >>
>>>>
>>
>>
>> --
>> ****************************************
>> 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
>
>

--
****************************************
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

Michael Giacomelli

Re: High speed spinning disc confocal with EMCCD camera

In reply to this post by Guy Cox-2

*****
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*****

Hi Jim,

I don't know if I'd say that the EMCCD is an advantage in efficiency.
Certainly the QE can be higher, but the small pixel size and the need
to collect in an imaging geometry means that the light
throughput/crosstalk will take a large hit if there is scattering. Of
course, parallel detection has its own advantages, particularly at
very high speeds.

Mike

On Fri, Jan 9, 2015 at 3:13 PM, James Pawley <[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.
> *****
>
>> *****
>> 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.
>> *****
>>
>> Basically we're seeing a convergence here. It's hard to see why these
>> schemes are going to give you any different image from doing an XZ scan in
>> conventional multiphoton.
>> Guy
>>
>> Guy Cox, Honorary Associate Professor
>> School of Medical Sciences
>>
>> Australian Centre for Microscopy and Microanalysis,
>> Madsen, F09, University of Sydney, NSW 2006
>>
>
>
> Well, I hope that we can all agree that there are some small differences.
>
> On the normal 2-photon side, the advantages would seem to be that you only
> need to arrange one lens facing the specimen and you can use non-descanned
> detection.
>
> The disadvantage is that you must work closer to saturation (at east during
> the pulse) which with some dyes, will cause much faster bleaching, and may
> affect all dies to some extent (in terms of excitations before death).
>
> On the 2-photon-from-the-side, the disadvantages might include the fact
> that the raw (straight from the CCD) image, z resolution is going to be
> quite a lot worse, you cannot correlate scattered fluorescence photons with
> their site of origin as well as with single-beam 2-photon. Finally, you have
> to arrange for 2 objectives to approach the specimen in the correct
> geometry.
>
> The advantage is that the imaging speed will potentially be faster and the
> damage per collected photon less because
>
> 1) At any instant you collect from a fuzzy line of illumination converging
> at NA 0.06, rather than a much smaller blobby circle. So you can work
> farther from saturation and still collect data fast.
>
> 2) As this line of excitation is quite wide (4µm?) and you are collecting
> the data onto the CCD using another lens that can work at much higher NA,
> the fuzzy line of illumination actually will cover a volume of the specimen
> that will be recorded on many lines of the CCD at any instant. This also
> helps to keep the required dose to any molecule low at any instant (reducing
> the change of re-exciting an already excited molecule) even though the data
> total rate is still high.
>
> 3) You will collect your data on a CCD a device that has a much higher
> effective QE than even the best PMT (although not much better than an MPPC,
> as long as the light signal is spread out to cover this detector
> approximately evenly). As detected signal is the limitation, higher QE means
> that again you can again reduce the excitation intensity. The LaVision
> Biotec also uses a CCD but only "illuminates" up to 64 spots, not 10-20
> lines.
>
> The proof here is in the pudding: I have not seen any images from normal 2P
> close to the 4D embryo images made with the side-scan 2P (or even SPIM).
> Although these side-scan images are essentially low resolution images, they
> show what the researcher needs and do so using a process that seems to allow
> the embryo to survive and grow.
>
> Both 2P systems have the disadvantage of collecting signal only during a
> brief period after each pulse and are somewhat less convenient than 1P when
> viewing more than one fluorophor.
>
> Does that sound fair?
>
> Jim Pawley
>
>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Andreas Bruckbauer
>> Sent: Friday, 9 January 2015 8:20 PM
>> To: [hidden email]
>> Subject: Re: High speed spinning disc confocal with EMCCD camera
>>
>> all these two-photon light sheet
>> *****
>> 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.
>> *****
>>
>>
>> John, Jim and others
>>
>> all these two-photon light sheet papers use a beam scanned in the
>> y-direction to produce the light sheet, this helps to increase the intensity
>> to achieve two-photon fluorescence and has the advantage of only cooking the
>> part of the sample which is temporally in the beam...
>>
>> One should also look at the 2011 Planchon et al. Nature Methods Bessel
>> beam paper where they use 2P and 1P excitation. Interestingly in the 2012
>> Gao et al. Cell paper they drop the two-photon mode in favor of a multi-beam
>> parallel one-photon mode which was further improved in the recent lattice
>> light sheet paper.
>>
>> best wishes
>>
>> Andreas
>>
>>
>>
>>
>>
>> -----Original Message-----
>> From: John Oreopoulos <[hidden email]>
>> To: CONFOCALMICROSCOPY <[hidden email]>
>> Sent: Fri, 9 Jan 2015 7:25
>> Subject: Re: High speed spinning disc confocal with EMCCD camera
>>
>>
>> *****
>> 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.
>> *****
>>
>> But Jim,
>>
>> 2-photon light sheet microscopy has been demonstrated a few times now:
>>
>> http://www.nature.com/nmeth/journal/v8/n9/full/nmeth.1652.html
>>
>> http://www.nature.com/nmeth/journal/v11/n6/full/nmeth.2963.html
>>
>> http://www.nature.com/cr/journal/vaop/ncurrent/full/cr2014124a.html
>>
>> John Oreopoulos
>>
>>
>> On 2015-01-08, at 10:41 PM, James Pawley 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 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
>>>
>>>> *****
>>>> 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
>>>>> >>>>
>>>>> >>>
>>>>> >>
>>>>>
>>>
>>>
>>> --
>>> ****************************************
>>> 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
>>
>>
>>
>
>
> --
> ****************************************
> 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

Andrew York

Re: High speed spinning disc confocal with EMCCD camera - commercial response

In reply to this post by Michael Giacomelli

*****
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>
> Its hard for me to say since I don't have any experience with temporal
> focusing, but I wonder if part of the problems you observed were related to
> that technique?

Temporal focus is just fast passive scanning, like the lavision technique
but with shoulder-to-shoulder spots. The typical diffraction-grating
implementation is equivalent to splitting your beam (or line focus) into a
bunch of lines (or spots) and delaying each one a different amount.

> From what I've read, getting tight axial confinement is
> fairly difficult often requiring relatively high power or a low rep rate
> laser for a given level of 2p excitation.

The degree of axial confinement is independent of the power; I guess you
mean you have to overfill the objective BFP pretty hard to get good
sectioning, wasting power? Line scanning temporal focusing should give
sectioning equivalent to point-scanning 2p, and that's what we measured (
http://www.nature.com/nmeth/journal/v8/n4/full/nmeth.1571.html ,
http://www.ncbi.nlm.nih.gov/pubmed/21317909 Fig 1c, Sup Fig 1, and Sup Note
1). It's lots of 2p power compared to point scanning because the scanning
is so much faster, but the required power nicely matches the output power
of the Coherent Chameleon.

> Furthermore, if you're doing
> parallel detection, you tend to have less efficient light collection,

I think I might misunderstand what you mean here; I don't think this is
true, unless the sample scatters a lot. The C. elegans embryo shell
scatters, but not much. I don't think this explains my experience.

Intuitively at least (and I could be wrong), I would
> expect that it would be very difficult to read the same level of
> contrast/power that a point scanning system achieves.
>

Check out Sup Note 1 from that link above; it seemed to me we had plenty of
power, mostly because of how ridiculously powerful the Chameleon is. We
could turn up the power high enough to bleach quantum dots.

An off-list friend directed me to this paper:
http://www.ncbi.nlm.nih.gov/pubmed/15884061
If I'm reading it right, the paper suggests that 2p illumination bleaches
rhodamine about 18x faster than 1p, for the same output signal rates. If
this similarly true for GFP, it lines up with my suspicions, and explains
my experience. If generally true, it would also be pretty important for
everyone else using 2p illumination! Anyone know of similar work on GFP?

-Andy

Michael Giacomelli

Re: High speed spinning disc confocal with EMCCD camera - commercial response

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To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
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>The degree of axial confinement is independent of the power; I guess you
mean you have to overfill the objective BFP pretty hard to get good
sectioning, wasting power?

What I was thinking was that there was a loss of spatial confinement
associated with TF and so you would need more average power to get the
same peak power, but looking online, I think I was confusing the
widefield and linescan equations.

Mike

On Fri, Jan 9, 2015 at 5:48 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.
> *****
>
>>
>> Its hard for me to say since I don't have any experience with temporal
>> focusing, but I wonder if part of the problems you observed were related to
>> that technique?
>
>
> Temporal focus is just fast passive scanning, like the lavision technique
> but with shoulder-to-shoulder spots. The typical diffraction-grating
> implementation is equivalent to splitting your beam (or line focus) into a
> bunch of lines (or spots) and delaying each one a different amount.
>
>
>> From what I've read, getting tight axial confinement is
>> fairly difficult often requiring relatively high power or a low rep rate
>> laser for a given level of 2p excitation.
>
>
> The degree of axial confinement is independent of the power; I guess you
> mean you have to overfill the objective BFP pretty hard to get good
> sectioning, wasting power? Line scanning temporal focusing should give
> sectioning equivalent to point-scanning 2p, and that's what we measured (
> http://www.nature.com/nmeth/journal/v8/n4/full/nmeth.1571.html ,
> http://www.ncbi.nlm.nih.gov/pubmed/21317909 Fig 1c, Sup Fig 1, and Sup Note
> 1). It's lots of 2p power compared to point scanning because the scanning
> is so much faster, but the required power nicely matches the output power
> of the Coherent Chameleon.
>
>
>> Furthermore, if you're doing
>> parallel detection, you tend to have less efficient light collection,
>
>
> I think I might misunderstand what you mean here; I don't think this is
> true, unless the sample scatters a lot. The C. elegans embryo shell
> scatters, but not much. I don't think this explains my experience.
>
> Intuitively at least (and I could be wrong), I would
>> expect that it would be very difficult to read the same level of
>> contrast/power that a point scanning system achieves.
>>
>
> Check out Sup Note 1 from that link above; it seemed to me we had plenty of
> power, mostly because of how ridiculously powerful the Chameleon is. We
> could turn up the power high enough to bleach quantum dots.
>
> An off-list friend directed me to this paper:
> http://www.ncbi.nlm.nih.gov/pubmed/15884061
> If I'm reading it right, the paper suggests that 2p illumination bleaches
> rhodamine about 18x faster than 1p, for the same output signal rates. If
> this similarly true for GFP, it lines up with my suspicions, and explains
> my experience. If generally true, it would also be pretty important for
> everyone else using 2p illumination! Anyone know of similar work on GFP?
>
> -Andy

Zdenek Svindrych

Re: High speed spinning disc confocal with EMCCD camera

In reply to this post by Andrew York

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Hi Andrew,
I don't know what exactly you mean by 'passive scanning' but my
understanding of the technique (see e.g. http://www.opticsinfobase.org/oe/
abstract.cfm?uri=oe-21-10-12951) is that it's a widefield two-photon
excitation which is *magically* confined to single plane. So there is no
scanning required to get a single plane. On the other hand the lavision
scanners, either the cloud scanner or the 64 point scanner, require quite
active scanning to get a useful 2D image.

The *magic* is a chromatic spreading of the beam. As long as the beam is
spread it may not cause efficient 2p excitation, because individual 'colors'
of the IR pulse are necessarily stretched in time. Only in the focus the
chromatic spreading vanishes, this helps to gain (some) optical sectioning.

Anyway, this method (as well as 2p spinning disc, http://www.pnas.org/
content/110/9/3399.full.pdf) is plagued by the low efficiency of the wide-
area 2p excitation, that is 1) the 3 W output of Chameleon is not enough to
get a decent FOV; 2) the 1p absorption of IR pulses starts to be a serious
problem.

Btw, for single-point scanning microscope, the Chameleon is really too
powerful. Have you tried 100% laser power? In my hands (bear in mind the
actual power depends in a strongly nonlinear manner on the scope settings):

30% - ablates the coverslip near the glass-water interface, lots of
microscopic glass chips all around the sample; accompanied by a nice 'cool
white' plasma emission.

10% - bleaches almost everything almost instantly (without getting too many
useful photons).

2% - good for fluorescent proteins but definitely not good for continuous
live cells/embryos imaging; sometimes signs of overheating are observed (air
bubbles, denatured structures, burst cells).
less than 1% - that's what we mostly use for lifetime imaging and sensitive
stuff, requires patience! Still I'm not confident the cells feel good.

Bottom line (does anyone remember the original topic?): I don't believe you
can go high speed with 2p excitation, no matter what degree of
parallelization you choose.

--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, USA
http://www.kcci.virginia.edu/workshop/index.php

---------- Původní zpráva ----------
Od: Andrew York <[hidden email]>
Komu: [hidden email]
Datum: 9. 1. 2015 18:24:56
Předmět: Re: High speed spinning disc confocal with EMCCD camera -
commercial response

"*****
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>
> Its hard for me to say since I don't have any experience with temporal
> focusing, but I wonder if part of the problems you observed were related
to
> that technique?

Temporal focus is just fast passive scanning, like the lavision technique
but with shoulder-to-shoulder spots. The typical diffraction-grating
implementation is equivalent to splitting your beam (or line focus) into a
bunch of lines (or spots) and delaying each one a different amount.

> From what I've read, getting tight axial confinement is
> fairly difficult often requiring relatively high power or a low rep rate
> laser for a given level of 2p excitation.

The degree of axial confinement is independent of the power; I guess you
mean you have to overfill the objective BFP pretty hard to get good
sectioning, wasting power? Line scanning temporal focusing should give
sectioning equivalent to point-scanning 2p, and that's what we measured (
http://www.nature.com/nmeth/journal/v8/n4/full/nmeth.1571.html ,
http://www.ncbi.nlm.nih.gov/pubmed/21317909 Fig 1c, Sup Fig 1, and Sup Note
1). It's lots of 2p power compared to point scanning because the scanning
is so much faster, but the required power nicely matches the output power
of the Coherent Chameleon.

> Furthermore, if you're doing
> parallel detection, you tend to have less efficient light collection,

I think I might misunderstand what you mean here; I don't think this is
true, unless the sample scatters a lot. The C. elegans embryo shell
scatters, but not much. I don't think this explains my experience.

Intuitively at least (and I could be wrong), I would
> expect that it would be very difficult to read the same level of
> contrast/power that a point scanning system achieves.
>

Check out Sup Note 1 from that link above; it seemed to me we had plenty of
power, mostly because of how ridiculously powerful the Chameleon is. We
could turn up the power high enough to bleach quantum dots.

An off-list friend directed me to this paper:
http://www.ncbi.nlm.nih.gov/pubmed/15884061
If I'm reading it right, the paper suggests that 2p illumination bleaches
rhodamine about 18x faster than 1p, for the same output signal rates. If
this similarly true for GFP, it lines up with my suspicions, and explains
my experience. If generally true, it would also be pretty important for
everyone else using 2p illumination! Anyone know of similar work on GFP?

-Andy"

Andrew York

Re: High speed spinning disc confocal with EMCCD camera

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>
> I don't know what exactly you mean by 'passive scanning' but my
> understanding of the technique (see e.g. http://www.opticsinfobase.org/oe/
> abstract.cfm?uri=oe-21-10-12951
> <http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-21-10-12951>) is
> that it's a widefield two-photon
> excitation which is *magically* confined to single plane.

The traditional Fourier-domain explanation of temporal focusing is correct
but opaque and misleading. I guess no one's published the clear, simple
time/space domain explanation?

Imagine your femtosecond pulse as a pancake of light, flying along in the
direction perpendicular to its length. It hits a reflective stairstep, like
this:
http://goo.gl/JNrBA0
and your pancake gets chopped into a bunch of "french fries". Each
french-fry spreads out in the lateral (x) direction, (since it's too
narrow to be collimated), but returns to a tight line focus if you image
the face of the grating into the sample (with large demagnification). Since
each "fry" forms an independent line-focus, at a slightly different time,
you get the same sectioning as line-focused 2p. I like this description
because it makes it really clear where the sectioning comes from; lateral
spreading of independent lines, not really spreading in time at all.

You can interpret this lateral spreading as the pulse getting longer, as
it propagates away from the grating, and shorter as it returns to an image
of the grating face, because any given GFP molecule will get hit by just
one "fry" when they're in focus, but by many "fries" one after another if
it's out of the focal plane and the fries have widened out in x. This
explanation is not wrong; it's just confusing, since none of the individual
chunks of energy (the fries) spreads out in time, at all.

A grating is equivalent to a stairstep in the limit where the step width
gets really small:
http://goo.gl/bTXDQf

This resolves the apparent paradox "how can a pulse get longer or shorter
as it propagates, if it's all going at the speed of light? How does some of
the light manage to lag behind, but catch up later?". The pulse isn't
getting longer or shorter; you're just (effectively) scanning a line-focus
through the sample on a picosecond time scale, as fast as one can possibly
scan.

Hopefully this clarifies what I meant by "fast, passive scanning".

> So there is no
> scanning required to get a single plane. On the other hand the lavision
> scanners, either the cloud scanner or the 64 point scanner, require quite
> active scanning to get a useful 2D image.
>

There's no scanning required, as long as you're ok with the same (lousy)
sectioning as a line-scanning 2p scope. In my experience it's better to put
a line focus on your grating, so you get sectioning equivalent to
point-focused illumination in your sample. Instead of a pancake getting
chopped into french fries, you now have a french fry getting chopped into
hash browns.

In this case, you have to mechanically scan your line focus up and down on
the grating, but multi-kHz 1-D scanning is easy. This has the added benefit
that a line focus gives about the right degree of parallelization for 2p;
if you spread out more, you run out of power and heat too much. If you
spread out less, you can't use all the power available in modern 2p lasers.

> The *magic* is a chromatic spreading of the beam. As long as the beam is
> spread it may not cause efficient 2p excitation, because individual
> 'colors'
> of the IR pulse are necessarily stretched in time. Only in the focus the
> chromatic spreading vanishes, this helps to gain (some) optical sectioning.
>

Totally agree, although the time/space interpretation fits more naturally
into my brain than the Fourier explanation. On the other hand, the Fourier
picture is what lead me to start thinking about temporal focusing in the
first place, but I had no contact with biologists back then, and no idea it
might be useful for microscopy until Silberberg and Xu published their work.

Anyway, this method (as well as 2p spinning disc, http://www.pnas.org/
> content/110/9/3399.full.pdf
> <http://www.pnas.org/content/110/9/3399.full.pdf>) is plagued by the low
> efficiency of the wide-
> area 2p excitation, that is 1) the 3 W output of Chameleon is not enough to
> get a decent FOV; 2) the 1p absorption of IR pulses starts to be a serious
> problem.
>

Totally agree, similar to our experience here:
http://www.pnas.org/content/111/14/5254
However, the same criticism does not apply to line-scanning temporal
focusing like we used here:
http://www.nature.com/nmeth/journal/v8/n4/full/nmeth.1571.html

> Btw, for single-point scanning microscope, the Chameleon is really too
> powerful. Have you tried 100% laser power? In my hands (bear in mind the
> actual power depends in a strongly nonlinear manner on the scope
> settings):
>
> 30% - ablates the coverslip near the glass-water interface, lots of
> microscopic glass chips all around the sample; accompanied by a nice 'cool
> white' plasma emission.
>
> 10% - bleaches almost everything almost instantly (without getting too many
> useful photons).
>
> 2% - good for fluorescent proteins but definitely not good for continuous
> live cells/embryos imaging; sometimes signs of overheating are observed
> (air
> bubbles, denatured structures, burst cells).
> less than 1% - that's what we mostly use for lifetime imaging and sensitive
> stuff, requires patience! Still I'm not confident the cells feel good.
>

Wow, that's crazy! I haven't done much single-point scanning 2p microscopy,
but it sounds like everyone should team up, buy just one Chameleon, and
split it between a few hundred point-scanning 2p microscopes. I guess two,
if we want multiple colors.

Bottom line (does anyone remember the original topic?): I don't believe you
> can go high speed with 2p excitation, no matter what degree of
> parallelization you choose.
>

Yeah, this has been my experience. I think line-scanning temporal focusing
is the best way to go for fast 2p (or the lavision approach, I have no
experience with that), but if my suspicions are right and 2p really
bleaches GFP faster than 1p for the same signal levels (
http://www.ncbi.nlm.nih.gov/pubmed/15884061 ), 2p is a tough sell.

Peter Rupprecht

Re: High speed spinning disc confocal with EMCCD camera

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>> The traditional Fourier-domain explanation of temporal focusing is correct but opaque and misleading. I guess no one's published the clear, simple time/space domain explanation?

At least in this paper : http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-19-6-4937 (page 4 of the pdf), there is an expression for the "scan speed" of the temporal focused beam. But I think that this speed is way too fast to be resolved by any detector for most cases.

Peter

Zdenek Svindrych-2

Re: High speed spinning disc confocal with EMCCD camera - OFF TOPIC

In reply to this post by Andrew York

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Dear Andrew,
thank you very much for the real space explanation and the nice drawings.
That clarified some aspects of time focusing a lot (while other aspect are
much more blurry now; it's still the same Fourier duality problem: once I've
managed to focus my ideas in real space, in the other space I'm lost...). I
didn't realize that the simple (phase) wavefront approach is helpful only
when the coherence length is large compared to path differences; short
pulses are clearly not the case.

As you've stated, some spreading in the back focal plane is necessary to get
narrow 'chips' in the sample plane. Incidentally, this spreading is
chromatic, i.e. you should see a thin rainbow line across your BFP in the
standard time focus setup (thought you may expect rather dull colors from
your Ti:S laser). Your line scan approach spreads the rainbow in the
perpendicular direction, filling the whole BFP. So it clearly must be
superior :-).
I agreee it is a scanning technique, you can achieve several tens of
picoseconds delay across your field of view. Very interesting.

Best, zdenek
--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, USA
http://www.kcci.virginia.edu/workshop/index.php

---------- Původní zpráva ----------
Od: Andrew York <[hidden email]>
Komu: [hidden email]
Datum: 10. 1. 2015 13:46:41
Předmět: Re: High speed spinning disc confocal with EMCCD camera

"*****
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The traditional Fourier-domain explanation of temporal focusing is correct
but opaque and misleading. I guess no one's published the clear, simple
time/space domain explanation?

Imagine your femtosecond pulse as a pancake of light, flying along in the
direction perpendicular to its length. It hits a reflective stairstep, like
this:
http://goo.gl/JNrBA0
and your pancake gets chopped into a bunch of "french fries". Each
french-fry spreads out in the lateral (x) direction, (since it's too
narrow to be collimated), but returns to a tight line focus if you image
the face of the grating into the sample (with large demagnification). Since
each "fry" forms an independent line-focus, at a slightly different time,
you get the same sectioning as line-focused 2p. I like this description
because it makes it really clear where the sectioning comes from; lateral
spreading of independent lines, not really spreading in time at all.

You can interpret this lateral spreading as the pulse getting longer, as
it propagates away from the grating, and shorter as it returns to an image
of the grating face, because any given GFP molecule will get hit by just
one "fry" when they're in focus, but by many "fries" one after another if
it's out of the focal plane and the fries have widened out in x. This
explanation is not wrong; it's just confusing, since none of the individual
chunks of energy (the fries) spreads out in time, at all.

A grating is equivalent to a stairstep in the limit where the step width
gets really small:
http://goo.gl/bTXDQf

This resolves the apparent paradox "how can a pulse get longer or shorter
as it propagates, if it's all going at the speed of light? How does some of
the light manage to lag behind, but catch up later?". The pulse isn't
getting longer or shorter; you're just (effectively) scanning a line-focus
through the sample on a picosecond time scale, as fast as one can possibly
scan.

Hopefully this clarifies what I meant by "fast, passive scanning".

There's no scanning required, as long as you're ok with the same (lousy)
sectioning as a line-scanning 2p scope. In my experience it's better to put
a line focus on your grating, so you get sectioning equivalent to
point-focused illumination in your sample. Instead of a pancake getting
chopped into french fries, you now have a french fry getting chopped into
hash browns.

In this case, you have to mechanically scan your line focus up and down on
the grating, but multi-kHz 1-D scanning is easy. This has the added benefit
that a line focus gives about the right degree of parallelization for 2p;
if you spread out more, you run out of power and heat too much. If you
spread out less, you can't use all the power available in modern 2p lasers.

Totally agree, although the time/space interpretation fits more naturally
into my brain than the Fourier explanation. On the other hand, the Fourier
picture is what lead me to start thinking about temporal focusing in the
first place, but I had no contact with biologists back then, and no idea it
might be useful for microscopy until Silberberg and Xu published their work.

Totally agree, similar to our experience here:
http://www.pnas.org/content/111/14/5254
However, the same criticism does not apply to line-scanning temporal
focusing like we used here:
http://www.nature.com/nmeth/journal/v8/n4/full/nmeth.1571.html

Wow, that's crazy! I haven't done much single-point scanning 2p microscopy,
but it sounds like everyone should team up, buy just one Chameleon, and
split it between a few hundred point-scanning 2p microscopes. I guess two,
if we want multiple colors.

Yeah, this has been my experience. I think line-scanning temporal focusing
is the best way to go for fast 2p (or the lavision approach, I have no
experience with that), but if my suspicions are right and 2p really
bleaches GFP faster than 1p for the same signal levels (
http://www.ncbi.nlm.nih.gov/pubmed/15884061 ), 2p is a tough sell."

James Pawley

Re: High speed spinning disc confocal with EMCCD camera

In reply to this post by John Oreopoulos

>*****
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>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>But Jim,
>
>2-photon light sheet microscopy has been demonstrated a few times now:
>
>http://www.nature.com/nmeth/journal/v8/n9/full/nmeth.1652.html
>
>http://www.nature.com/nmeth/journal/v11/n6/full/nmeth.2963.html
>
>http://www.nature.com/cr/journal/vaop/ncurrent/full/cr2014124a.html
>
>John Oreopoulos

Thank you for this.

I don't have access to all these refs but the one
I do know about employs a SCANNING light sheet.
This drops the volume excited at any time by a
factor equal to the number of lines. In addition,
the specimens were very highly stained and the
scanned "light sheet" seems likely (from the NA
and wavelength) to be very thick (4µm?), two
factors that allow you get a large signal from
large objects like nuclei with even a very low
level of excitation (and hence low bleaching and
toxicity?). The low probability of any one
molecule being excited probably also helps avoid
the "2P bleaching penalty" shown by Piston and
others with single-beam instruments at excitation
rates closer to singlet saturation. Although I
believe that it is only surmise, this penalty is
thought to be caused by "one-plus-one"
excitations (or in the 2-photon case "two plus
one" or maybe "two-plus-two") in which the
already excited molecule is further excited by
the acquisition of the energy from an additional
photon before it can decay. Clearly such a
mechanism would be less likely to be important
when working at excitation levels that only
excite one molecule in a hundred, than those that
require each molecule to be excited many times
during the course of a single pixel.

But I concede that the 2-P LASM results on
embryos are exactly what is needed. Well done CIT!

Jim Pawley

>
>On 2015-01-08, at 10:41 PM, James Pawley 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 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
>>
>>> *****
>>> 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
>>>> >>>>
>>>> >>>
>>>> >>
>>>>
>>
>>
>> --
>> ****************************************
>> 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

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