Webinar: Advanced quantitative analysis of tissue morphogenesis using Imaris and Matlab (commercial posting)

[Daniel McRitchie]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Webinar: Advanced quantitative analysis of tissue morphogenesis using Imaris
and Matlab (commercial posting)

Speaker:

Willy Suppato, PhD
Ecole Polytechnique
CNS, France

Abstract:

Multidimensional study of tissue morphogenesis tremendously benefits from
recent advances in live microscopy and computational analysis. The study of
cellular processes shaping an embryo and the control of embryonic
morphogenesis can be investigated in vivo with higher spatial and temporal
resolution, from the subcellular scale to the level of entire tissue or organism,
and with richer quantitative measurements...

Read the full abstract here - http://goo.gl/ZxSvR

Date: 28th February, 15:00 GMT (16:00 Central European/ 11:00 Eastern US)

Relevant for researchers interested in:

1. Developmental biology, image analysis and bioimaging 2. Visualization of large
3D/4D data sets 3. Brainbow, drosophila and zebrafish experimental models.

Register Here - http://goo.gl/mMVUI

[Tim Feinstein-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hello all,

There is an interesting demonstration of multiphoton spinning disc confocal imaging in the latest PNAS by Yuko Mimori-Kosue from RIKEN.  It shows the expected Z-resolution, depth and s/n improvements (maybe a little better than due to pinhole refinement), but their last paragraph is a doozy.    

"In putting the system to practical use, the available output laser power is the limiting factor for the illumination area and the maximum imaging depth. Even with the two-photon lasers having the highest levels of output power currently available, we could trigger two-photon absorption for only ∼40-μm-diameter areas of GFP-expressing animals (less than 10% of the effective frame size with a 60× objective; Fig. S1B). Lasers with roughly 5–10 times the power would be required. The higher laser power is also required to excite red fluorescent proteins, which can be excited at wavelengths between 1,000 and 1,200 nm, at which the output power of the existing mode-locked laser decreases to ∼15% of peak power. "

Is there any chance of seeing a ti-sapphire with 5 to 10-fold more power?  Would it cost 5-10 times as much?  It sounds like a nice approach but maybe not so much if you need a military LASER to use it.  On the other hand maybe resonant two-photon (or slit-scanning, with sacrifices) can already deliver similar performance.  Thoughts appreciated.  

All the best,


TF

Timothy Feinstein, PhD
Visiting Research Associate
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

[sunandod]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hello All
We are intending to set a live cell imaging set up at our department. I
am interested to carry out FRET/FLIM studies.
Would someone help me in suggesting about the camera ?

We would go for Epi set up. Does not funding for confocal....

Thank you advance
Sunando


--
Dr. Sunando Datta
Assistant Professor
Department of Biological Sciences
Indian Institute of Science Education and Research Bhopal
ITI Gas Rahat Building
Govindpura Industrial Area
Bhopal-462023
Madhya Pradesh
India

[Craig Brideau]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

If you could sacrifice tunability, fiber lasers are pretty much there, at
least for 1040nm.  Time-bandwidth-products (the company, not the unit of
merit for pulsed lasers) makes a death ray called the Fortis that outputs
50W at 50 MHz...

Craig


On Fri, Feb 15, 2013 at 9:37 AM, Tim Feinstein <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello all,
>
> There is an interesting demonstration of multiphoton spinning disc
> confocal imaging in the latest PNAS by Yuko Mimori-Kosue from RIKEN.  It
> shows the expected Z-resolution, depth and s/n improvements (maybe a little
> better than due to pinhole refinement), but their last paragraph is a doozy.
>
> "In putting the system to practical use, the available output laser power
> is the limiting factor for the illumination area and the maximum imaging
> depth. Even with the two-photon lasers having the highest levels of output
> power currently available, we could trigger two-photon absorption for only
> ∼40-μm-diameter areas of GFP-expressing animals (less than 10% of the
> effective frame size with a 60× objective; Fig. S1B). Lasers with roughly
> 5–10 times the power would be required. The higher laser power is also
> required to excite red fluorescent proteins, which can be excited at
> wavelengths between 1,000 and 1,200 nm, at which the output power of the
> existing mode-locked laser decreases to ∼15% of peak power. "
>
> Is there any chance of seeing a ti-sapphire with 5 to 10-fold more power?
>  Would it cost 5-10 times as much?  It sounds like a nice approach but
> maybe not so much if you need a military LASER to use it.  On the other
> hand maybe resonant two-photon (or slit-scanning, with sacrifices) can
> already deliver similar performance.  Thoughts appreciated.
>
> All the best,
>
>
> TF
>
> Timothy Feinstein, PhD
> Visiting Research Associate
> Laboratory for GPCR Biology
> Dept. of Pharmacology & Chemical Biology
> University of Pittsburgh, School of Medicine
> BST W1301, 200 Lothrop St.
> Pittsburgh, PA  15261
>

[Unruh, Jay]

I would imagine that quite a bit could be gained by going to a shorter pulsed laser and performing dispersion compensation.  I would guess that the micro lens array introduces a significant amount of dispersion.  Of course many of the shorter pulsed lasers aren't tunable either.  Large field of view applications with longer pulsed lasers may result in an undesired amount of heating anyway.  Del Mar ventures (no commercial interest) actually sells a DIY kit for a <20 fs laser for much less than a single box tunable system.

Jay

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Friday, February 15, 2013 11:46 AM
To: [hidden email]
Subject: Re: multiphoton spinning disc

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

If you could sacrifice tunability, fiber lasers are pretty much there, at least for 1040nm.  Time-bandwidth-products (the company, not the unit of merit for pulsed lasers) makes a death ray called the Fortis that outputs 50W at 50 MHz...

Craig


On Fri, Feb 15, 2013 at 9:37 AM, Tim Feinstein <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello all,
>
> There is an interesting demonstration of multiphoton spinning disc
> confocal imaging in the latest PNAS by Yuko Mimori-Kosue from RIKEN.  
> It shows the expected Z-resolution, depth and s/n improvements (maybe
> a little better than due to pinhole refinement), but their last paragraph is a doozy.
>
> "In putting the system to practical use, the available output laser
> power is the limiting factor for the illumination area and the maximum
> imaging depth. Even with the two-photon lasers having the highest
> levels of output power currently available, we could trigger
> two-photon absorption for only ∼40-μm-diameter areas of GFP-expressing
> animals (less than 10% of the effective frame size with a 60×
> objective; Fig. S1B). Lasers with roughly
> 5–10 times the power would be required. The higher laser power is also
> required to excite red fluorescent proteins, which can be excited at
> wavelengths between 1,000 and 1,200 nm, at which the output power of
> the existing mode-locked laser decreases to ∼15% of peak power. "
>
> Is there any chance of seeing a ti-sapphire with 5 to 10-fold more power?
>  Would it cost 5-10 times as much?  It sounds like a nice approach but
> maybe not so much if you need a military LASER to use it.  On the
> other hand maybe resonant two-photon (or slit-scanning, with
> sacrifices) can already deliver similar performance.  Thoughts appreciated.
>
> All the best,
>
>
> TF
>
> Timothy Feinstein, PhD
> Visiting Research Associate
> Laboratory for GPCR Biology
> Dept. of Pharmacology & Chemical Biology University of Pittsburgh,
> School of Medicine BST W1301, 200 Lothrop St.
> Pittsburgh, PA  15261
>

[Zdenek Svindrych]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi guys,

it is a simple matter, multiphoton is not suitable for spinning disc as it's not suitable for widefield.
It's a nonlinear process and with the same average intensity of illumination the signal will be the higher the higher is the peak intensity. To get intense peak illumination you can focus it spatially (to a diffraction-limited spot) or 'temporally' (with fs pulses); one has to use both approaches to get appreciable results with common fluorescent dyes...

Shortening the pulses is vital, but you cannot go to 1fs - the spectrum would be too broad. I don't think the microlenses could stretch the pulses, because they are fairly thin. The do destroy the wavefronts a bit (coz the are so small...), but this is cleaned up by the pinholes...

Simply put, in a regular confocal you are creating one diffraction-limited spot, in a spinning disc you work with thousand spots. So you would need thousand times more power (assuming the same pulse duration). Bu then the thermal effects (assuming the contribution of the multiphoton processes is negligible) also increase thousand times...

And now the solutions:
1) use less pinholes - you hardly can modify your disc, so you decrease FOV instead, like in that paper.
2) use better dyes - there are special up-conversion dyes that work even in widefield and without femtoseconds, so some could be found that would work here.
3) more powerful pulses - not necessarily shorter, if I got the money I would find someone to build a pulse picker and an amplifier for me. I think 80 kHz repetition with thousand times the peak power could work and the scanning artifacts wouldn't be too severe (you may imagine this can't work with regular confocal...)

Sorry for the lengthy post, bu I didn't want to disclose my brightest ideas on the first few lines :-).

Cheers,

zdenek

---------- Původní zpráva ----------
Od: Unruh, Jay
Datum: 15. 2. 2013
Předmět: Re: multiphoton spinning disc

I would imagine that quite a bit could be gained by going to a shorter pulsed laser and performing dispersion compensation.  I would guess that the micro lens array introduces a significant amount of dispersion.  Of course many of the shorter pulsed lasers aren't tunable either.  Large field of view applications with longer pulsed lasers may result in an undesired amount of heating anyway.  Del Mar ventures (no commercial interest) actually sells a DIY kit for a <20 fs laser for much less than a single box tunable system.

Jay

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Friday, February 15, 2013 11:46 AM
To: [hidden email]
Subject: Re: multiphoton spinning disc

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

If you could sacrifice tunability, fiber lasers are pretty much there, at least for 1040nm.  Time-bandwidth-products (the company, not the unit of merit for pulsed lasers) makes a death ray called the Fortis that outputs 50W at 50 MHz...

Craig


On Fri, Feb 15, 2013 at 9:37 AM, Tim Feinstein  wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello all,
>
> There is an interesting demonstration of multiphoton spinning disc
> confocal imaging in the latest PNAS by Yuko Mimori-Kosue from RIKEN.  
> It shows the expected Z-resolution, depth and s/n improvements (maybe
> a little better than due to pinhole refinement), but their last paragraph is a doozy.
>
> "In putting the system to practical use, the available output laser
> power is the limiting factor for the illumination area and the maximum
> imaging depth. Even with the two-photon lasers having the highest
> levels of output power currently available, we could trigger
> two-photon absorption for only ∼40-μm-diameter areas of GFP-expressing
> animals (less than 10% of the effective frame size with a 60×
> objective; Fig. S1B). Lasers with roughly
> 5–10 times the power would be required. The higher laser power is also
> required to excite red fluorescent proteins, which can be excited at
> wavelengths between 1,000 and 1,200 nm, at which the output power of
> the existing mode-locked laser decreases to ∼15% of peak power. "
>
> Is there any chance of seeing a ti-sapphire with 5 to 10-fold more power?
>  Would it cost 5-10 times as much?  It sounds like a nice approach but
> maybe not so much if you need a military LASER to use it.  On the
> other hand maybe resonant two-photon (or slit-scanning, with
> sacrifices) can already deliver similar performance.  Thoughts appreciated.
>
> All the best,
>
>
> TF
>
> Timothy Feinstein, PhD
> Visiting Research Associate
> Laboratory for GPCR Biology
> Dept. of Pharmacology & Chemical Biology University of Pittsburgh,
> School of Medicine BST W1301, 200 Lothrop St.
> Pittsburgh, PA  15261
>

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****


"multiphoton is ... not suitable for widefield."

Gary Brooker and the US Patent Office disagree:

http://www.google.com/patents/US7468837?

*Patent number*: 7468837
*Filing date*: Dec 18, 2006
*Issue date*: Dec 23, 2008

Wide-field multi-photon microscope having simultaneous confocal imaging
over at least two pixels.




On 2/16/2013 2:04 AM, Zdenek Svindrych wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi guys,
>
> it is a simple matter, multiphoton is not suitable for spinning disc as it's not suitable for widefield.
> It's a nonlinear process and with the same average intensity of illumination the signal will be the higher the higher is the peak intensity. To get intense peak illumination you can focus it spatially (to a diffraction-limited spot) or 'temporally' (with fs pulses); one has to use both approaches to get appreciable results with common fluorescent dyes...
>
> Shortening the pulses is vital, but you cannot go to 1fs - the spectrum would be too broad. I don't think the microlenses could stretch the pulses, because they are fairly thin. The do destroy the wavefronts a bit (coz the are so small...), but this is cleaned up by the pinholes...
>
> Simply put, in a regular confocal you are creating one diffraction-limited spot, in a spinning disc you work with thousand spots. So you would need thousand times more power (assuming the same pulse duration). Bu then the thermal effects (assuming the contribution of the multiphoton processes is negligible) also increase thousand times...
>
> And now the solutions:
> 1) use less pinholes - you hardly can modify your disc, so you decrease FOV instead, like in that paper.
> 2) use better dyes - there are special up-conversion dyes that work even in widefield and without femtoseconds, so some could be found that would work here.
> 3) more powerful pulses - not necessarily shorter, if I got the money I would find someone to build a pulse picker and an amplifier for me. I think 80 kHz repetition with thousand times the peak power could work and the scanning artifacts wouldn't be too severe (you may imagine this can't work with regular confocal...)
>
> Sorry for the lengthy post, bu I didn't want to disclose my brightest ideas on the first few lines :-).
>
> Cheers,
>
> zdenek
>
> ---------- Původní zpráva ----------
> Od: Unruh, Jay
> Datum: 15. 2. 2013
> Předmět: Re: multiphoton spinning disc
>
> I would imagine that quite a bit could be gained by going to a shorter pulsed laser and performing dispersion compensation.  I would guess that the micro lens array introduces a significant amount of dispersion.  Of course many of the shorter pulsed lasers aren't tunable either.  Large field of view applications with longer pulsed lasers may result in an undesired amount of heating anyway.  Del Mar ventures (no commercial interest) actually sells a DIY kit for a<20 fs laser for much less than a single box tunable system.
>
> Jay
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
> Sent: Friday, February 15, 2013 11:46 AM
> To: [hidden email]
> Subject: Re: multiphoton spinning disc
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If you could sacrifice tunability, fiber lasers are pretty much there, at least for 1040nm.  Time-bandwidth-products (the company, not the unit of merit for pulsed lasers) makes a death ray called the Fortis that outputs 50W at 50 MHz...
>
> Craig
>
>
> On Fri, Feb 15, 2013 at 9:37 AM, Tim Feinstein  wrote:
>
>    
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello all,
>>
>> There is an interesting demonstration of multiphoton spinning disc
>> confocal imaging in the latest PNAS by Yuko Mimori-Kosue from RIKEN.
>> It shows the expected Z-resolution, depth and s/n improvements (maybe
>> a little better than due to pinhole refinement), but their last paragraph is a doozy.
>>
>> "In putting the system to practical use, the available output laser
>> power is the limiting factor for the illumination area and the maximum
>> imaging depth. Even with the two-photon lasers having the highest
>> levels of output power currently available, we could trigger
>> two-photon absorption for only ∼40-μm-diameter areas of GFP-expressing
>> animals (less than 10% of the effective frame size with a 60×
>> objective; Fig. S1B). Lasers with roughly
>> 5–10 times the power would be required. The higher laser power is also
>> required to excite red fluorescent proteins, which can be excited at
>> wavelengths between 1,000 and 1,200 nm, at which the output power of
>> the existing mode-locked laser decreases to ∼15% of peak power. "
>>
>> Is there any chance of seeing a ti-sapphire with 5 to 10-fold more power?
>>   Would it cost 5-10 times as much?  It sounds like a nice approach but
>> maybe not so much if you need a military LASER to use it.  On the
>> other hand maybe resonant two-photon (or slit-scanning, with
>> sacrifices) can already deliver similar performance.  Thoughts appreciated.
>>
>> All the best,
>>
>>
>> TF
>>
>> Timothy Feinstein, PhD
>> Visiting Research Associate
>> Laboratory for GPCR Biology
>> Dept. of Pharmacology&  Chemical Biology University of Pittsburgh,
>> School of Medicine BST W1301, 200 Lothrop St.
>> Pittsburgh, PA  15261
>>
>>      

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

)See:

Buranachai C, Kamiyama D, Chiba A, Williams BD, Clegg RM. Rapid frequency-domain FLIM spinning disk confocal microscope:
lifetime resolution, image improvement and wavelet analysis. J Fluoresc. 2008 Sep;18(5):929-42.
doi: 10.1007/s10895-008-0332-3. PubMed PMID: 18324453.

Akira Chiba here at UMiami has two spinning disk FLIM microscopes (see
above for details or contact Akira). See also
http://www.miami.edu/index.php/features/propelling_proteomics-1/
http://ispinproject.org/

If you cannot afford a Yokogawa spinning disk, check out the X-Light at
http://www.biovis.com/x-light.htm


If you are using very thin cells (low autofluorescence also good - see
PubMed 23285248), non-fluorescent culture media, then spinning disk (or
DMD or similar multi-point) not needed. See also Tom Jovin's PAM scope,
which can do FLIM in widefield or optical sectioning modes:

Hanley QS, Lidke KA, Heintzmann R, Arndt-Jovin DJ, Jovin TM. Fluorescence lifetime imaging in an optically sectioning
programmable array microscope (PAM). Cytometry A. 2005 Oct;67(2):112-8. PubMed PMID: 16163693.

Tom also has nice reviews on FRET:

Jares-Erijman EA, Jovin TM. Imaging molecular interactions in living cells by
FRET microscopy. Curr Opin Chem Biol. 2006 Oct;10(5):409-16. Epub 2006 Sep 1.
Review. PubMed PMID: 16949332.

Jares-Erijman EA, Jovin TM. FRET imaging. Nat Biotechnol. 2003
Nov;21(11):1387-95. Review. PubMed PMID: 14595367.



Enjoy,

George


On 2/15/2013 12:13 PM, sunando wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello All
> We are intending to set a live cell imaging set up at our department.
> I am interested to carry out FRET/FLIM studies.
> Would someone help me in suggesting about the camera ?
>
> We would go for Epi set up. Does not funding for confocal....
>
> Thank you advance
> Sunando
>
>

[samuel connell]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Commercial Response:

Hi Sunando,

As George mentioned, Akira Chiba and others have set up systems with
Intelligent Imaging Innovations (3i) to execute spinning disc confocal,
wide-field, or TIRF FLIM systems. Importantly as your suggested, all three
of these experimental paradigms are camera based. 3i is currently selling a
frequency domain FLIM camera, packaged with our SlideBook software and
LaserStack, with significant improvements in both QE and resolution
compared to the previous industry standard. If you are interested in a
discussion on how this approach would fit into your department's needs,
feel free to contact me off list.

FYI: 3i also delivers TCSPC FLIM systems in conjunction with our 2-Photon
system offerings.

Kindest Regards,
--
Sam

------------------------

Samuel A. Connell

Senior Applications Scientist

Intelligent Imaging Innovations, Inc

3250 Ocean Park Blvd, Suite 202

Santa Monica, CA  90405

Office: [phone] x6926

Cell:    [phone]

[hidden email]

www.intelligent-imaging.com


On Sat, Feb 16, 2013 at 6:36 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<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> )See:
>
> Buranachai C, Kamiyama D, Chiba A, Williams BD, Clegg RM. Rapid
> frequency-domain FLIM spinning disk confocal microscope:
> lifetime resolution, image improvement and wavelet analysis. J Fluoresc.
> 2008 Sep;18(5):929-42.
> doi: 10.1007/s10895-008-0332-3. PubMed PMID: 18324453.
>
> Akira Chiba here at UMiami has two spinning disk FLIM microscopes (see
> above for details or contact Akira). See also
> http://www.miami.edu/index.**php/features/propelling_**proteomics-1/<http://www.miami.edu/index.php/features/propelling_proteomics-1/>
> http://ispinproject.org/
>
> If you cannot afford a Yokogawa spinning disk, check out the X-Light at
> http://www.biovis.com/x-light.**htm <http://www.biovis.com/x-light.htm>
>
>
> If you are using very thin cells (low autofluorescence also good - see
> PubMed 23285248), non-fluorescent culture media, then spinning disk (or DMD
> or similar multi-point) not needed. See also Tom Jovin's PAM scope, which
> can do FLIM in widefield or optical sectioning modes:
>
> Hanley QS, Lidke KA, Heintzmann R, Arndt-Jovin DJ, Jovin TM. Fluorescence
> lifetime imaging in an optically sectioning
> programmable array microscope (PAM). Cytometry A. 2005 Oct;67(2):112-8.
> PubMed PMID: 16163693.
>
> Tom also has nice reviews on FRET:
>
> Jares-Erijman EA, Jovin TM. Imaging molecular interactions in living cells
> by
> FRET microscopy. Curr Opin Chem Biol. 2006 Oct;10(5):409-16. Epub 2006 Sep
> 1.
> Review. PubMed PMID: 16949332.
>
> Jares-Erijman EA, Jovin TM. FRET imaging. Nat Biotechnol. 2003
> Nov;21(11):1387-95. Review. PubMed PMID: 14595367.
>
>
>
> Enjoy,
>
> George
>
>
> On 2/15/2013 12:13 PM, sunando wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>> *****
>>
>> Hello All
>> We are intending to set a live cell imaging set up at our department. I
>> am interested to carry out FRET/FLIM studies.
>> Would someone help me in suggesting about the camera ?
>>
>> We would go for Epi set up. Does not funding for confocal....
>>
>> Thank you advance
>> Sunando
>>
>>
>>

[Periasamy, Ammasi (ap3t)]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Sunando
Not sure why you need only the spinning disk based FLIM system to image FLIM-FRET in live cells.
We use and train in the FRET workshop both photon counting and camera based FLIM system for live cells. Camera based system is frequency domain, wide-field microscope and photon counting mode is time domain. You can use two-photon based system for photon counting or confocal based photon counting system. Becker & Hickl, Germany, sells stand alone confocal FLIM-FRET system for very reasonable price.

If you would like to explore and get trained on the FLIM-FRET system attend our workshop. Few companies system will be used to train the workshop participants including Becker & Hickl, Lambert instruments and ISS.
The workshop is during March 11-16, 2013
http://www.kcci.virginia.edu/workshop/workshop2013/index.php
Hope this helps.

Dr. Ammasi Periasamy
Professor & Center Director
W.M. Keck Center for Cellular Imaging (KCCI)
(A University Imaging Center)
Biology, University of Virginia
Mail or FedEx: 485 McCormick Rd.
Charlottesville, VA 22904.
Office Location: Physical Life Sciences Building (B005)
90, Geldard Drive, Charlottesville, VA 22904
Voice: 434-243-7602 (Office); 982-4869 (lab)
Fax:[phone]; Email:[hidden email]
http://www.kcci.virginia.edu/contact/peri.php
************************
12th Annual Workshop on FRET Microscopy, March 11-16, 2013
http://www.kcci.virginia.edu/workshop/workshop2013/index.php
*************************


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of samuel connell
Sent: Saturday, February 16, 2013 11:38 AM
To: [hidden email]
Subject: Re: Live cell set up with FLIM

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Commercial Response:

Hi Sunando,

As George mentioned, Akira Chiba and others have set up systems with Intelligent Imaging Innovations (3i) to execute spinning disc confocal, wide-field, or TIRF FLIM systems. Importantly as your suggested, all three of these experimental paradigms are camera based. 3i is currently selling a frequency domain FLIM camera, packaged with our SlideBook software and LaserStack, with significant improvements in both QE and resolution compared to the previous industry standard. If you are interested in a discussion on how this approach would fit into your department's needs, feel free to contact me off list.

FYI: 3i also delivers TCSPC FLIM systems in conjunction with our 2-Photon system offerings.

Kindest Regards,
--
Sam

------------------------

Samuel A. Connell

Senior Applications Scientist

Intelligent Imaging Innovations, Inc

3250 Ocean Park Blvd, Suite 202

Santa Monica, CA  90405

Office: [phone] x6926

Cell:    [phone]

[hidden email]

www.intelligent-imaging.com


On Sat, Feb 16, 2013 at 6:36 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<http://lists.u
> mn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> )See:
>
> Buranachai C, Kamiyama D, Chiba A, Williams BD, Clegg RM. Rapid
> frequency-domain FLIM spinning disk confocal microscope:
> lifetime resolution, image improvement and wavelet analysis. J Fluoresc.
> 2008 Sep;18(5):929-42.
> doi: 10.1007/s10895-008-0332-3. PubMed PMID: 18324453.
>
> Akira Chiba here at UMiami has two spinning disk FLIM microscopes (see
> above for details or contact Akira). See also
> http://www.miami.edu/index.**php/features/propelling_**proteomics-1/<h
> ttp://www.miami.edu/index.php/features/propelling_proteomics-1/>
> http://ispinproject.org/
>
> If you cannot afford a Yokogawa spinning disk, check out the X-Light
> at http://www.biovis.com/x-light.**htm 
> <http://www.biovis.com/x-light.htm>
>
>
> If you are using very thin cells (low autofluorescence also good - see
> PubMed 23285248), non-fluorescent culture media, then spinning disk
> (or DMD or similar multi-point) not needed. See also Tom Jovin's PAM
> scope, which can do FLIM in widefield or optical sectioning modes:
>
> Hanley QS, Lidke KA, Heintzmann R, Arndt-Jovin DJ, Jovin TM.
> Fluorescence lifetime imaging in an optically sectioning programmable
> array microscope (PAM). Cytometry A. 2005 Oct;67(2):112-8.
> PubMed PMID: 16163693.
>
> Tom also has nice reviews on FRET:
>
> Jares-Erijman EA, Jovin TM. Imaging molecular interactions in living
> cells by FRET microscopy. Curr Opin Chem Biol. 2006 Oct;10(5):409-16.
> Epub 2006 Sep 1.
> Review. PubMed PMID: 16949332.
>
> Jares-Erijman EA, Jovin TM. FRET imaging. Nat Biotechnol. 2003
> Nov;21(11):1387-95. Review. PubMed PMID: 14595367.
>
>
>
> Enjoy,
>
> George
>
>
> On 2/15/2013 12:13 PM, sunando wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.
>> umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>> *****
>>
>> Hello All
>> We are intending to set a live cell imaging set up at our department.
>> I am interested to carry out FRET/FLIM studies.
>> Would someone help me in suggesting about the camera ?
>>
>> We would go for Epi set up. Does not funding for confocal....
>>
>> Thank you advance
>> Sunando
>>
>>
>>

[Guy Cox-2]

Hellwarth R and Christensen P. 1974, Nonlinear microscopic examination of structure in polycrystalline ZnSe.  Optics Communications 12, 318-322  

This was widefield SHG microscopy.  If you can do that I guess you can do widefield MPE as well.  Frankly I can't see either as having any serious practical application.

                                                   Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: Sunday, 17 February 2013 1:21 AM
To: [hidden email]
Subject: Re: multiphoton spinning disc

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****


"multiphoton is ... not suitable for widefield."

Gary Brooker and the US Patent Office disagree:

http://www.google.com/patents/US7468837?

*Patent number*: 7468837
*Filing date*: Dec 18, 2006
*Issue date*: Dec 23, 2008

Wide-field multi-photon microscope having simultaneous confocal imaging
over at least two pixels.




On 2/16/2013 2:04 AM, Zdenek Svindrych wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi guys,
>
> it is a simple matter, multiphoton is not suitable for spinning disc as it's not suitable for widefield.
> It's a nonlinear process and with the same average intensity of illumination the signal will be the higher the higher is the peak intensity. To get intense peak illumination you can focus it spatially (to a diffraction-limited spot) or 'temporally' (with fs pulses); one has to use both approaches to get appreciable results with common fluorescent dyes...
>
> Shortening the pulses is vital, but you cannot go to 1fs - the spectrum would be too broad. I don't think the microlenses could stretch the pulses, because they are fairly thin. The do destroy the wavefronts a bit (coz the are so small...), but this is cleaned up by the pinholes...
>
> Simply put, in a regular confocal you are creating one diffraction-limited spot, in a spinning disc you work with thousand spots. So you would need thousand times more power (assuming the same pulse duration). Bu then the thermal effects (assuming the contribution of the multiphoton processes is negligible) also increase thousand times...
>
> And now the solutions:
> 1) use less pinholes - you hardly can modify your disc, so you decrease FOV instead, like in that paper.
> 2) use better dyes - there are special up-conversion dyes that work even in widefield and without femtoseconds, so some could be found that would work here.
> 3) more powerful pulses - not necessarily shorter, if I got the money I would find someone to build a pulse picker and an amplifier for me. I think 80 kHz repetition with thousand times the peak power could work and the scanning artifacts wouldn't be too severe (you may imagine this can't work with regular confocal...)
>
> Sorry for the lengthy post, bu I didn't want to disclose my brightest ideas on the first few lines :-).
>
> Cheers,
>
> zdenek
>
> ---------- Původní zpráva ----------
> Od: Unruh, Jay
> Datum: 15. 2. 2013
> Předmět: Re: multiphoton spinning disc
>
> I would imagine that quite a bit could be gained by going to a shorter pulsed laser and performing dispersion compensation.  I would guess that the micro lens array introduces a significant amount of dispersion.  Of course many of the shorter pulsed lasers aren't tunable either.  Large field of view applications with longer pulsed lasers may result in an undesired amount of heating anyway.  Del Mar ventures (no commercial interest) actually sells a DIY kit for a<20 fs laser for much less than a single box tunable system.
>
> Jay
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
> Sent: Friday, February 15, 2013 11:46 AM
> To: [hidden email]
> Subject: Re: multiphoton spinning disc
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If you could sacrifice tunability, fiber lasers are pretty much there, at least for 1040nm.  Time-bandwidth-products (the company, not the unit of merit for pulsed lasers) makes a death ray called the Fortis that outputs 50W at 50 MHz...
>
> Craig
>
>
> On Fri, Feb 15, 2013 at 9:37 AM, Tim Feinstein  wrote:
>
>    
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello all,
>>
>> There is an interesting demonstration of multiphoton spinning disc
>> confocal imaging in the latest PNAS by Yuko Mimori-Kosue from RIKEN.
>> It shows the expected Z-resolution, depth and s/n improvements (maybe
>> a little better than due to pinhole refinement), but their last paragraph is a doozy.
>>
>> "In putting the system to practical use, the available output laser
>> power is the limiting factor for the illumination area and the maximum
>> imaging depth. Even with the two-photon lasers having the highest
>> levels of output power currently available, we could trigger
>> two-photon absorption for only ∼40-μm-diameter areas of GFP-expressing
>> animals (less than 10% of the effective frame size with a 60×
>> objective; Fig. S1B). Lasers with roughly
>> 5–10 times the power would be required. The higher laser power is also
>> required to excite red fluorescent proteins, which can be excited at
>> wavelengths between 1,000 and 1,200 nm, at which the output power of
>> the existing mode-locked laser decreases to ∼15% of peak power. "
>>
>> Is there any chance of seeing a ti-sapphire with 5 to 10-fold more power?
>>   Would it cost 5-10 times as much?  It sounds like a nice approach but
>> maybe not so much if you need a military LASER to use it.  On the
>> other hand maybe resonant two-photon (or slit-scanning, with
>> sacrifices) can already deliver similar performance.  Thoughts appreciated.
>>
>> All the best,
>>
>>
>> TF
>>
>> Timothy Feinstein, PhD
>> Visiting Research Associate
>> Laboratory for GPCR Biology
>> Dept. of Pharmacology&  Chemical Biology University of Pittsburgh,
>> School of Medicine BST W1301, 200 Lothrop St.
>> Pittsburgh, PA  15261
>>
>>      

[Craig Brideau]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I agree with George; it's just a matter of having enough power/energy to
distribute amongst the beamlets on the disk.  Unless there is an AOM in the
system or the like compression probably wouldn't be much of an issue as
well.  Basically you can just brute-force it by using a lot of power into
the scanning system.  Given that current Ti:Saphs are overpowered for point
scanning as it is, it should be feasible to get a sufficiently energetic
system for spinning disk use.

Craig


On Sun, Feb 17, 2013 at 12:03 AM, Guy Cox <[hidden email]> wrote:

> Hellwarth R and Christensen P. 1974, Nonlinear microscopic examination of
> structure in polycrystalline ZnSe.  Optics Communications 12, 318-322
>
> This was widefield SHG microscopy.  If you can do that I guess you can do
> widefield MPE as well.  Frankly I can't see either as having any serious
> practical application.
>
>                                                    Guy
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of George McNamara
> Sent: Sunday, 17 February 2013 1:21 AM
> To: [hidden email]
> Subject: Re: multiphoton spinning disc
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> "multiphoton is ... not suitable for widefield."
>
> Gary Brooker and the US Patent Office disagree:
>
> http://www.google.com/patents/US7468837?
>
> *Patent number*: 7468837
> *Filing date*: Dec 18, 2006
> *Issue date*: Dec 23, 2008
>
> Wide-field multi-photon microscope having simultaneous confocal imaging
> over at least two pixels.
>
>
>
>
> On 2/16/2013 2:04 AM, Zdenek Svindrych wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi guys,
> >
> > it is a simple matter, multiphoton is not suitable for spinning disc as
> it's not suitable for widefield.
> > It's a nonlinear process and with the same average intensity of
> illumination the signal will be the higher the higher is the peak
> intensity. To get intense peak illumination you can focus it spatially (to
> a diffraction-limited spot) or 'temporally' (with fs pulses); one has to
> use both approaches to get appreciable results with common fluorescent
> dyes...
> >
> > Shortening the pulses is vital, but you cannot go to 1fs - the spectrum
> would be too broad. I don't think the microlenses could stretch the pulses,
> because they are fairly thin. The do destroy the wavefronts a bit (coz the
> are so small...), but this is cleaned up by the pinholes...
> >
> > Simply put, in a regular confocal you are creating one
> diffraction-limited spot, in a spinning disc you work with thousand spots.
> So you would need thousand times more power (assuming the same pulse
> duration). Bu then the thermal effects (assuming the contribution of the
> multiphoton processes is negligible) also increase thousand times...
> >
> > And now the solutions:
> > 1) use less pinholes - you hardly can modify your disc, so you decrease
> FOV instead, like in that paper.
> > 2) use better dyes - there are special up-conversion dyes that work even
> in widefield and without femtoseconds, so some could be found that would
> work here.
> > 3) more powerful pulses - not necessarily shorter, if I got the money I
> would find someone to build a pulse picker and an amplifier for me. I think
> 80 kHz repetition with thousand times the peak power could work and the
> scanning artifacts wouldn't be too severe (you may imagine this can't work
> with regular confocal...)
> >
> > Sorry for the lengthy post, bu I didn't want to disclose my brightest
> ideas on the first few lines :-).
> >
> > Cheers,
> >
> > zdenek
> >
> > ---------- Původní zpráva ----------
> > Od: Unruh, Jay
> > Datum: 15. 2. 2013
> > Předmět: Re: multiphoton spinning disc
> >
> > I would imagine that quite a bit could be gained by going to a shorter
> pulsed laser and performing dispersion compensation.  I would guess that
> the micro lens array introduces a significant amount of dispersion.  Of
> course many of the shorter pulsed lasers aren't tunable either.  Large
> field of view applications with longer pulsed lasers may result in an
> undesired amount of heating anyway.  Del Mar ventures (no commercial
> interest) actually sells a DIY kit for a<20 fs laser for much less than a
> single box tunable system.
> >
> > Jay
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Craig Brideau
> > Sent: Friday, February 15, 2013 11:46 AM
> > To: [hidden email]
> > Subject: Re: multiphoton spinning disc
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > If you could sacrifice tunability, fiber lasers are pretty much there,
> at least for 1040nm.  Time-bandwidth-products (the company, not the unit of
> merit for pulsed lasers) makes a death ray called the Fortis that outputs
> 50W at 50 MHz...
> >
> > Craig
> >
> >
> > On Fri, Feb 15, 2013 at 9:37 AM, Tim Feinstein  wrote:
> >
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Hello all,
> >>
> >> There is an interesting demonstration of multiphoton spinning disc
> >> confocal imaging in the latest PNAS by Yuko Mimori-Kosue from RIKEN.
> >> It shows the expected Z-resolution, depth and s/n improvements (maybe
> >> a little better than due to pinhole refinement), but their last
> paragraph is a doozy.
> >>
> >> "In putting the system to practical use, the available output laser
> >> power is the limiting factor for the illumination area and the maximum
> >> imaging depth. Even with the two-photon lasers having the highest
> >> levels of output power currently available, we could trigger
> >> two-photon absorption for only ∼40-μm-diameter areas of GFP-expressing
> >> animals (less than 10% of the effective frame size with a 60×
> >> objective; Fig. S1B). Lasers with roughly
> >> 5–10 times the power would be required. The higher laser power is also
> >> required to excite red fluorescent proteins, which can be excited at
> >> wavelengths between 1,000 and 1,200 nm, at which the output power of
> >> the existing mode-locked laser decreases to ∼15% of peak power. "
> >>
> >> Is there any chance of seeing a ti-sapphire with 5 to 10-fold more
> power?
> >>   Would it cost 5-10 times as much?  It sounds like a nice approach but
> >> maybe not so much if you need a military LASER to use it.  On the
> >> other hand maybe resonant two-photon (or slit-scanning, with
> >> sacrifices) can already deliver similar performance.  Thoughts
> appreciated.
> >>
> >> All the best,
> >>
> >>
> >> TF
> >>
> >> Timothy Feinstein, PhD
> >> Visiting Research Associate
> >> Laboratory for GPCR Biology
> >> Dept. of Pharmacology&  Chemical Biology University of Pittsburgh,
> >> School of Medicine BST W1301, 200 Lothrop St.
> >> Pittsburgh, PA  15261
> >>
> >>
>

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

My thinking for Gary Brooker's widefield multiphoton is not to use it on
a spinning disk system, but rather on Andrew York & Hari Shroff's MSIM2
nanoscope - a much faster design than their 2012 Nature Methods paper
(keep an eye out for their work). Using it on Tom Jovin's PAM might also
be useful. If I have my math correct, a Chameleon Ultra II MP laser plus
Chameleon OPO-VIS on MSIM2 would also enable both WF-MPEF 2photon and
wide tuning range 1photon nanoscopy.

On 2/17/2013 3:14 PM, Craig Brideau wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I agree with George; it's just a matter of having enough power/energy to
> distribute amongst the beamlets on the disk.  Unless there is an AOM in the
> system or the like compression probably wouldn't be much of an issue as
> well.  Basically you can just brute-force it by using a lot of power into
> the scanning system.  Given that current Ti:Saphs are overpowered for point
> scanning as it is, it should be feasible to get a sufficiently energetic
> system for spinning disk use.
>
> Craig
>
>
> On Sun, Feb 17, 2013 at 12:03 AM, Guy Cox<[hidden email]>  wrote:
>
>    
>> Hellwarth R and Christensen P. 1974, Nonlinear microscopic examination of
>> structure in polycrystalline ZnSe.  Optics Communications 12, 318-322
>>
>> This was widefield SHG microscopy.  If you can do that I guess you can do
>> widefield MPE as well.  Frankly I can't see either as having any serious
>> practical application.
>>
>>                                                     Guy
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of George McNamara
>> Sent: Sunday, 17 February 2013 1:21 AM
>> To: [hidden email]
>> Subject: Re: multiphoton spinning disc
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>>
>> "multiphoton is ... not suitable for widefield."
>>
>> Gary Brooker and the US Patent Office disagree:
>>
>> http://www.google.com/patents/US7468837?
>>
>> *Patent number*: 7468837
>> *Filing date*: Dec 18, 2006
>> *Issue date*: Dec 23, 2008
>>
>> Wide-field multi-photon microscope having simultaneous confocal imaging
>> over at least two pixels.
>>
>>
>>
>>
>> On 2/16/2013 2:04 AM, Zdenek Svindrych wrote:
>>      
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi guys,
>>>
>>> it is a simple matter, multiphoton is not suitable for spinning disc as
>>>        
>> it's not suitable for widefield.
>>      
>>> It's a nonlinear process and with the same average intensity of
>>>        
>> illumination the signal will be the higher the higher is the peak
>> intensity. To get intense peak illumination you can focus it spatially (to
>> a diffraction-limited spot) or 'temporally' (with fs pulses); one has to
>> use both approaches to get appreciable results with common fluorescent
>> dyes...
>>      
>>> Shortening the pulses is vital, but you cannot go to 1fs - the spectrum
>>>        
>> would be too broad. I don't think the microlenses could stretch the pulses,
>> because they are fairly thin. The do destroy the wavefronts a bit (coz the
>> are so small...), but this is cleaned up by the pinholes...
>>      
>>> Simply put, in a regular confocal you are creating one
>>>        
>> diffraction-limited spot, in a spinning disc you work with thousand spots.
>> So you would need thousand times more power (assuming the same pulse
>> duration). Bu then the thermal effects (assuming the contribution of the
>> multiphoton processes is negligible) also increase thousand times...
>>      
>>> And now the solutions:
>>> 1) use less pinholes - you hardly can modify your disc, so you decrease
>>>        
>> FOV instead, like in that paper.
>>      
>>> 2) use better dyes - there are special up-conversion dyes that work even
>>>        
>> in widefield and without femtoseconds, so some could be found that would
>> work here.
>>      
>>> 3) more powerful pulses - not necessarily shorter, if I got the money I
>>>        
>> would find someone to build a pulse picker and an amplifier for me. I think
>> 80 kHz repetition with thousand times the peak power could work and the
>> scanning artifacts wouldn't be too severe (you may imagine this can't work
>> with regular confocal...)
>>      
>>> Sorry for the lengthy post, bu I didn't want to disclose my brightest
>>>        
>> ideas on the first few lines :-).
>>      
>>> Cheers,
>>>
>>> zdenek
>>>
>>> ---------- Původní zpráva ----------
>>> Od: Unruh, Jay
>>> Datum: 15. 2. 2013
>>> Předmět: Re: multiphoton spinning disc
>>>
>>> I would imagine that quite a bit could be gained by going to a shorter
>>>        
>> pulsed laser and performing dispersion compensation.  I would guess that
>> the micro lens array introduces a significant amount of dispersion.  Of
>> course many of the shorter pulsed lasers aren't tunable either.  Large
>> field of view applications with longer pulsed lasers may result in an
>> undesired amount of heating anyway.  Del Mar ventures (no commercial
>> interest) actually sells a DIY kit for a<20 fs laser for much less than a
>> single box tunable system.
>>      
>>> Jay
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:[hidden email]]
>>>        
>> On Behalf Of Craig Brideau
>>      
>>> Sent: Friday, February 15, 2013 11:46 AM
>>> To: [hidden email]
>>> Subject: Re: multiphoton spinning disc
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> If you could sacrifice tunability, fiber lasers are pretty much there,
>>>        
>> at least for 1040nm.  Time-bandwidth-products (the company, not the unit of
>> merit for pulsed lasers) makes a death ray called the Fortis that outputs
>> 50W at 50 MHz...
>>      
>>> Craig
>>>
>>>
>>> On Fri, Feb 15, 2013 at 9:37 AM, Tim Feinstein  wrote:
>>>
>>>
>>>        
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Hello all,
>>>>
>>>> There is an interesting demonstration of multiphoton spinning disc
>>>> confocal imaging in the latest PNAS by Yuko Mimori-Kosue from RIKEN.
>>>> It shows the expected Z-resolution, depth and s/n improvements (maybe
>>>> a little better than due to pinhole refinement), but their last
>>>>          
>> paragraph is a doozy.
>>      
>>>> "In putting the system to practical use, the available output laser
>>>> power is the limiting factor for the illumination area and the maximum
>>>> imaging depth. Even with the two-photon lasers having the highest
>>>> levels of output power currently available, we could trigger
>>>> two-photon absorption for only ∼40-μm-diameter areas of GFP-expressing
>>>> animals (less than 10% of the effective frame size with a 60×
>>>> objective; Fig. S1B). Lasers with roughly
>>>> 5–10 times the power would be required. The higher laser power is also
>>>> required to excite red fluorescent proteins, which can be excited at
>>>> wavelengths between 1,000 and 1,200 nm, at which the output power of
>>>> the existing mode-locked laser decreases to ∼15% of peak power. "
>>>>
>>>> Is there any chance of seeing a ti-sapphire with 5 to 10-fold more
>>>>          
>> power?
>>      
>>>>    Would it cost 5-10 times as much?  It sounds like a nice approach but
>>>> maybe not so much if you need a military LASER to use it.  On the
>>>> other hand maybe resonant two-photon (or slit-scanning, with
>>>> sacrifices) can already deliver similar performance.  Thoughts
>>>>          
>> appreciated.
>>      
>>>> All the best,
>>>>
>>>>
>>>> TF
>>>>
>>>> Timothy Feinstein, PhD
>>>> Visiting Research Associate
>>>> Laboratory for GPCR Biology
>>>> Dept. of Pharmacology&   Chemical Biology University of Pittsburgh,
>>>> School of Medicine BST W1301, 200 Lothrop St.
>>>> Pittsburgh, PA  15261
>>>>
>>>>
>>>>          
>>      
>    

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****


Zdenek: "it is a simple matter, multiphoton is not suitable for spinning
disc as it's not suitable for widefield. "

Shimozawa et al: "Second, the generation of out-of-focus light is
prevented by two-photon excitation that achieves selective-plane
illumination. </pubmed/23401517>"

*Shimozawa* T, Yamagata K, Kondo T, Hayashi S, Shitamukai A, Konno D,
Matsuzaki F, Takayama J, Onami S, Nakayama H, Kosugi Y, Watanabe TM,
Fujita K, Mimori-Kiyosue Y. Improving spinning disk confocal microscopy
by preventing pinhole cross-talk for intravital imaging.
</pubmed/23401517>* *Proc Natl Acad Sci U S A. 2013 Feb 11.  PMID: 23401517

A recent key requirement in life sciences is the observation of
biological processes in their natural in vivo context. However, imaging
techniques that allow fast imaging with higher resolution in 3D thick
specimens are still limited. Spinning disk confocal microscopy using a
Yokogawa Confocal Scanner Unit, which offers high-speed multipoint
confocal live imaging, has been found to have wide utility among cell
biologists. A conventional Confocal Scanner Unit configuration, however,
is not optimized for thick specimens, for which the background noise
attributed to "pinhole cross-talk," which is unintended pinhole
transmission of out-of-focus light, limits overall performance in focal
discrimination and reduces confocal capability. Here, we improve
spinning disk confocal microscopy by eliminating pinhole cross-talk.
First, the amount of pinhole cross-talk is reduced by increasing the
interpinhole distance. Second, the generation of out-of-focus light is
prevented by two-photon excitation that achieves selective-plane
illumination. We evaluate the effect of these modifications and test the
applicability to the live imaging of green fluorescent
protein-expressing model animals. As demonstrated by visualizing the
fine details of the 3D cell shape and submicron-size cytoskeletal
structures inside animals, these strategies dramatically improve
higher-resolution intravital imaging. </pubmed/23401517>*
*

On 2/16/2013 2:04 AM, Zdenek Svindrych wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi guys,
>
> it is a simple matter, multiphoton is not suitable for spinning disc as it's not suitable for widefield.
> It's a nonlinear process and with the same average intensity of illumination the signal will be the higher the higher is the peak intensity. To get intense peak illumination you can focus it spatially (to a diffraction-limited spot) or 'temporally' (with fs pulses); one has to use both approaches to get appreciable results with common fluorescent dyes...
>
> Shortening the pulses is vital, but you cannot go to 1fs - the spectrum would be too broad. I don't think the microlenses could stretch the pulses, because they are fairly thin. The do destroy the wavefronts a bit (coz the are so small...), but this is cleaned up by the pinholes...
>
> Simply put, in a regular confocal you are creating one diffraction-limited spot, in a spinning disc you work with thousand spots. So you would need thousand times more power (assuming the same pulse duration). Bu then the thermal effects (assuming the contribution of the multiphoton processes is negligible) also increase thousand times...
>
> And now the solutions:
> 1) use less pinholes - you hardly can modify your disc, so you decrease FOV instead, like in that paper.
> 2) use better dyes - there are special up-conversion dyes that work even in widefield and without femtoseconds, so some could be found that would work here.
> 3) more powerful pulses - not necessarily shorter, if I got the money I would find someone to build a pulse picker and an amplifier for me. I think 80 kHz repetition with thousand times the peak power could work and the scanning artifacts wouldn't be too severe (you may imagine this can't work with regular confocal...)
>
> Sorry for the lengthy post, bu I didn't want to disclose my brightest ideas on the first few lines :-).
>
> Cheers,
>
> zdenek
>
> ---------- Původní zpráva ----------
> Od: Unruh, Jay
> Datum: 15. 2. 2013
> Předmět: Re: multiphoton spinning disc
>
> I would imagine that quite a bit could be gained by going to a shorter pulsed laser and performing dispersion compensation.  I would guess that the micro lens array introduces a significant amount of dispersion.  Of course many of the shorter pulsed lasers aren't tunable either.  Large field of view applications with longer pulsed lasers may result in an undesired amount of heating anyway.  Del Mar ventures (no commercial interest) actually sells a DIY kit for a<20 fs laser for much less than a single box tunable system.
>
> Jay
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
> Sent: Friday, February 15, 2013 11:46 AM
> To: [hidden email]
> Subject: Re: multiphoton spinning disc
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If you could sacrifice tunability, fiber lasers are pretty much there, at least for 1040nm.  Time-bandwidth-products (the company, not the unit of merit for pulsed lasers) makes a death ray called the Fortis that outputs 50W at 50 MHz...
>
> Craig
>
>
> On Fri, Feb 15, 2013 at 9:37 AM, Tim Feinstein  wrote:
>
>    
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello all,
>>
>> There is an interesting demonstration of multiphoton spinning disc
>> confocal imaging in the latest PNAS by Yuko Mimori-Kosue from RIKEN.
>> It shows the expected Z-resolution, depth and s/n improvements (maybe
>> a little better than due to pinhole refinement), but their last paragraph is a doozy.
>>
>> "In putting the system to practical use, the available output laser
>> power is the limiting factor for the illumination area and the maximum
>> imaging depth. Even with the two-photon lasers having the highest
>> levels of output power currently available, we could trigger
>> two-photon absorption for only ∼40-μm-diameter areas of GFP-expressing
>> animals (less than 10% of the effective frame size with a 60×
>> objective; Fig. S1B). Lasers with roughly
>> 5–10 times the power would be required. The higher laser power is also
>> required to excite red fluorescent proteins, which can be excited at
>> wavelengths between 1,000 and 1,200 nm, at which the output power of
>> the existing mode-locked laser decreases to ∼15% of peak power. "
>>
>> Is there any chance of seeing a ti-sapphire with 5 to 10-fold more power?
>>   Would it cost 5-10 times as much?  It sounds like a nice approach but
>> maybe not so much if you need a military LASER to use it.  On the
>> other hand maybe resonant two-photon (or slit-scanning, with
>> sacrifices) can already deliver similar performance.  Thoughts appreciated.
>>
>> All the best,
>>
>>
>> TF
>>
>> Timothy Feinstein, PhD
>> Visiting Research Associate
>> Laboratory for GPCR Biology
>> Dept. of Pharmacology&  Chemical Biology University of Pittsburgh,
>> School of Medicine BST W1301, 200 Lothrop St.
>> Pittsburgh, PA  15261
>>
>>      

[Zdenek Svindrych]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Thanks to the List I am aware of this paper.

I have spent some time doing 2PE and SHG on a scanning confocal. Though our
setup was not tuned optimally (a prechirp would really help), we were often
limited by the absorbed heat. So increasing the power to get larger FOV  of
the 2P spinning disc might be problematic.

The work reported in the paper is great, no doubt (I'd also like to design
my own disc :-). But I think it will be only useful with new effective up-
converting dyes or something the like.

Cheers,
zdenek
P.S.:
I didn't mean unsuitable, i just thing it is not the preferred combination,
at least for now.


---------- Původní zpráva ----------
Od: George McNamara <[hidden email]>
Datum: 25. 2. 2013
Předmět: Re: multiphoton spinning disc

"*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****


Zdenek: "it is a simple matter, multiphoton is not suitable for spinning
disc as it's not suitable for widefield. "

Shimozawa et al: "Second, the generation of out-of-focus light is
prevented by two-photon excitation that achieves selective-plane
illumination. </pubmed/23401517>"

*Shimozawa* T, Yamagata K, Kondo T, Hayashi S, Shitamukai A, Konno D,
Matsuzaki F, Takayama J, Onami S, Nakayama H, Kosugi Y, Watanabe TM,
Fujita K, Mimori-Kiyosue Y. Improving spinning disk confocal microscopy
by preventing pinhole cross-talk for intravital imaging.
</pubmed/23401517>* *Proc Natl Acad Sci U S A. 2013 Feb 11. PMID: 23401517

A recent key requirement in life sciences is the observation of
biological processes in their natural in vivo context. However, imaging
techniques that allow fast imaging with higher resolution in 3D thick
specimens are still limited. Spinning disk confocal microscopy using a
Yokogawa Confocal Scanner Unit, which offers high-speed multipoint
confocal live imaging, has been found to have wide utility among cell
biologists. A conventional Confocal Scanner Unit configuration, however,
is not optimized for thick specimens, for which the background noise
attributed to "pinhole cross-talk," which is unintended pinhole
transmission of out-of-focus light, limits overall performance in focal
discrimination and reduces confocal capability. Here, we improve
spinning disk confocal microscopy by eliminating pinhole cross-talk.
First, the amount of pinhole cross-talk is reduced by increasing the
interpinhole distance. Second, the generation of out-of-focus light is
prevented by two-photon excitation that achieves selective-plane
illumination. We evaluate the effect of these modifications and test the
applicability to the live imaging of green fluorescent
protein-expressing model animals. As demonstrated by visualizing the
fine details of the 3D cell shape and submicron-size cytoskeletal
structures inside animals, these strategies dramatically improve
higher-resolution intravital imaging. </pubmed/23401517>*
*

On 2/16/2013 2:04 AM, Zdenek Svindrych wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi guys,
>
> it is a simple matter, multiphoton is not suitable for spinning disc as
it's not suitable for widefield.
> It's a nonlinear process and with the same average intensity of
illumination the signal will be the higher the higher is the peak intensity.
To get intense peak illumination you can focus it spatially (to a
diffraction-limited spot) or 'temporally' (with fs pulses); one has to use
both approaches to get appreciable results with common fluorescent dyes...
>
> Shortening the pulses is vital, but you cannot go to 1fs - the spectrum
would be too broad. I don't think the microlenses could stretch the pulses,
because they are fairly thin. The do destroy the wavefronts a bit (coz the
are so small...), but this is cleaned up by the pinholes...
>
> Simply put, in a regular confocal you are creating one diffraction-limited
spot, in a spinning disc you work with thousand spots. So you would need
thousand times more power (assuming the same pulse duration). Bu then the
thermal effects (assuming the contribution of the multiphoton processes is
negligible) also increase thousand times...
>
> And now the solutions:
> 1) use less pinholes - you hardly can modify your disc, so you decrease
FOV instead, like in that paper.
> 2) use better dyes - there are special up-conversion dyes that work even
in widefield and without femtoseconds, so some could be found that would
work here.
> 3) more powerful pulses - not necessarily shorter, if I got the money I
would find someone to build a pulse picker and an amplifier for me. I think
80 kHz repetition with thousand times the peak power could work and the
scanning artifacts wouldn't be too severe (you may imagine this can't work
with regular confocal...)
>
> Sorry for the lengthy post, bu I didn't want to disclose my brightest
ideas on the first few lines :-).

>
> Cheers,
>
> zdenek
>
> ---------- Původní zpráva ----------
> Od: Unruh, Jay
> Datum: 15. 2. 2013
> Předmět: Re: multiphoton spinning disc
>
> I would imagine that quite a bit could be gained by going to a shorter

pulsed laser and performing dispersion compensation. I would guess that the
micro lens array introduces a significant amount of dispersion. Of course
many of the shorter pulsed lasers aren't tunable either. Large field of view
applications with longer pulsed lasers may result in an undesired amount of
heating anyway. Del Mar ventures (no commercial interest) actually sells a
DIY kit for a<20 fs laser for much less than a single box tunable system.
>
> Jay
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Craig Brideau

> Sent: Friday, February 15, 2013 11:46 AM
> To: [hidden email]
> Subject: Re: multiphoton spinning disc
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If you could sacrifice tunability, fiber lasers are pretty much there, at

least for 1040nm. Time-bandwidth-products (the company, not the unit of
merit for pulsed lasers) makes a death ray called the Fortis that outputs 50
W at 50 MHz...

>
> Craig
>
>
> On Fri, Feb 15, 2013 at 9:37 AM, Tim Feinstein wrote:
>
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello all,
>>
>> There is an interesting demonstration of multiphoton spinning disc
>> confocal imaging in the latest PNAS by Yuko Mimori-Kosue from RIKEN.
>> It shows the expected Z-resolution, depth and s/n improvements (maybe
>> a little better than due to pinhole refinement), but their last paragraph

is a doozy.

>>
>> "In putting the system to practical use, the available output laser
>> power is the limiting factor for the illumination area and the maximum
>> imaging depth. Even with the two-photon lasers having the highest
>> levels of output power currently available, we could trigger
>> two-photon absorption for only ∼40-μm-diameter areas of GFP-expressing
>> animals (less than 10% of the effective frame size with a 60×
>> objective; Fig. S1B). Lasers with roughly
>> 5–10 times the power would be required. The higher laser power is also
>> required to excite red fluorescent proteins, which can be excited at
>> wavelengths between 1,000 and 1,200 nm, at which the output power of
>> the existing mode-locked laser decreases to ∼15% of peak power. "
>>
>> Is there any chance of seeing a ti-sapphire with 5 to 10-fold more power?
>> Would it cost 5-10 times as much? It sounds like a nice approach but
>> maybe not so much if you need a military LASER to use it. On the
>> other hand maybe resonant two-photon (or slit-scanning, with
>> sacrifices) can already deliver similar performance. Thoughts

appreciated.

>>
>> All the best,
>>
>>
>> TF
>>
>> Timothy Feinstein, PhD
>> Visiting Research Associate
>> Laboratory for GPCR Biology
>> Dept. of Pharmacology& Chemical Biology University of Pittsburgh,
>> School of Medicine BST W1301, 200 Lothrop St.
>> Pittsburgh, PA 15261
>>
>>"

[Guy Cox-2]

The plain fact remains that if you want to do multipoint multiphoton imaging there is a particular instrument available to do just that - the LaVision Biotech system.  Because there is a path difference between each point and the next cross-talk is totally eliminated.  This does not apply to a spinning-disk system.  I have no connection with LaVision whatsoever - never even owned one of their instruments - but it must be obvious that if you want to run a particular course you'll do best using the correct horse.  

                                             Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: Monday, 25 February 2013 12:45 PM
To: [hidden email]
Subject: Re: multiphoton spinning disc

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****


Zdenek: "it is a simple matter, multiphoton is not suitable for spinning disc as it's not suitable for widefield. "

Shimozawa et al: "Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. </pubmed/23401517>"

*Shimozawa* T, Yamagata K, Kondo T, Hayashi S, Shitamukai A, Konno D, Matsuzaki F, Takayama J, Onami S, Nakayama H, Kosugi Y, Watanabe TM, Fujita K, Mimori-Kiyosue Y. Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging.
</pubmed/23401517>* *Proc Natl Acad Sci U S A. 2013 Feb 11.  PMID: 23401517

A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to "pinhole cross-talk," which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk.
First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging. </pubmed/23401517>*
*

On 2/16/2013 2:04 AM, Zdenek Svindrych wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi guys,
>
> it is a simple matter, multiphoton is not suitable for spinning disc as it's not suitable for widefield.
> It's a nonlinear process and with the same average intensity of illumination the signal will be the higher the higher is the peak intensity. To get intense peak illumination you can focus it spatially (to a diffraction-limited spot) or 'temporally' (with fs pulses); one has to use both approaches to get appreciable results with common fluorescent dyes...
>
> Shortening the pulses is vital, but you cannot go to 1fs - the spectrum would be too broad. I don't think the microlenses could stretch the pulses, because they are fairly thin. The do destroy the wavefronts a bit (coz the are so small...), but this is cleaned up by the pinholes...
>
> Simply put, in a regular confocal you are creating one diffraction-limited spot, in a spinning disc you work with thousand spots. So you would need thousand times more power (assuming the same pulse duration). Bu then the thermal effects (assuming the contribution of the multiphoton processes is negligible) also increase thousand times...
>
> And now the solutions:
> 1) use less pinholes - you hardly can modify your disc, so you decrease FOV instead, like in that paper.
> 2) use better dyes - there are special up-conversion dyes that work even in widefield and without femtoseconds, so some could be found that would work here.
> 3) more powerful pulses - not necessarily shorter, if I got the money
> I would find someone to build a pulse picker and an amplifier for me.
> I think 80 kHz repetition with thousand times the peak power could
> work and the scanning artifacts wouldn't be too severe (you may
> imagine this can't work with regular confocal...)
>
> Sorry for the lengthy post, bu I didn't want to disclose my brightest ideas on the first few lines :-).
>
> Cheers,
>
> zdenek
>
> ---------- Původní zpráva ----------
> Od: Unruh, Jay
> Datum: 15. 2. 2013
> Předmět: Re: multiphoton spinning disc
>
> I would imagine that quite a bit could be gained by going to a shorter pulsed laser and performing dispersion compensation.  I would guess that the micro lens array introduces a significant amount of dispersion.  Of course many of the shorter pulsed lasers aren't tunable either.  Large field of view applications with longer pulsed lasers may result in an undesired amount of heating anyway.  Del Mar ventures (no commercial interest) actually sells a DIY kit for a<20 fs laser for much less than a single box tunable system.
>
> Jay
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Craig Brideau
> Sent: Friday, February 15, 2013 11:46 AM
> To: [hidden email]
> Subject: Re: multiphoton spinning disc
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If you could sacrifice tunability, fiber lasers are pretty much there, at least for 1040nm.  Time-bandwidth-products (the company, not the unit of merit for pulsed lasers) makes a death ray called the Fortis that outputs 50W at 50 MHz...
>
> Craig
>
>
> On Fri, Feb 15, 2013 at 9:37 AM, Tim Feinstein  wrote:
>
>    
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello all,
>>
>> There is an interesting demonstration of multiphoton spinning disc
>> confocal imaging in the latest PNAS by Yuko Mimori-Kosue from RIKEN.
>> It shows the expected Z-resolution, depth and s/n improvements (maybe
>> a little better than due to pinhole refinement), but their last paragraph is a doozy.
>>
>> "In putting the system to practical use, the available output laser
>> power is the limiting factor for the illumination area and the
>> maximum imaging depth. Even with the two-photon lasers having the
>> highest levels of output power currently available, we could trigger
>> two-photon absorption for only ∼40-μm-diameter areas of
>> GFP-expressing animals (less than 10% of the effective frame size
>> with a 60× objective; Fig. S1B). Lasers with roughly
>> 5–10 times the power would be required. The higher laser power is
>> also required to excite red fluorescent proteins, which can be
>> excited at wavelengths between 1,000 and 1,200 nm, at which the
>> output power of the existing mode-locked laser decreases to ∼15% of peak power. "
>>
>> Is there any chance of seeing a ti-sapphire with 5 to 10-fold more power?
>>   Would it cost 5-10 times as much?  It sounds like a nice approach
>> but maybe not so much if you need a military LASER to use it.  On the
>> other hand maybe resonant two-photon (or slit-scanning, with
>> sacrifices) can already deliver similar performance.  Thoughts appreciated.
>>
>> All the best,
>>
>>
>> TF
>>
>> Timothy Feinstein, PhD
>> Visiting Research Associate
>> Laboratory for GPCR Biology
>> Dept. of Pharmacology&  Chemical Biology University of Pittsburgh,
>> School of Medicine BST W1301, 200 Lothrop St.
>> Pittsburgh, PA  15261
>>
>>      

[Craig Brideau]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Chirped mirrors are a quick way to get a bit of pulse compression with
minimal effort (and cost) in a situation like this.
As for video-rate and 2-photon imaging, I'll stick to resonant scanners
before trying to do it with spinning disk.

Craig


On Mon, Feb 25, 2013 at 3:23 AM, Zdenek Svindrych <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thanks to the List I am aware of this paper.
>
> I have spent some time doing 2PE and SHG on a scanning confocal. Though our
> setup was not tuned optimally (a prechirp would really help), we were often
> limited by the absorbed heat. So increasing the power to get larger FOV  of
> the 2P spinning disc might be problematic.
>
> The work reported in the paper is great, no doubt (I'd also like to design
> my own disc :-). But I think it will be only useful with new effective up-
> converting dyes or something the like.
>
> Cheers,
> zdenek
> P.S.:
> I didn't mean unsuitable, i just thing it is not the preferred combination,
> at least for now.
>
>
> ---------- Původní zpráva ----------
> Od: George McNamara <[hidden email]>
> Datum: 25. 2. 2013
> Předmět: Re: multiphoton spinning disc
>
> "*****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> Zdenek: "it is a simple matter, multiphoton is not suitable for spinning
> disc as it's not suitable for widefield. "
>
> Shimozawa et al: "Second, the generation of out-of-focus light is
> prevented by two-photon excitation that achieves selective-plane
> illumination. </pubmed/23401517>"
>
> *Shimozawa* T, Yamagata K, Kondo T, Hayashi S, Shitamukai A, Konno D,
> Matsuzaki F, Takayama J, Onami S, Nakayama H, Kosugi Y, Watanabe TM,
> Fujita K, Mimori-Kiyosue Y. Improving spinning disk confocal microscopy
> by preventing pinhole cross-talk for intravital imaging.
> </pubmed/23401517>* *Proc Natl Acad Sci U S A. 2013 Feb 11. PMID: 23401517
>
> A recent key requirement in life sciences is the observation of
> biological processes in their natural in vivo context. However, imaging
> techniques that allow fast imaging with higher resolution in 3D thick
> specimens are still limited. Spinning disk confocal microscopy using a
> Yokogawa Confocal Scanner Unit, which offers high-speed multipoint
> confocal live imaging, has been found to have wide utility among cell
> biologists. A conventional Confocal Scanner Unit configuration, however,
> is not optimized for thick specimens, for which the background noise
> attributed to "pinhole cross-talk," which is unintended pinhole
> transmission of out-of-focus light, limits overall performance in focal
> discrimination and reduces confocal capability. Here, we improve
> spinning disk confocal microscopy by eliminating pinhole cross-talk.
> First, the amount of pinhole cross-talk is reduced by increasing the
> interpinhole distance. Second, the generation of out-of-focus light is
> prevented by two-photon excitation that achieves selective-plane
> illumination. We evaluate the effect of these modifications and test the
> applicability to the live imaging of green fluorescent
> protein-expressing model animals. As demonstrated by visualizing the
> fine details of the 3D cell shape and submicron-size cytoskeletal
> structures inside animals, these strategies dramatically improve
> higher-resolution intravital imaging. </pubmed/23401517>*
> *
>
> On 2/16/2013 2:04 AM, Zdenek Svindrych wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi guys,
> >
> > it is a simple matter, multiphoton is not suitable for spinning disc as
> it's not suitable for widefield.
> > It's a nonlinear process and with the same average intensity of
> illumination the signal will be the higher the higher is the peak
> intensity.
> To get intense peak illumination you can focus it spatially (to a
> diffraction-limited spot) or 'temporally' (with fs pulses); one has to use
> both approaches to get appreciable results with common fluorescent dyes...
> >
> > Shortening the pulses is vital, but you cannot go to 1fs - the spectrum
> would be too broad. I don't think the microlenses could stretch the pulses,
> because they are fairly thin. The do destroy the wavefronts a bit (coz the
> are so small...), but this is cleaned up by the pinholes...
> >
> > Simply put, in a regular confocal you are creating one
> diffraction-limited
> spot, in a spinning disc you work with thousand spots. So you would need
> thousand times more power (assuming the same pulse duration). Bu then the
> thermal effects (assuming the contribution of the multiphoton processes is
> negligible) also increase thousand times...
> >
> > And now the solutions:
> > 1) use less pinholes - you hardly can modify your disc, so you decrease
> FOV instead, like in that paper.
> > 2) use better dyes - there are special up-conversion dyes that work even
> in widefield and without femtoseconds, so some could be found that would
> work here.
> > 3) more powerful pulses - not necessarily shorter, if I got the money I
> would find someone to build a pulse picker and an amplifier for me. I think
> 80 kHz repetition with thousand times the peak power could work and the
> scanning artifacts wouldn't be too severe (you may imagine this can't work
> with regular confocal...)
> >
> > Sorry for the lengthy post, bu I didn't want to disclose my brightest
> ideas on the first few lines :-).
> >
> > Cheers,
> >
> > zdenek
> >
> > ---------- Původní zpráva ----------
> > Od: Unruh, Jay
> > Datum: 15. 2. 2013
> > Předmět: Re: multiphoton spinning disc
> >
> > I would imagine that quite a bit could be gained by going to a shorter
> pulsed laser and performing dispersion compensation. I would guess that the
> micro lens array introduces a significant amount of dispersion. Of course
> many of the shorter pulsed lasers aren't tunable either. Large field of
> view
> applications with longer pulsed lasers may result in an undesired amount of
> heating anyway. Del Mar ventures (no commercial interest) actually sells a
> DIY kit for a<20 fs laser for much less than a single box tunable system.
> >
> > Jay
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Craig Brideau
> > Sent: Friday, February 15, 2013 11:46 AM
> > To: [hidden email]
> > Subject: Re: multiphoton spinning disc
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > If you could sacrifice tunability, fiber lasers are pretty much there, at
> least for 1040nm. Time-bandwidth-products (the company, not the unit of
> merit for pulsed lasers) makes a death ray called the Fortis that outputs
> 50
> W at 50 MHz...
> >
> > Craig
> >
> >
> > On Fri, Feb 15, 2013 at 9:37 AM, Tim Feinstein wrote:
> >
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Hello all,
> >>
> >> There is an interesting demonstration of multiphoton spinning disc
> >> confocal imaging in the latest PNAS by Yuko Mimori-Kosue from RIKEN.
> >> It shows the expected Z-resolution, depth and s/n improvements (maybe
> >> a little better than due to pinhole refinement), but their last
> paragraph
> is a doozy.
> >>
> >> "In putting the system to practical use, the available output laser
> >> power is the limiting factor for the illumination area and the maximum
> >> imaging depth. Even with the two-photon lasers having the highest
> >> levels of output power currently available, we could trigger
> >> two-photon absorption for only ∼40-μm-diameter areas of GFP-expressing
> >> animals (less than 10% of the effective frame size with a 60×
> >> objective; Fig. S1B). Lasers with roughly
> >> 5–10 times the power would be required. The higher laser power is also
> >> required to excite red fluorescent proteins, which can be excited at
> >> wavelengths between 1,000 and 1,200 nm, at which the output power of
> >> the existing mode-locked laser decreases to ∼15% of peak power. "
> >>
> >> Is there any chance of seeing a ti-sapphire with 5 to 10-fold more
> power?
> >> Would it cost 5-10 times as much? It sounds like a nice approach but
> >> maybe not so much if you need a military LASER to use it. On the
> >> other hand maybe resonant two-photon (or slit-scanning, with
> >> sacrifices) can already deliver similar performance. Thoughts
> appreciated.
> >>
> >> All the best,
> >>
> >>
> >> TF
> >>
> >> Timothy Feinstein, PhD
> >> Visiting Research Associate
> >> Laboratory for GPCR Biology
> >> Dept. of Pharmacology& Chemical Biology University of Pittsburgh,
> >> School of Medicine BST W1301, 200 Lothrop St.
> >> Pittsburgh, PA 15261
> >>
> >>"
>