High speed spinning disc confocal with EMCCD camera

[James 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.
*****

>*****
>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: [phone] | 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_JSBGnaEcBqKN1nFKNqOL1R
>>ax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalmic
>>roscopy
>>Post images on
>>http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFKNqOLwF
>>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_JSBGnaEcBqKN1nFKNqOL1
>>>Rax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalm
>>>icroscopy
>>>Post images on
>>>http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFKNqOLw
>>>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_JSBGnaEcBqKN1nFKNqOL
>>>>1Rax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfoca
>>>>lmicroscopy
>>>>  Post images on
>>>>http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFKNqOL
>>>>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 [phone], email <[hidden email]>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146

[Andrew York]

*****
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.
*****

I'm guessing the z-piezo is the speed limiting factor for most systems, but
let's assume everyone's using Wilson-style remote refocusing and changing
z-planes is really fast. With tons of illumination power, I'd bet the next
speed limiting factor is signal starvation due to saturation of
fluorescence excitation, so total speed is going to be proportional to the
volume of the sample that's giving useful signal. Single-point-scanning
confocal will be slowest, widefield/SPIM (with a true sheet) will be
fastest, and parallel-point-scanning/line-scanning/SPIM (with a digitally
scanned pencil to make a sheet) will be in between. SPIM gets bonus points
since it's not generating any "bad" signal to degrade the SNR of the "good"
signal, and widefield gets a penalty in most samples due to poor sectioning
degrading in-focus SNR.

 Now let's suppose the sample is really densely tagged, so saturation isn't
limiting. I'd be surprised if scan speed were a major limit for anything
but non-resonant single-point confocal, so next important limiting factor
is probably data flow rate. Confocal, SPIM, and instant SIM-type methods
measure one pixel per voxel. Digital-postprocessing SIM-type methods
measure ~15 pixels per voxel, and Airyscan measures ~30. Typical computers
choke on much more than 1 GB/s, so this is probably the practical limit for
most techniques. I'm only experienced with sCMOS/EMCCD cameras, so I'm
assuming single-pixel detectors for confocal can spit out tons of data per
second; please let me know if I'm wrong about this.

On Sat, Jan 3, 2015 at 5:12 PM, Feinstein, Timothy <
[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 all,
>
> Does anyone think it would be possible to tabulate a ¹speed limit' for the
> various options discussed?  I know it sounds near impossible to come up
> with a standard basis for comparison, but let¹s 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: [phone] | 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_JSBGnaEcBqKN1nFKNqOL1R
> >ax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalmic
> >roscopy
> >Post images on
> >
> http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFKNqOLwF
> >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_JSBGnaEcBqKN1nFKNqOL1
> >>Rax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalm
> >>icroscopy
> >>Post images on
> >>
> http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFKNqOLw
> >>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_JSBGnaEcBqKN1nFKNqOL
> >>>1Rax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfoca
> >>>lmicroscopy
> >>> Post images on
> >>>
> http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFKNqOL
> >>>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]
> >>>
>

[Guy Cox-2]

*****
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: [phone] | 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 [phone], email <[hidden email]> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146

[sivaram mylavarapu]

*****
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.
*****

Dear colleagues,

Thank you all for your inputs over the past few weeks to my original query
regarding combinations of high-speed microscopes with high sensitivity
cameras for live cell time lapse imaging - all are highly appreciated. The
compilation suggested by Tim Feinstein here would serve as an extremely
useful source for end users including me. If the list will indeed be
compiled and could also include cameras based on a hierarchy of sensitivity
(EMCCD, sCMOS, others) to go with the illumination technologies, it would
be of great help.

Warm regards for the New Year!

Sivaram.

On Sun, Jan 4, 2015 at 3:42 AM, Feinstein, Timothy <
[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 all,
>
> Does anyone think it would be possible to tabulate a ¹speed limit' for the
> various options discussed?  I know it sounds near impossible to come up
> with a standard basis for comparison, but let¹s 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: [phone] | 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_JSBGnaEcBqKN1nFKNqOL1R
> >ax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalmic
> >roscopy
> >Post images on
> >
> http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFKNqOLwF
> >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_JSBGnaEcBqKN1nFKNqOL1
> >>Rax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalm
> >>icroscopy
> >>Post images on
> >>
> http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFKNqOLw
> >>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_JSBGnaEcBqKN1nFKNqOL
> >>>1Rax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfoca
> >>>lmicroscopy
> >>> Post images on
> >>>
> http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFKNqOL
> >>>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]
> >>>
>

--
Sivaram Mylavarapu.
[hidden email]

[Andrew York]

*****
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: [phone] | 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 [phone], email <[hidden email]> NEW! NEW!
> AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
>

[Guy Cox-2]

Look, I'm not trying to sell any solution, I'm just contributing to the discussion.  Of course the 2P cross-section is smaller - 2P would not be depth-selective and (moderately) super-resolution if it were not.  But IR is MUCH less damaging to living tissue than UV/blue.  As to photo-bleaching, I think you are wrong BUT you can be right!  Years ago, at a conference in Taiwan, a speaker presented data that FITC bleached much more rapidly in 2P then in 1P.  The problem was he hadn't considered the photochemistry.  Fluorescein is a symmetrical molecule, and as such 2P excitation cannot occur at half the 1P frequency.  The excitation curves had been published, but he hadn't looked at them.  If he'd excited at the (published) 2P maximum he would not have found this.  Exciting ANY fluorochrome to above the S1 level will naturally increase bleaching.  

Fluorescence lifetimes of common fluorochromes range from 1.1ns (eosin) to 3.41ns (Texas Red).  So your have a point, but it is not tenfold.  

Not being able to saturate excitation is a good thing, not a bad thing!  I am perpetually telling people to reduce their excitation to 50% and check that their excitation decreases by the same amount - if not they are saturating (and destroying) their fluorochrome.  Nobody ever follows my advice - they just want a bright (and usually saturated and therefore uninformative) image.

                                   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 Andrew York
Sent: Monday, 5 January 2015 5:15 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.
*****

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: [phone] | 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_JSBGnaEcBqKN1nFK
> >>Nq
> >>OL1R
> >>ax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfo
> >>ca
> >>lmic
> >>roscopy
> >>Post images on
> >>http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nFK
> >>Nq 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_JSBGnaEcBqKN1nF
> >>>KN
> >>>qOL1
> >>>Rax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dcon
> >>>fo
> >>>calm
> >>>icroscopy
> >>>Post images on
> >>>http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1nF
> >>>KN 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_JSBGnaEcBqKN1n
> >>>>FK
> >>>>NqOL
> >>>>1Rax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dc
> >>>>on
> >>>>foca
> >>>>lmicroscopy
> >>>>  Post images on
> >>>>http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1n
> >>>>FK 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 [phone], email <[hidden email]> NEW! NEW!
> AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
>

[Guy Cox-2]

Michael,

         Both you and I have been telling users this for years.  Likewise, if they want to image a mitochondrion, they gain nothing by going beyond Nyquist, yet they still zoom it up to 1024 x 768.  These people are supposed to be scientists - but are they?  A microscope is a scientific instrument, not a black box.

                              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: Cammer, Michael [mailto:[hidden email]]
Sent: Tuesday, 6 January 2015 2:57 AM
To: Guy Cox
Subject: RE: High speed spinning disc confocal with EMCCD camera - commercial response

I constantly tell people doing live imaging with the multiphoton who want to track cells to turn down the excitation and do running averages or other noise filtering later.  I show them examples but when I leave the room they invariably crank up the light because they want to see a perfect  low noise high contrast image right now and then they are disappointed when the experiment doesn't work either because the sample bleaches or the cells stop moving.
=========================================================================
 Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
                          Cell:  914-309-3270     Temporary location:  SK2-7
          http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Monday, January 05, 2015 10:45 AM
To: [hidden email]
Subject: Re: High speed spinning disc confocal with EMCCD camera - commercial response


Not being able to saturate excitation is a good thing, not a bad thing!  I am perpetually telling people to reduce their excitation to 50% and check that their excitation decreases by the same amount - if not they are saturating (and destroying) their fluorochrome.  Nobody ever follows my advice - they just want a bright (and usually saturated and therefore uninformative) image.

                                   Guy

 
Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis, Madsen, F09, University of Sydney, NSW 2006

[George McNamara]

*****
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 Guy,

Nyquist is dead.

For mitochondria way beyond, see Shim et al 2012,
http://www.ncbi.nlm.nih.gov/pubmed/22891300
http://www.pnas.org/content/109/35/13978.long   (open access)

also several papers from Hell and colleagues, such as;

http://www.ncbi.nlm.nih.gov/pubmed/20205711
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838807/pdf/1757-5036-3-4.pdf

and
30 nm isotropic resolution (isoSTED)
http://www.ncbi.nlm.nih.gov/pubmed/19459703

George
p.s. http://en.wikipedia.org/wiki/Harry_Nyquist
*Harry Nyquist* (/né/ Harry Theodor Nyqvist; /
<http://en.wikipedia.org/wiki/Help:IPA_for_English>ˈ
<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>n
<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>aɪ
<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>k
<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>w
<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>ɪ
<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>s
<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>t
<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>/
<http://en.wikipedia.org/wiki/Help:IPA_for_English>, Swedish: [nʏːkvɪst]
<http://en.wikipedia.org/wiki/Help:IPA_for_Swedish_and_Norwegian>;
February 7, 1889 – April 4, 1976)

On 1/5/2015 10:10 AM, Guy Cox wrote:

> Michael,
>
>           Both you and I have been telling users this for years.  Likewise, if they want to image a mitochondrion, they gain nothing by going beyond Nyquist, yet they still zoom it up to 1024 x 768.  These people are supposed to be scientists - but are they?  A microscope is a scientific instrument, not a black box.
>
>                                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: Cammer, Michael [mailto:[hidden email]]
> Sent: Tuesday, 6 January 2015 2:57 AM
> To: Guy Cox
> Subject: RE: High speed spinning disc confocal with EMCCD camera - commercial response
>
> I constantly tell people doing live imaging with the multiphoton who want to track cells to turn down the excitation and do running averages or other noise filtering later.  I show them examples but when I leave the room they invariably crank up the light because they want to see a perfect  low noise high contrast image right now and then they are disappointed when the experiment doesn't work either because the sample bleaches or the cells stop moving.
> =========================================================================
>   Michael Cammer, Microscopy Core&  Skirball Institute, NYU Langone Medical Center
>                            Cell:  914-309-3270     Temporary location:  SK2-7
>            http://ocs.med.nyu.edu/microscopy&  http://microscopynotes.com/
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
> Sent: Monday, January 05, 2015 10:45 AM
> To: [hidden email]
> Subject: Re: High speed spinning disc confocal with EMCCD camera - commercial response
>
>
> Not being able to saturate excitation is a good thing, not a bad thing!  I am perpetually telling people to reduce their excitation to 50% and check that their excitation decreases by the same amount - if not they are saturating (and destroying) their fluorochrome.  Nobody ever follows my advice - they just want a bright (and usually saturated and therefore uninformative) image.
>
>                                     Guy
>
>
> Guy Cox, Honorary Associate Professor
> School of Medical Sciences
>
> Australian Centre for Microscopy and Microanalysis, Madsen, F09, University of Sydney, NSW 2006
>
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42

[Guy Cox-2]

Hang on, you think I don’t know this??  Huh??  That wasn’t what I was talking about.

                                                     Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006

From: George McNamara [mailto:[hidden email]]
Sent: Tuesday, 6 January 2015 4:28 PM
To: Confocal Microscopy List
Cc: Guy Cox
Subject: Re: High speed spinning disc confocal with EMCCD camera - commercial response

Hi Guy,

Nyquist is dead.

For mitochondria way beyond, see Shim et al 2012,
http://www.ncbi.nlm.nih.gov/pubmed/22891300
http://www.pnas.org/content/109/35/13978.long   (open access)

also several papers from Hell and colleagues, such as;

http://www.ncbi.nlm.nih.gov/pubmed/20205711
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838807/pdf/1757-5036-3-4.pdf

and
30 nm isotropic resolution (isoSTED)
http://www.ncbi.nlm.nih.gov/pubmed/19459703

George
p.s. http://en.wikipedia.org/wiki/Harry_Nyquist
Harry Nyquist (né Harry Theodor Nyqvist; /<http://en.wikipedia.org/wiki/Help:IPA_for_English>ˈ<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>n<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>aɪ<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>k<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>w<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>ɪ<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>s<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>t<http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>/<http://en.wikipedia.org/wiki/Help:IPA_for_English>, Swedish: [nʏːkvɪst]<http://en.wikipedia.org/wiki/Help:IPA_for_Swedish_and_Norwegian>; February 7, 1889 – April 4, 1976)

On 1/5/2015 10:10 AM, Guy Cox wrote:

Michael,



         Both you and I have been telling users this for years.  Likewise, if they want to image a mitochondrion, they gain nothing by going beyond Nyquist, yet they still zoom it up to 1024 x 768.  These people are supposed to be scientists - but are they?  A microscope is a scientific instrument, not a black box.



                              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: Cammer, Michael [mailto:[hidden email]]

Sent: Tuesday, 6 January 2015 2:57 AM

To: Guy Cox

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



I constantly tell people doing live imaging with the multiphoton who want to track cells to turn down the excitation and do running averages or other noise filtering later.  I show them examples but when I leave the room they invariably crank up the light because they want to see a perfect  low noise high contrast image right now and then they are disappointed when the experiment doesn't work either because the sample bleaches or the cells stop moving.

=========================================================================

 Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center

                          Cell:  914-309-3270     Temporary location:  SK2-7

          http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/





-----Original Message-----

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox

Sent: Monday, January 05, 2015 10:45 AM

To: [hidden email]<mailto:[hidden email]>

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





Not being able to saturate excitation is a good thing, not a bad thing!  I am perpetually telling people to reduce their excitation to 50% and check that their excitation decreases by the same amount - if not they are saturating (and destroying) their fluorochrome.  Nobody ever follows my advice - they just want a bright (and usually saturated and therefore uninformative) image.



                                   Guy





Guy Cox, Honorary Associate Professor

School of Medical Sciences



Australian Centre for Microscopy and Microanalysis, Madsen, F09, University of Sydney, NSW 2006










--







George McNamara, Ph.D.

Single Cells Analyst

L.J.N. Cooper Lab

University of Texas M.D. Anderson Cancer Center

Houston, TX 77054

Tattletales http://works.bepress.com/gmcnamara/42

[Michael Giacomelli]

*****
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 George,

You probably mean "diffraction" rather than "Nyquist".  The Nyquist limit
applies even to superresolution systems, whereas the diffraction limit does
not.  Guy's point is that one only needs to sample densely enough to
satisfy the Nyquist critiera (for a diffraction or subdiffraction) system.
Going further helps SNR (it is really just a form of averaging) but does
nothing for resolution.

Mike

On Tue, Jan 6, 2015 at 12:28 AM, George McNamara <[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 Guy,
>
> Nyquist is dead.
>
> For mitochondria way beyond, see Shim et al 2012,
> http://www.ncbi.nlm.nih.gov/pubmed/22891300
> http://www.pnas.org/content/109/35/13978.long   (open access)
>
> also several papers from Hell and colleagues, such as;
>
> http://www.ncbi.nlm.nih.gov/pubmed/20205711
> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838807/pdf/1757-5036-3-4.pdf
>
> and
> 30 nm isotropic resolution (isoSTED)
> http://www.ncbi.nlm.nih.gov/pubmed/19459703
>
> George
> p.s. http://en.wikipedia.org/wiki/Harry_Nyquist
> *Harry Nyquist* (/né/ Harry Theodor Nyqvist; / <
> http://en.wikipedia.org/wiki/Help:IPA_for_English>ˈ <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>n <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>aɪ <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>k <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>w <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>ɪ <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>s <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>t <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>/ <
> http://en.wikipedia.org/wiki/Help:IPA_for_English>, Swedish: [nʏːkvɪst] <
> http://en.wikipedia.org/wiki/Help:IPA_for_Swedish_and_Norwegian>;
> February 7, 1889 – April 4, 1976)
>
> On 1/5/2015 10:10 AM, Guy Cox wrote:
>
>> Michael,
>>
>>           Both you and I have been telling users this for years.
>> Likewise, if they want to image a mitochondrion, they gain nothing by going
>> beyond Nyquist, yet they still zoom it up to 1024 x 768.  These people are
>> supposed to be scientists - but are they?  A microscope is a scientific
>> instrument, not a black box.
>>
>>                                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: Cammer, Michael [mailto:[hidden email]]
>> Sent: Tuesday, 6 January 2015 2:57 AM
>> To: Guy Cox
>> Subject: RE: High speed spinning disc confocal with EMCCD camera -
>> commercial response
>>
>> I constantly tell people doing live imaging with the multiphoton who want
>> to track cells to turn down the excitation and do running averages or other
>> noise filtering later.  I show them examples but when I leave the room they
>> invariably crank up the light because they want to see a perfect  low noise
>> high contrast image right now and then they are disappointed when the
>> experiment doesn't work either because the sample bleaches or the cells
>> stop moving.
>> =========================================================================
>>   Michael Cammer, Microscopy Core&  Skirball Institute, NYU Langone
>> Medical Center
>>                            Cell:  914-309-3270     Temporary location:
>> SK2-7
>>            http://ocs.med.nyu.edu/microscopy&
>> http://microscopynotes.com/
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Guy Cox
>> Sent: Monday, January 05, 2015 10:45 AM
>> To: [hidden email]
>> Subject: Re: High speed spinning disc confocal with EMCCD camera -
>> commercial response
>>
>>
>> Not being able to saturate excitation is a good thing, not a bad thing!
>> I am perpetually telling people to reduce their excitation to 50% and check
>> that their excitation decreases by the same amount - if not they are
>> saturating (and destroying) their fluorochrome.  Nobody ever follows my
>> advice - they just want a bright (and usually saturated and therefore
>> uninformative) image.
>>
>>                                     Guy
>>
>>
>> Guy Cox, Honorary Associate Professor
>> School of Medical Sciences
>>
>> Australian Centre for Microscopy and Microanalysis, Madsen, F09,
>> University of Sydney, NSW 2006
>>
>>
>>
>>
>
>
> --
>
>
>
> George McNamara, Ph.D.
> Single Cells Analyst
> L.J.N. Cooper Lab
> University of Texas M.D. Anderson Cancer Center
> Houston, TX 77054
> Tattletales http://works.bepress.com/gmcnamara/42
>

[Michael Giacomelli]

*****
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: [phone] | 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 [phone], email <[hidden email]> NEW! NEW!
> > AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
> >
>

[Andrew York]

*****
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: [phone] | 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 [phone], email <[hidden email]> NEW!
> NEW!
> > > AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
> > >
> >
>

[Andrea Latini]

*****
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.
*****

hello Mike, All
being working on R2PI technique (Resonant Two Photons Inonization), for long time, I'm
wondering on how much the single phon absorption is taking place compared to the
two photons one.
in other words, do you have any estimate for the 1phothon/2photons cross section
ratio?

thanks.

Andrea
CrestOptics
www.crestoptics.com



On Tue, 6 Jan 2015 15:16:22 -0500, 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: [phone] | 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 [phone], email <[hidden email]> NEW! NEW!
>> > AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
>> >
>>

[John Oreopoulos]

*****
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: [phone] | 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 [phone], email <[hidden email]> NEW!
>> NEW!
>>>> AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
>>>>
>>>
>>

[Guy Cox-2]

*****
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.
*****

According to my photochemist colleague Scott Kable the difference is ~ 10 orders of magnitude.  

                                        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 Andrea Latini
Sent: Wednesday, 7 January 2015 9:12 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.
*****

hello Mike, All
being working on R2PI technique (Resonant Two Photons Inonization), for long time, I'm wondering on how much the single phon absorption is taking place compared to the two photons one.
in other words, do you have any estimate for the 1phothon/2photons cross section ratio?

thanks.

Andrea
CrestOptics
www.crestoptics.com



On Tue, 6 Jan 2015 15:16:22 -0500, 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: [phone] | Email: [hidden email]
>> > >
>> > >
>> > >
>> > >
>> > >
>> > >
>> > >
>> > >On 12/30/14, 2:36 AM, "Andrea Latini" <[hidden email]> wrote:
>> > >
>> > >>*****
>> > >>To join, leave or search the confocal microscopy listserv, go to:
>> > >>http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1
>> > >>nFKNq
>> > >>OL1R
>> > >>ax-qpJw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-

bin%2fwa%3fA0%3dconfoca

>> > >>lmic
>> > >>roscopy
>> > >>Post images on
>> > >>http://scanmail.trustwave.com/?c=129&d=09ai1Hwf389A_JSBGnaEcBqKN1
>> > >>nFKNq 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_JSBGnaEcBqKN
>> > >>>1nFKN
>> > >>>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_JSBGnaEcBqKN
>> > >>>1nFKN 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_JSBGnaEcBqK
>> > >>>>N1nFK
>> > >>>>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_JSBGnaEcBqK
>> > >>>>N1nFK 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 [phone], email <[hidden email]> NEW! NEW!
>> > AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
>> >
>>

[Andreas Bruckbauer]

*****
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,
This has gone slightly off topic now, but Nyquist might be the next barrier to fall, see compressed sensing which has already been used in microscopy and I think looks very promising:

Faster STORM using compressed sensing.
Zhu L, Zhang W, Elnatan D, Huang B.
Nat Methods. 2012 Apr 22;9(7):721-3. doi: 10.1038/nmeth.1978.

Single-shot compressed ultrafast photography at one hundred billion frames per second
Liang Gao,
 Jinyang Liang,
 Chiye Li
 & Lihong V. Wang
Nature  516, 74–77 (04 December 2014)

Andreas

-----Original Message-----
From: "Michael Giacomelli" <[hidden email]>
Sent: ‎06/‎01/‎2015 19:47
To: "[hidden email]" <[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.
*****

Hi George,

You probably mean "diffraction" rather than "Nyquist".  The Nyquist limit
applies even to superresolution systems, whereas the diffraction limit does
not.  Guy's point is that one only needs to sample densely enough to
satisfy the Nyquist critiera (for a diffraction or subdiffraction) system.
Going further helps SNR (it is really just a form of averaging) but does
nothing for resolution.

Mike

On Tue, Jan 6, 2015 at 12:28 AM, George McNamara <[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 Guy,
>
> Nyquist is dead.
>
> For mitochondria way beyond, see Shim et al 2012,
> http://www.ncbi.nlm.nih.gov/pubmed/22891300
> http://www.pnas.org/content/109/35/13978.long   (open access)
>
> also several papers from Hell and colleagues, such as;
>
> http://www.ncbi.nlm.nih.gov/pubmed/20205711
> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838807/pdf/1757-5036-3-4.pdf
>
> and
> 30 nm isotropic resolution (isoSTED)
> http://www.ncbi.nlm.nih.gov/pubmed/19459703
>
> George
> p.s. http://en.wikipedia.org/wiki/Harry_Nyquist
> *Harry Nyquist* (/né/ Harry Theodor Nyqvist; / <
> http://en.wikipedia.org/wiki/Help:IPA_for_English>ˈ <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>n <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>aɪ <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>k <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>w <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>ɪ <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>s <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>t <
> http://en.wikipedia.org/wiki/Help:IPA_for_English#Key>/ <
> http://en.wikipedia.org/wiki/Help:IPA_for_English>, Swedish: [nʏːkvɪst] <
> http://en.wikipedia.org/wiki/Help:IPA_for_Swedish_and_Norwegian>;
> February 7, 1889 – April 4, 1976)
>
> On 1/5/2015 10:10 AM, Guy Cox wrote:
>
>> Michael,
>>
>>           Both you and I have been telling users this for years.
>> Likewise, if they want to image a mitochondrion, they gain nothing by going
>> beyond Nyquist, yet they still zoom it up to 1024 x 768.  These people are
>> supposed to be scientists - but are they?  A microscope is a scientific
>> instrument, not a black box.
>>
>>                                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: Cammer, Michael [mailto:[hidden email]]
>> Sent: Tuesday, 6 January 2015 2:57 AM
>> To: Guy Cox
>> Subject: RE: High speed spinning disc confocal with EMCCD camera -
>> commercial response
>>
>> I constantly tell people doing live imaging with the multiphoton who want
>> to track cells to turn down the excitation and do running averages or other
>> noise filtering later.  I show them examples but when I leave the room they
>> invariably crank up the light because they want to see a perfect  low noise
>> high contrast image right now and then they are disappointed when the
>> experiment doesn't work either because the sample bleaches or the cells
>> stop moving.
>> =========================================================================
>>   Michael Cammer, Microscopy Core&  Skirball Institute, NYU Langone
>> Medical Center
>>                            Cell:  914-309-3270     Temporary location:
>> SK2-7
>>            http://ocs.med.nyu.edu/microscopy&
>> http://microscopynotes.com/
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Guy Cox
>> Sent: Monday, January 05, 2015 10:45 AM
>> To: [hidden email]
>> Subject: Re: High speed spinning disc confocal with EMCCD camera -
>> commercial response
>>
>>
>> Not being able to saturate excitation is a good thing, not a bad thing!
>> I am perpetually telling people to reduce their excitation to 50% and check
>> that their excitation decreases by the same amount - if not they are
>> saturating (and destroying) their fluorochrome.  Nobody ever follows my
>> advice - they just want a bright (and usually saturated and therefore
>> uninformative) image.
>>
>>                                     Guy
>>
>>
>> Guy Cox, Honorary Associate Professor
>> School of Medical Sciences
>>
>> Australian Centre for Microscopy and Microanalysis, Madsen, F09,
>> University of Sydney, NSW 2006
>>
>>
>>
>>
>
>
> --
>
>
>
> George McNamara, Ph.D.
> Single Cells Analyst
> L.J.N. Cooper Lab
> University of Texas M.D. Anderson Cancer Center
> Houston, TX 77054
> Tattletales http://works.bepress.com/gmcnamara/42
>

[Michael Giacomelli]

*****
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,

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?  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.  Furthermore, if you're doing
parallel detection, you tend to have less efficient light collection, which
then implies that you need to excite even more fluorophore (and thus incur
more photo damage).  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.

Mike

On Tue, Jan 6, 2015 at 5:05 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.
> *****
>
>  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: [phone] | 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 [phone], email <[hidden email]> NEW!
> > NEW!
> > > > AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
> > > >
> > >
> >
>

[Andrew York]

*****
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: [phone] | 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 [phone], email <[hidden email]> NEW!
> >> NEW!
> >>>> AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
> >>>>
> >>>
> >>
>

[James 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.
*****

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: [phone] | 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 [phone], 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 [phone], email <[hidden email]>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146

[John Oreopoulos]

*****
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: [phone] | 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 [phone], 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 [phone], email <[hidden email]>
> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146