Live cell imaging with multiphoton confocal

[Knecht, David]

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I have limited experience with multi-photon confocal microscopes.  I tried imaging live cells with one a while back and was unimpressed with the results as compared to a standard laser scanning instrument.  If you had a new single photon instrument and a new multi-photon instrument sitting side by side in your facility (and I presume some of you do), which would you choose for imaging  fixed cells or live cells in culture (not trying to go deep into tissue)?  Thanks- Dave

Dr. David Knecht    
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[Vladimir Ghukasyan-2]

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Hi David,

For this kind of application MP has no advantage other than no
out-of-focus photobleaching. CLSM is a better solution.

With regards,
Vladimir Ghukasyan
Confocal and Multiphoton Imaging Facility
Neuroscience Center
University of North Carolina
115 Mason Farm Rd.,
Chapel Hill 27599-7250
Tel.: [phone]


On Fri, Apr 22, 2011 at 1:29 PM, David Knecht charter
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I have limited experience with multi-photon confocal microscopes.  I tried imaging live cells with one a while back and was unimpressed with the results as compared to a standard laser scanning instrument.  If you had a new single photon instrument and a new multi-photon instrument sitting side by side in your facility (and I presume some of you do), which would you choose for imaging  fixed cells or live cells in culture (not trying to go deep into tissue)?  Thanks- Dave
>
> Dr. David Knecht
> Department of Molecular and Cell Biology
> Co-head Flow Cytometry and Confocal Microscopy Facility
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)
>

[Armstrong, Brian]

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David, I think from a theoretical viewpoint the multiphoton would have worse resolution because of the higher lambda (d = 900nm * 0.61 / NA), but better resolution due to smaller focal spot of 2P, so the resolution should be roughly equal to CLSM.

In practice (in my hands at least) CLSM images appear to have much better resolution. I would choose CLSM over 2P for the application you describe.
Part of the problem as I see it comes from the wide excitation band of 2PE, meaning that you are exciting many fluorophores with a single wavelength, and you do not have this problem when using a "vis" laser.

Also, I think per unit area, in the focal spot, the 2PE will kill live cells faster than 1PE.

Cheers,  

Brian D Armstrong PhD
Assistant Research Professor
Light Microscopy Core
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872

http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht charter
Sent: Friday, April 22, 2011 10:29 AM
To: [hidden email]
Subject: Live cell imaging with multiphoton confocal

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I have limited experience with multi-photon confocal microscopes.  I tried imaging live cells with one a while back and was unimpressed with the results as compared to a standard laser scanning instrument.  If you had a new single photon instrument and a new multi-photon instrument sitting side by side in your facility (and I presume some of you do), which would you choose for imaging  fixed cells or live cells in culture (not trying to go deep into tissue)?  Thanks- Dave

Dr. David Knecht    
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)


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[Kate Luby-Phelps]

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Multiphoton has no clear advantage for cells in culture. For cell culture samples,
we use two photon  only to excite DAPI or other UV and near-UV fluors since we
had to make a choice between the Ti:S and a 405 laser on our scope.

Kate Luby-Phelps

[Lemasters, John J.]

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Multiphoton microscopy is helpful to excite UV-absorbing fluorophores like NAD(P)H that cannot otherwise be excited without specialized lasers and optics.

--
John J. Lemasters, MD, PhD
Professor and GlaxoSmithKline Distinguished Endowed Chair
Director, Center for Cell Death, Injury and Regeneration
Departments of Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular Biology
Medical University of South Carolina
QF213 Quadrangle Building
280 Calhoun Street, MSC 140
Charleston, SC 29425

Office: 843-792-2153
Lab: 843-792-3530
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kate Luby-Phelps
Sent: Saturday, April 23, 2011 11:22 AM
To: [hidden email]
Subject: Re: Live cell imaging with multiphoton confocal

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Multiphoton has no clear advantage for cells in culture. For cell culture samples, we use two photon  only to excite DAPI or other UV and near-UV fluors since we had to make a choice between the Ti:S and a 405 laser on our scope.

Kate Luby-Phelps

[Mark Cannell]

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Err, is a Ti:S not a very "specialized laser" ?
;-)

>
> Multiphoton microscopy is helpful to excite UV-absorbing  
> fluorophores like NAD(P)H that cannot otherwise be excited without  
> specialized lasers and optics.
>

[Knecht, David]

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Is there any disadvantage to multiphoton for cultured cells (besides cost and complexity)? Cell viability?  Phototoxicity?  Dave

On Apr 23, 2011, at 11:21 AM, Kate Luby-Phelps wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Multiphoton has no clear advantage for cells in culture. For cell culture samples,
> we use two photon  only to excite DAPI or other UV and near-UV fluors since we
> had to make a choice between the Ti:S and a 405 laser on our scope.
>
> Kate Luby-Phelps

Dr. David Knecht    
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[Lemasters, John J.]

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Hi David and Mark,

Good points. By specialized laser and optics, I meant a UV argon laser and UV quartz optics. I had such a beast once to image NADH, but multiphoton is much easier and more versatile.

Regarding multiphoton photodamage, David Piston has a paper showing that 2 photon imaging is more damaging than 1 photon imaging for cells in culture and that the multiphoton photodamage has a 3 photon power profile. Hence most folks will tell you to use conventional 1 photon imaging for cells in culture -- but you'll still need 2-photon microscopy to image things like NADH and NADPH unless, of course, you have a UV laser and UV optics.

John

--
John J. Lemasters, MD, PhD
Professor and GlaxoSmithKline Distinguished Endowed Chair
Director, Center for Cell Death, Injury and Regeneration
Departments of Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular Biology
Medical University of South Carolina
QF213 Quadrangle Building
280 Calhoun Street, MSC 140
Charleston, SC 29425

Office: 843-792-2153
Lab: 843-792-3530
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht charter
Sent: Saturday, April 23, 2011 6:03 PM
To: [hidden email]
Subject: Re: Live cell imaging with multiphoton confocal

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Is there any disadvantage to multiphoton for cultured cells (besides cost and complexity)? Cell viability?  Phototoxicity?  Dave

On Apr 23, 2011, at 11:21 AM, Kate Luby-Phelps wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Multiphoton has no clear advantage for cells in culture. For cell
> culture samples, we use two photon  only to excite DAPI or other UV
> and near-UV fluors since we had to make a choice between the Ti:S and a 405 laser on our scope.
>
> Kate Luby-Phelps

Dr. David Knecht    
Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[Lemasters, John J.]

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P.S. UV-transmitting optics don't have to be quartz. Many lens used for UV illumination of Fura2 or Indo1 transmit UV but are still not chromatically corrected in the UV as is desirable if not necessary for point scanning confocal.

--
John J. Lemasters, MD, PhD
Professor and GlaxoSmithKline Distinguished Endowed Chair
Director, Center for Cell Death, Injury and Regeneration
Departments of Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular Biology
Medical University of South Carolina
QF213 Quadrangle Building
280 Calhoun Street, MSC 140
Charleston, SC 29425

Office: 843-792-2153
Lab: 843-792-3530
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Lemasters, John
Sent: Saturday, April 23, 2011 9:21 PM
To: [hidden email]
Subject: Re: Live cell imaging with multiphoton confocal

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Hi David and Mark,

Good points. By specialized laser and optics, I meant a UV argon laser and UV quartz optics. I had such a beast once to image NADH, but multiphoton is much easier and more versatile.

Regarding multiphoton photodamage, David Piston has a paper showing that 2 photon imaging is more damaging than 1 photon imaging for cells in culture and that the multiphoton photodamage has a 3 photon power profile. Hence most folks will tell you to use conventional 1 photon imaging for cells in culture -- but you'll still need 2-photon microscopy to image things like NADH and NADPH unless, of course, you have a UV laser and UV optics.

John

--
John J. Lemasters, MD, PhD
Professor and GlaxoSmithKline Distinguished Endowed Chair Director, Center for Cell Death, Injury and Regeneration Departments of Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular Biology Medical University of South Carolina
QF213 Quadrangle Building
280 Calhoun Street, MSC 140
Charleston, SC 29425

Office: 843-792-2153
Lab: 843-792-3530
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht charter
Sent: Saturday, April 23, 2011 6:03 PM
To: [hidden email]
Subject: Re: Live cell imaging with multiphoton confocal

*****
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Is there any disadvantage to multiphoton for cultured cells (besides cost and complexity)? Cell viability?  Phototoxicity?  Dave

On Apr 23, 2011, at 11:21 AM, Kate Luby-Phelps wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Multiphoton has no clear advantage for cells in culture. For cell
> culture samples, we use two photon  only to excite DAPI or other UV
> and near-UV fluors since we had to make a choice between the Ti:S and a 405 laser on our scope.
>
> Kate Luby-Phelps

Dr. David Knecht    
Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[George McNamara]

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Hi David,

The tuning range of Ti:Sapphire (ex. 690-1040 nm) used to be problematic
for exciting far red and infrared fluorophores. There were some
exceptions, such as Alexa Fluor 488m 568, 594, and 633 all exciting well
around 755 nm (Fig 5D of Dickinson et al 2003 J Biomed Optics 8:
329-338), but since you are ok with cost and complexity, adding an
OPO(s) and/or multiple MP lasers, gives you full range (see
http://www.coherent.com/products/?1570/Chameleon-OPO-Tuning-Range for OPO).

Depending on cell type and physiological needs, you may be able to
suppress phototoxicity by lowering O2 - cell culture at ~18% O2 is
something of an artifact for many cell types - by using catalase/glucose
oxidase/glucose or Oxyrase (for the latter, www.oxyrase.com, see
Waterman-Storer 1993 and Mikhailov 1995 Cell Motil Cytokseleton).

Enjoy,

George

On 4/23/2011 6:03 PM, David Knecht charter wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Is there any disadvantage to multiphoton for cultured cells (besides cost and complexity)? Cell viability?  Phototoxicity?  Dave
>
> On Apr 23, 2011, at 11:21 AM, Kate Luby-Phelps wrote:
>
>    
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Multiphoton has no clear advantage for cells in culture. For cell culture samples,
>> we use two photon  only to excite DAPI or other UV and near-UV fluors since we
>> had to make a choice between the Ti:S and a 405 laser on our scope.
>>
>> Kate Luby-Phelps
>>      
> Dr. David Knecht
> Department of Molecular and Cell Biology
> Co-head Flow Cytometry and Confocal Microscopy Facility
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[Cameron, Lisa]

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Hello -

Question for those of you who have a Leica SP5X (the white light laser version)
-

What has been your experience with maintenance and need for service on the white
light laser?
Do people recommend getting the service contract on the laser once the warrantee
at purchase is over?

I run a microscopy facility and have service contracts on all the equipment and
do feel it's a good idea especially in a facility. However Leica has made the
white light laser service contract separate from the main confocal service
contract and the price of both more than doubles the yearly cost of the SP5
alone.

Thanks for any input (and feel free to contact me off list).

- Lisa

---------------------------------------
Lisa Cameron
Dana Farber Cancer Institute
Boston, MA
[hidden email]


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[Craig Brideau]

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I second the previous lister's opinions.  At the end of the day MP is really
only good for thick tissue sections.  If you are looking at cell layers just
use a conventional 1P confocal.  The key advantage of 2P is penetration
depth, which is moot when you are looking at cells.  That said, John's
comments about shorter UV imaging are still valid; it's easier to get a NIR
Ti:Saph beam through conventional optics than a very UV beam.  This only
applies if you are using dyes that need very UV excitation of course.

Craig




On Sun, Apr 24, 2011 at 6:29 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
> *****
>
> Hi David,
>
> The tuning range of Ti:Sapphire (ex. 690-1040 nm) used to be problematic
> for exciting far red and infrared fluorophores. There were some exceptions,
> such as Alexa Fluor 488m 568, 594, and 633 all exciting well around 755 nm
> (Fig 5D of Dickinson et al 2003 J Biomed Optics 8: 329-338), but since you
> are ok with cost and complexity, adding an OPO(s) and/or multiple MP lasers,
> gives you full range (see
> http://www.coherent.com/products/?1570/Chameleon-OPO-Tuning-Range for
> OPO).
>
> Depending on cell type and physiological needs, you may be able to suppress
> phototoxicity by lowering O2 - cell culture at ~18% O2 is something of an
> artifact for many cell types - by using catalase/glucose oxidase/glucose or
> Oxyrase (for the latter, www.oxyrase.com, see Waterman-Storer 1993 and
> Mikhailov 1995 Cell Motil Cytokseleton).
>
> Enjoy,
>
> George
>
>
> On 4/23/2011 6:03 PM, David Knecht charter wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Is there any disadvantage to multiphoton for cultured cells (besides cost
>> and complexity)? Cell viability?  Phototoxicity?  Dave
>>
>> On Apr 23, 2011, at 11:21 AM, Kate Luby-Phelps wrote:
>>
>>
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Multiphoton has no clear advantage for cells in culture. For cell culture
>>> samples,
>>> we use two photon  only to excite DAPI or other UV and near-UV fluors
>>> since we
>>> had to make a choice between the Ti:S and a 405 laser on our scope.
>>>
>>> Kate Luby-Phelps
>>>
>>>
>> Dr. David Knecht
>> Department of Molecular and Cell Biology
>> Co-head Flow Cytometry and Confocal Microscopy Facility
>> U-3125
>> 91 N. Eagleville Rd.
>> University of Connecticut
>> Storrs, CT 06269
>> 860-486-2200
>> 860-486-4331 (fax)
>>
>>
>>
>
>
> --
>
>
> George McNamara, PhD
> Analytical Imaging Core Facility
> University of Miami
>

[Rosemary.White]

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Dear all,

Haven't followed this discussion closely, but would one advantage of 2P lie
in the ability to uncage caged compounds - fluorescent tracers, for example,
within a single cell?  We've used UV uncaging and while you get most
uncaging in the target cell, you also get cones of uncaging above and below
the plane of focus.  Seems that 2P would overcome this problem.

Rosemary White

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
E [hidden email]



On 28/04/11 10:10 AM, "Craig Brideau" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I second the previous lister's opinions.  At the end of the day MP is really
> only good for thick tissue sections.  If you are looking at cell layers just
> use a conventional 1P confocal.  The key advantage of 2P is penetration
> depth, which is moot when you are looking at cells.  That said, John's
> comments about shorter UV imaging are still valid; it's easier to get a NIR
> Ti:Saph beam through conventional optics than a very UV beam.  This only
> applies if you are using dyes that need very UV excitation of course.
>
> Craig
>
>
>
>
> On Sun, Apr 24, 2011 at 6:29 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
>> *****
>>
>> Hi David,
>>
>> The tuning range of Ti:Sapphire (ex. 690-1040 nm) used to be problematic
>> for exciting far red and infrared fluorophores. There were some exceptions,
>> such as Alexa Fluor 488m 568, 594, and 633 all exciting well around 755 nm
>> (Fig 5D of Dickinson et al 2003 J Biomed Optics 8: 329-338), but since you
>> are ok with cost and complexity, adding an OPO(s) and/or multiple MP lasers,
>> gives you full range (see
>> http://www.coherent.com/products/?1570/Chameleon-OPO-Tuning-Range for
>> OPO).
>>
>> Depending on cell type and physiological needs, you may be able to suppress
>> phototoxicity by lowering O2 - cell culture at ~18% O2 is something of an
>> artifact for many cell types - by using catalase/glucose oxidase/glucose or
>> Oxyrase (for the latter, www.oxyrase.com, see Waterman-Storer 1993 and
>> Mikhailov 1995 Cell Motil Cytokseleton).
>>
>> Enjoy,
>>
>> George
>>
>>
>> On 4/23/2011 6:03 PM, David Knecht charter wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Is there any disadvantage to multiphoton for cultured cells (besides cost
>>> and complexity)? Cell viability?  Phototoxicity?  Dave
>>>
>>> On Apr 23, 2011, at 11:21 AM, Kate Luby-Phelps wrote:
>>>
>>>
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Multiphoton has no clear advantage for cells in culture. For cell culture
>>>> samples,
>>>> we use two photon  only to excite DAPI or other UV and near-UV fluors
>>>> since we
>>>> had to make a choice between the Ti:S and a 405 laser on our scope.
>>>>
>>>> Kate Luby-Phelps
>>>>
>>>>
>>> Dr. David Knecht
>>> Department of Molecular and Cell Biology
>>> Co-head Flow Cytometry and Confocal Microscopy Facility
>>> U-3125
>>> 91 N. Eagleville Rd.
>>> University of Connecticut
>>> Storrs, CT 06269
>>> 860-486-2200
>>> 860-486-4331 (fax)
>>>
>>>
>>>
>>
>>
>> --
>>
>>
>> George McNamara, PhD
>> Analytical Imaging Core Facility
>> University of Miami
>>

[Mark Cannell]

*****
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Actually, that is a very good use of 2P and it works really well  
(shameless self citation follows)
See:


Cannell, M.B., Jacobs, M., Donaldson, P. & Soeller, C. (2004) Probing  
microscopic diffusion by 2-photon flash photolysis: Visualization of  
isotropic and anisotropic diffusion between lens fiber cells. Micr.  
Res. Tech. 63(1) 50-57

  Soeller, C., Jacobs, M.D., Jones, K.T., Ellis-Davies, G.C.R.,  
Donaldson, P.J. and Cannell, M.B. (2003)  Probing microscopic  
transport and release fluxes in cells by two-photon flash photolysis.  
J. Biomed. Opt. 8:418-427

Soeller, C. & Cannell, M.B. (2002) Estimation of the sarcoplasmic  
reticulum Ca2+ release flux underlying Ca2+ sparks. Biophys. J. 82.  
2396-2414.

Cheers


On 28/04/2011, at 12:48 PM, Rosemary White wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> Haven't followed this discussion closely, but would one advantage of  
> 2P lie
> in the ability to uncage caged compounds - fluorescent tracers, for  
> example,
> within a single cell?  We've used UV uncaging and while you get most
> uncaging in the target cell, you also get cones of uncaging above  
> and below
> the plane of focus.  Seems that 2P would overcome this problem.
>
> Rosemary White
>
> Dr Rosemary White
> CSIRO Plant Industry
> GPO Box 1600
> Canberra, ACT 2601
> Australia
>
> T 61 2 6246 5475
> F 61 2 6246 5334
> E [hidden email]
>
>
>
> On 28/04/11 10:10 AM, "Craig Brideau" <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I second the previous lister's opinions.  At the end of the day MP  
>> is really
>> only good for thick tissue sections.  If you are looking at cell  
>> layers just
>> use a conventional 1P confocal.  The key advantage of 2P is  
>> penetration
>> depth, which is moot when you are looking at cells.  That said,  
>> John's
>> comments about shorter UV imaging are still valid; it's easier to  
>> get a NIR
>> Ti:Saph beam through conventional optics than a very UV beam.  This  
>> only
>> applies if you are using dyes that need very UV excitation of course.
>>
>> Craig
>>
>>
>>
>>
>> On Sun, Apr 24, 2011 at 6:29 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
>>> *****
>>>
>>> Hi David,
>>>
>>> The tuning range of Ti:Sapphire (ex. 690-1040 nm) used to be  
>>> problematic
>>> for exciting far red and infrared fluorophores. There were some  
>>> exceptions,
>>> such as Alexa Fluor 488m 568, 594, and 633 all exciting well  
>>> around 755 nm
>>> (Fig 5D of Dickinson et al 2003 J Biomed Optics 8: 329-338), but  
>>> since you
>>> are ok with cost and complexity, adding an OPO(s) and/or multiple  
>>> MP lasers,
>>> gives you full range (see
>>> http://www.coherent.com/products/?1570/Chameleon-OPO-Tuning-Range 
>>> for
>>> OPO).
>>>
>>> Depending on cell type and physiological needs, you may be able to  
>>> suppress
>>> phototoxicity by lowering O2 - cell culture at ~18% O2 is  
>>> something of an
>>> artifact for many cell types - by using catalase/glucose oxidase/
>>> glucose or
>>> Oxyrase (for the latter, www.oxyrase.com, see Waterman-Storer 1993  
>>> and
>>> Mikhailov 1995 Cell Motil Cytokseleton).
>>>
>>> Enjoy,
>>>
>>> George
>>>
>>>
>>> On 4/23/2011 6:03 PM, David Knecht charter wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Is there any disadvantage to multiphoton for cultured cells  
>>>> (besides cost
>>>> and complexity)? Cell viability?  Phototoxicity?  Dave
>>>>
>>>> On Apr 23, 2011, at 11:21 AM, Kate Luby-Phelps wrote:
>>>>
>>>>
>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> Multiphoton has no clear advantage for cells in culture. For  
>>>>> cell culture
>>>>> samples,
>>>>> we use two photon  only to excite DAPI or other UV and near-UV  
>>>>> fluors
>>>>> since we
>>>>> had to make a choice between the Ti:S and a 405 laser on our  
>>>>> scope.
>>>>>
>>>>> Kate Luby-Phelps
>>>>>
>>>>>
>>>> Dr. David Knecht
>>>> Department of Molecular and Cell Biology
>>>> Co-head Flow Cytometry and Confocal Microscopy Facility
>>>> U-3125
>>>> 91 N. Eagleville Rd.
>>>> University of Connecticut
>>>> Storrs, CT 06269
>>>> 860-486-2200
>>>> 860-486-4331 (fax)
>>>>
>>>>
>>>>
>>>
>>>
>>> --
>>>
>>>
>>> George McNamara, PhD
>>> Analytical Imaging Core Facility
>>> University of Miami
>>>

[Craig Brideau]

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

How much uncaging occurs outside the focal region in single photon?  If you
don't have your laser turned up to much the energy density should only
exceed the uncaging threshold near the focal point, yes?

Craig


On Wed, Apr 27, 2011 at 6:48 PM, Rosemary White <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> Haven't followed this discussion closely, but would one advantage of 2P lie
> in the ability to uncage caged compounds - fluorescent tracers, for
> example,
> within a single cell?  We've used UV uncaging and while you get most
> uncaging in the target cell, you also get cones of uncaging above and below
> the plane of focus.  Seems that 2P would overcome this problem.
>
> Rosemary White
>
> Dr Rosemary White
> CSIRO Plant Industry
> GPO Box 1600
> Canberra, ACT 2601
> Australia
>
> T 61 2 6246 5475
> F 61 2 6246 5334
> E [hidden email]
>
>
>
> On 28/04/11 10:10 AM, "Craig Brideau" <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > I second the previous lister's opinions.  At the end of the day MP is
> really
> > only good for thick tissue sections.  If you are looking at cell layers
> just
> > use a conventional 1P confocal.  The key advantage of 2P is penetration
> > depth, which is moot when you are looking at cells.  That said, John's
> > comments about shorter UV imaging are still valid; it's easier to get a
> NIR
> > Ti:Saph beam through conventional optics than a very UV beam.  This only
> > applies if you are using dyes that need very UV excitation of course.
> >
> > Craig
> >
> >
> >
> >
> > On Sun, Apr 24, 2011 at 6:29 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
> >> *****
> >>
> >> Hi David,
> >>
> >> The tuning range of Ti:Sapphire (ex. 690-1040 nm) used to be problematic
> >> for exciting far red and infrared fluorophores. There were some
> exceptions,
> >> such as Alexa Fluor 488m 568, 594, and 633 all exciting well around 755
> nm
> >> (Fig 5D of Dickinson et al 2003 J Biomed Optics 8: 329-338), but since
> you
> >> are ok with cost and complexity, adding an OPO(s) and/or multiple MP
> lasers,
> >> gives you full range (see
> >> http://www.coherent.com/products/?1570/Chameleon-OPO-Tuning-Range for
> >> OPO).
> >>
> >> Depending on cell type and physiological needs, you may be able to
> suppress
> >> phototoxicity by lowering O2 - cell culture at ~18% O2 is something of
> an
> >> artifact for many cell types - by using catalase/glucose oxidase/glucose
> or
> >> Oxyrase (for the latter, www.oxyrase.com, see Waterman-Storer 1993 and
> >> Mikhailov 1995 Cell Motil Cytokseleton).
> >>
> >> Enjoy,
> >>
> >> George
> >>
> >>
> >> On 4/23/2011 6:03 PM, David Knecht charter wrote:
> >>
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>> *****
> >>>
> >>> Is there any disadvantage to multiphoton for cultured cells (besides
> cost
> >>> and complexity)? Cell viability?  Phototoxicity?  Dave
> >>>
> >>> On Apr 23, 2011, at 11:21 AM, Kate Luby-Phelps wrote:
> >>>
> >>>
> >>>
> >>>> *****
> >>>> To join, leave or search the confocal microscopy listserv, go to:
> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>>> *****
> >>>>
> >>>> Multiphoton has no clear advantage for cells in culture. For cell
> culture
> >>>> samples,
> >>>> we use two photon  only to excite DAPI or other UV and near-UV fluors
> >>>> since we
> >>>> had to make a choice between the Ti:S and a 405 laser on our scope.
> >>>>
> >>>> Kate Luby-Phelps
> >>>>
> >>>>
> >>> Dr. David Knecht
> >>> Department of Molecular and Cell Biology
> >>> Co-head Flow Cytometry and Confocal Microscopy Facility
> >>> U-3125
> >>> 91 N. Eagleville Rd.
> >>> University of Connecticut
> >>> Storrs, CT 06269
> >>> 860-486-2200
> >>> 860-486-4331 (fax)
> >>>
> >>>
> >>>
> >>
> >>
> >> --
> >>
> >>
> >> George McNamara, PhD
> >> Analytical Imaging Core Facility
> >> University of Miami
> >>
>

[Mark Cannell]

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

At low powers the uncaging is linear in intensity so there should be  
no threshold.  So, the uncaging volume at low powers is the same as  
the PSF and for 2P its the 2P PSF. If ground state depletion develops  
the FWHM of the focal uncaging spot grows (of course).

Mark

On 28/04/2011, at 2:18 PM, Craig Brideau wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> How much uncaging occurs outside the focal region in single photon?  
> If you
> don't have your laser turned up to much the energy density should only
> exceed the uncaging threshold near the focal point, yes?
>
> Craig
>
>
> On Wed, Apr 27, 2011 at 6:48 PM, Rosemary White <[hidden email]
> >wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear all,
>>
>> Haven't followed this discussion closely, but would one advantage  
>> of 2P lie
>> in the ability to uncage caged compounds - fluorescent tracers, for
>> example,
>> within a single cell?  We've used UV uncaging and while you get most
>> uncaging in the target cell, you also get cones of uncaging above  
>> and below
>> the plane of focus.  Seems that 2P would overcome this problem.
>>
>> Rosemary White
>>
>> Dr Rosemary White
>> CSIRO Plant Industry
>> GPO Box 1600
>> Canberra, ACT 2601
>> Australia
>>
>> T 61 2 6246 5475
>> F 61 2 6246 5334
>> E [hidden email]
>>
>>
>>
>> On 28/04/11 10:10 AM, "Craig Brideau" <[hidden email]>  
>> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> I second the previous lister's opinions.  At the end of the day MP  
>>> is
>> really
>>> only good for thick tissue sections.  If you are looking at cell  
>>> layers
>> just
>>> use a conventional 1P confocal.  The key advantage of 2P is  
>>> penetration
>>> depth, which is moot when you are looking at cells.  That said,  
>>> John's
>>> comments about shorter UV imaging are still valid; it's easier to  
>>> get a
>> NIR
>>> Ti:Saph beam through conventional optics than a very UV beam.  
>>> This only
>>> applies if you are using dyes that need very UV excitation of  
>>> course.
>>>
>>> Craig
>>>
>>>
>>>
>>>
>>> On Sun, Apr 24, 2011 at 6:29 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
>>>> *****
>>>>
>>>> Hi David,
>>>>
>>>> The tuning range of Ti:Sapphire (ex. 690-1040 nm) used to be  
>>>> problematic
>>>> for exciting far red and infrared fluorophores. There were some
>> exceptions,
>>>> such as Alexa Fluor 488m 568, 594, and 633 all exciting well  
>>>> around 755
>> nm
>>>> (Fig 5D of Dickinson et al 2003 J Biomed Optics 8: 329-338), but  
>>>> since
>> you
>>>> are ok with cost and complexity, adding an OPO(s) and/or multiple  
>>>> MP
>> lasers,
>>>> gives you full range (see
>>>> http://www.coherent.com/products/?1570/Chameleon-OPO-Tuning-Range 
>>>> for
>>>> OPO).
>>>>
>>>> Depending on cell type and physiological needs, you may be able to
>> suppress
>>>> phototoxicity by lowering O2 - cell culture at ~18% O2 is  
>>>> something of
>> an
>>>> artifact for many cell types - by using catalase/glucose oxidase/
>>>> glucose
>> or
>>>> Oxyrase (for the latter, www.oxyrase.com, see Waterman-Storer  
>>>> 1993 and
>>>> Mikhailov 1995 Cell Motil Cytokseleton).
>>>>
>>>> Enjoy,
>>>>
>>>> George
>>>>
>>>>
>>>> On 4/23/2011 6:03 PM, David Knecht charter wrote:
>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> Is there any disadvantage to multiphoton for cultured cells  
>>>>> (besides
>> cost
>>>>> and complexity)? Cell viability?  Phototoxicity?  Dave
>>>>>
>>>>> On Apr 23, 2011, at 11:21 AM, Kate Luby-Phelps wrote:
>>>>>
>>>>>
>>>>>
>>>>>> *****
>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>> *****
>>>>>>
>>>>>> Multiphoton has no clear advantage for cells in culture. For cell
>> culture
>>>>>> samples,
>>>>>> we use two photon  only to excite DAPI or other UV and near-UV  
>>>>>> fluors
>>>>>> since we
>>>>>> had to make a choice between the Ti:S and a 405 laser on our  
>>>>>> scope.
>>>>>>
>>>>>> Kate Luby-Phelps
>>>>>>
>>>>>>
>>>>> Dr. David Knecht
>>>>> Department of Molecular and Cell Biology
>>>>> Co-head Flow Cytometry and Confocal Microscopy Facility
>>>>> U-3125
>>>>> 91 N. Eagleville Rd.
>>>>> University of Connecticut
>>>>> Storrs, CT 06269
>>>>> 860-486-2200
>>>>> 860-486-4331 (fax)
>>>>>
>>>>>
>>>>>
>>>>
>>>>
>>>> --
>>>>
>>>>
>>>> George McNamara, PhD
>>>> Analytical Imaging Core Facility
>>>> University of Miami
>>>>
>>

[Sudipta Maiti]

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

Other than UV dyes, native chromophores , such as NaDh, serotoni etc., are
only accessible through multiphoton for live cell UV imaging. I can send a
large list of our work in this area (off the list)if anyone cares to know
about it.
Sudipta

which are On Wed, 27 Apr 2011 18:10:16 -0600, Craig Brideau wrote

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I second the previous lister's opinions.  At the end of the day MP
> is really only good for thick tissue sections.  If you are looking
> at cell layers just use a conventional 1P confocal.  The key
> advantage of 2P is penetration depth, which is moot when you are
> looking at cells.  That said, John's comments about shorter UV
> imaging are still valid; it's easier to get a NIR Ti:Saph beam
> through conventional optics than a very UV beam.  This only applies
> if you are using dyes that need very UV excitation of course.
>
> Craig
>
> On Sun, Apr 24, 2011 at 6:29 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
> > *****
> >
> > Hi David,
> >
> > The tuning range of Ti:Sapphire (ex. 690-1040 nm) used to be problematic
> > for exciting far red and infrared fluorophores. There were some

exceptions,
> > such as Alexa Fluor 488m 568, 594, and 633 all exciting well around 755 nm
> > (Fig 5D of Dickinson et al 2003 J Biomed Optics 8: 329-338), but since you
> > are ok with cost and complexity, adding an OPO(s) and/or multiple MP
lasers,
> > gives you full range (see
> > http://www.coherent.com/products/?1570/Chameleon-OPO-Tuning-Range for
> > OPO).
> >
> > Depending on cell type and physiological needs, you may be able to
suppress
> > phototoxicity by lowering O2 - cell culture at ~18% O2 is something of an
> > artifact for many cell types - by using catalase/glucose oxidase/glucose
or

> > Oxyrase (for the latter, www.oxyrase.com, see Waterman-Storer 1993 and
> > Mikhailov 1995 Cell Motil Cytokseleton).
> >
> > Enjoy,
> >
> > George
> >
> >
> > On 4/23/2011 6:03 PM, David Knecht charter wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Is there any disadvantage to multiphoton for cultured cells (besides cost
> >> and complexity)? Cell viability?  Phototoxicity?  Dave
> >>
> >> On Apr 23, 2011, at 11:21 AM, Kate Luby-Phelps wrote:
> >>
> >>
> >>
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>> *****
> >>>
> >>> Multiphoton has no clear advantage for cells in culture. For cell

culture

> >>> samples,
> >>> we use two photon  only to excite DAPI or other UV and near-UV fluors
> >>> since we
> >>> had to make a choice between the Ti:S and a 405 laser on our scope.
> >>>
> >>> Kate Luby-Phelps
> >>>
> >>>
> >> Dr. David Knecht
> >> Department of Molecular and Cell Biology
> >> Co-head Flow Cytometry and Confocal Microscopy Facility
> >> U-3125
> >> 91 N. Eagleville Rd.
> >> University of Connecticut
> >> Storrs, CT 06269
> >> 860-486-2200
> >> 860-486-4331 (fax)
> >>
> >>
> >>
> >
> >
> > --
> >
> >
> > George McNamara, PhD
> > Analytical Imaging Core Facility
> > University of Miami
> >

Dr. Sudipta Maiti
Associate Professor
Dept. of Chemical Sciences
Tata Institute of Fundamental Research
Homi Bhabha Raod, Colaba, Mumbai 400005
Ph. 91-22-2278-2716 / 2539
Fax: [phone]
alternate e-mail: [hidden email]
url: biophotonics.wetpaint.com

[Craig Brideau]

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

So if it is linear with intensity (1P), should it roll off to 1/4 in terms
of distance from the ideal focal point?  Intensity drops to 1/4 at 1 unit
distance from a point source, so I'm approximating the center of the focal
point here.  Am I right in my thinking?

Craig



On Wed, Apr 27, 2011 at 8:38 PM, Mark Cannell <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> At low powers the uncaging is linear in intensity so there should be no
> threshold.  So, the uncaging volume at low powers is the same as the PSF and
> for 2P its the 2P PSF. If ground state depletion develops the FWHM of the
> focal uncaging spot grows (of course).
>
> Mark
>
>
> On 28/04/2011, at 2:18 PM, Craig Brideau wrote:
>
>  *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> How much uncaging occurs outside the focal region in single photon?  If
>> you
>> don't have your laser turned up to much the energy density should only
>> exceed the uncaging threshold near the focal point, yes?
>>
>> Craig
>>
>>
>> On Wed, Apr 27, 2011 at 6:48 PM, Rosemary White <[hidden email]
>> >wrote:
>>
>>  *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear all,
>>>
>>> Haven't followed this discussion closely, but would one advantage of 2P
>>> lie
>>> in the ability to uncage caged compounds - fluorescent tracers, for
>>> example,
>>> within a single cell?  We've used UV uncaging and while you get most
>>> uncaging in the target cell, you also get cones of uncaging above and
>>> below
>>> the plane of focus.  Seems that 2P would overcome this problem.
>>>
>>> Rosemary White
>>>
>>> Dr Rosemary White
>>> CSIRO Plant Industry
>>> GPO Box 1600
>>> Canberra, ACT 2601
>>> Australia
>>>
>>> T 61 2 6246 5475
>>> F 61 2 6246 5334
>>> E [hidden email]
>>>
>>>
>>>
>>> On 28/04/11 10:10 AM, "Craig Brideau" <[hidden email]> wrote:
>>>
>>>  *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> I second the previous lister's opinions.  At the end of the day MP is
>>>>
>>> really
>>>
>>>> only good for thick tissue sections.  If you are looking at cell layers
>>>>
>>> just
>>>
>>>> use a conventional 1P confocal.  The key advantage of 2P is penetration
>>>> depth, which is moot when you are looking at cells.  That said, John's
>>>> comments about shorter UV imaging are still valid; it's easier to get a
>>>>
>>> NIR
>>>
>>>> Ti:Saph beam through conventional optics than a very UV beam.  This only
>>>> applies if you are using dyes that need very UV excitation of course.
>>>>
>>>> Craig
>>>>
>>>>
>>>>
>>>>
>>>> On Sun, Apr 24, 2011 at 6:29 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
>>>>> *****
>>>>>
>>>>> Hi David,
>>>>>
>>>>> The tuning range of Ti:Sapphire (ex. 690-1040 nm) used to be
>>>>> problematic
>>>>> for exciting far red and infrared fluorophores. There were some
>>>>>
>>>> exceptions,
>>>
>>>> such as Alexa Fluor 488m 568, 594, and 633 all exciting well around 755
>>>>>
>>>> nm
>>>
>>>> (Fig 5D of Dickinson et al 2003 J Biomed Optics 8: 329-338), but since
>>>>>
>>>> you
>>>
>>>> are ok with cost and complexity, adding an OPO(s) and/or multiple MP
>>>>>
>>>> lasers,
>>>
>>>> gives you full range (see
>>>>> http://www.coherent.com/products/?1570/Chameleon-OPO-Tuning-Range for
>>>>> OPO).
>>>>>
>>>>> Depending on cell type and physiological needs, you may be able to
>>>>>
>>>> suppress
>>>
>>>> phototoxicity by lowering O2 - cell culture at ~18% O2 is something of
>>>>>
>>>> an
>>>
>>>> artifact for many cell types - by using catalase/glucose oxidase/glucose
>>>>>
>>>> or
>>>
>>>> Oxyrase (for the latter, www.oxyrase.com, see Waterman-Storer 1993 and
>>>>> Mikhailov 1995 Cell Motil Cytokseleton).
>>>>>
>>>>> Enjoy,
>>>>>
>>>>> George
>>>>>
>>>>>
>>>>> On 4/23/2011 6:03 PM, David Knecht charter wrote:
>>>>>
>>>>>  *****
>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>> *****
>>>>>>
>>>>>> Is there any disadvantage to multiphoton for cultured cells (besides
>>>>>>
>>>>> cost
>>>
>>>> and complexity)? Cell viability?  Phototoxicity?  Dave
>>>>>>
>>>>>> On Apr 23, 2011, at 11:21 AM, Kate Luby-Phelps wrote:
>>>>>>
>>>>>>
>>>>>>
>>>>>>  *****
>>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>>> *****
>>>>>>>
>>>>>>> Multiphoton has no clear advantage for cells in culture. For cell
>>>>>>>
>>>>>> culture
>>>
>>>> samples,
>>>>>>> we use two photon  only to excite DAPI or other UV and near-UV fluors
>>>>>>> since we
>>>>>>> had to make a choice between the Ti:S and a 405 laser on our scope.
>>>>>>>
>>>>>>> Kate Luby-Phelps
>>>>>>>
>>>>>>>
>>>>>>>  Dr. David Knecht
>>>>>> Department of Molecular and Cell Biology
>>>>>> Co-head Flow Cytometry and Confocal Microscopy Facility
>>>>>> U-3125
>>>>>> 91 N. Eagleville Rd.
>>>>>> University of Connecticut
>>>>>> Storrs, CT 06269
>>>>>> 860-486-2200
>>>>>> 860-486-4331 (fax)
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>
>>>>> --
>>>>>
>>>>>
>>>>> George McNamara, PhD
>>>>> Analytical Imaging Core Facility
>>>>> University of Miami
>>>>>
>>>>>
>>>

[Mark Cannell]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Yes but the area is increased by a similar amount so the total  
released does not fall with distance (assuming no inner filtering  
effects etc)

Cheers
On 29/04/2011, at 7:41 AM, Craig Brideau wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> So if it is linear with intensity (1P), should it roll off to 1/4 in  
> terms
> of distance from the ideal focal point?  Intensity drops to 1/4 at 1  
> unit
> distance from a point source, so I'm approximating the center of the  
> focal
> point here.  Am I right in my thinking?
>
> Craig
>
>
>
> On Wed, Apr 27, 2011 at 8:38 PM, Mark Cannell <[hidden email]
> >wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> At low powers the uncaging is linear in intensity so there should  
>> be no
>> threshold.  So, the uncaging volume at low powers is the same as  
>> the PSF and
>> for 2P its the 2P PSF. If ground state depletion develops the FWHM  
>> of the
>> focal uncaging spot grows (of course).
>>
>> Mark
>>
>>
>> On 28/04/2011, at 2:18 PM, Craig Brideau wrote:
>>
>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> How much uncaging occurs outside the focal region in single  
>>> photon?  If
>>> you
>>> don't have your laser turned up to much the energy density should  
>>> only
>>> exceed the uncaging threshold near the focal point, yes?
>>>
>>> Craig
>>>
>>>
>>> On Wed, Apr 27, 2011 at 6:48 PM, Rosemary White <[hidden email]
>>>> wrote:
>>>
>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Dear all,
>>>>
>>>> Haven't followed this discussion closely, but would one advantage  
>>>> of 2P
>>>> lie
>>>> in the ability to uncage caged compounds - fluorescent tracers, for
>>>> example,
>>>> within a single cell?  We've used UV uncaging and while you get  
>>>> most
>>>> uncaging in the target cell, you also get cones of uncaging above  
>>>> and
>>>> below
>>>> the plane of focus.  Seems that 2P would overcome this problem.
>>>>
>>>> Rosemary White
>>>>
>>>> Dr Rosemary White
>>>> CSIRO Plant Industry
>>>> GPO Box 1600
>>>> Canberra, ACT 2601
>>>> Australia
>>>>
>>>> T 61 2 6246 5475
>>>> F 61 2 6246 5334
>>>> E [hidden email]
>>>>
>>>>
>>>>
>>>> On 28/04/11 10:10 AM, "Craig Brideau" <[hidden email]>  
>>>> wrote:
>>>>
>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> I second the previous lister's opinions.  At the end of the day  
>>>>> MP is
>>>>>
>>>> really
>>>>
>>>>> only good for thick tissue sections.  If you are looking at cell  
>>>>> layers
>>>>>
>>>> just
>>>>
>>>>> use a conventional 1P confocal.  The key advantage of 2P is  
>>>>> penetration
>>>>> depth, which is moot when you are looking at cells.  That said,  
>>>>> John's
>>>>> comments about shorter UV imaging are still valid; it's easier  
>>>>> to get a
>>>>>
>>>> NIR
>>>>
>>>>> Ti:Saph beam through conventional optics than a very UV beam.  
>>>>> This only
>>>>> applies if you are using dyes that need very UV excitation of  
>>>>> course.
>>>>>
>>>>> Craig
>>>>>
>>>>>
>>>>>
>>>>>
>>>>> On Sun, Apr 24, 2011 at 6:29 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
>>>>>> *****
>>>>>>
>>>>>> Hi David,
>>>>>>
>>>>>> The tuning range of Ti:Sapphire (ex. 690-1040 nm) used to be
>>>>>> problematic
>>>>>> for exciting far red and infrared fluorophores. There were some
>>>>>>
>>>>> exceptions,
>>>>
>>>>> such as Alexa Fluor 488m 568, 594, and 633 all exciting well  
>>>>> around 755
>>>>>>
>>>>> nm
>>>>
>>>>> (Fig 5D of Dickinson et al 2003 J Biomed Optics 8: 329-338), but  
>>>>> since
>>>>>>
>>>>> you
>>>>
>>>>> are ok with cost and complexity, adding an OPO(s) and/or  
>>>>> multiple MP
>>>>>>
>>>>> lasers,
>>>>
>>>>> gives you full range (see
>>>>>> http://www.coherent.com/products/?1570/Chameleon-OPO-Tuning- 
>>>>>> Range for
>>>>>> OPO).
>>>>>>
>>>>>> Depending on cell type and physiological needs, you may be able  
>>>>>> to
>>>>>>
>>>>> suppress
>>>>
>>>>> phototoxicity by lowering O2 - cell culture at ~18% O2 is  
>>>>> something of
>>>>>>
>>>>> an
>>>>
>>>>> artifact for many cell types - by using catalase/glucose oxidase/
>>>>> glucose
>>>>>>
>>>>> or
>>>>
>>>>> Oxyrase (for the latter, www.oxyrase.com, see Waterman-Storer  
>>>>> 1993 and
>>>>>> Mikhailov 1995 Cell Motil Cytokseleton).
>>>>>>
>>>>>> Enjoy,
>>>>>>
>>>>>> George
>>>>>>
>>>>>>
>>>>>> On 4/23/2011 6:03 PM, David Knecht charter wrote:
>>>>>>
>>>>>> *****
>>>>>>> To join, leave or search the confocal microscopy listserv, go  
>>>>>>> to:
>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>>> *****
>>>>>>>
>>>>>>> Is there any disadvantage to multiphoton for cultured cells  
>>>>>>> (besides
>>>>>>>
>>>>>> cost
>>>>
>>>>> and complexity)? Cell viability?  Phototoxicity?  Dave
>>>>>>>
>>>>>>> On Apr 23, 2011, at 11:21 AM, Kate Luby-Phelps wrote:
>>>>>>>
>>>>>>>
>>>>>>>
>>>>>>> *****
>>>>>>>> To join, leave or search the confocal microscopy listserv, go  
>>>>>>>> to:
>>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>>>> *****
>>>>>>>>
>>>>>>>> Multiphoton has no clear advantage for cells in culture. For  
>>>>>>>> cell
>>>>>>>>
>>>>>>> culture
>>>>
>>>>> samples,
>>>>>>>> we use two photon  only to excite DAPI or other UV and near-
>>>>>>>> UV fluors
>>>>>>>> since we
>>>>>>>> had to make a choice between the Ti:S and a 405 laser on our  
>>>>>>>> scope.
>>>>>>>>
>>>>>>>> Kate Luby-Phelps
>>>>>>>>
>>>>>>>>
>>>>>>>> Dr. David Knecht
>>>>>>> Department of Molecular and Cell Biology
>>>>>>> Co-head Flow Cytometry and Confocal Microscopy Facility
>>>>>>> U-3125
>>>>>>> 91 N. Eagleville Rd.
>>>>>>> University of Connecticut
>>>>>>> Storrs, CT 06269
>>>>>>> 860-486-2200
>>>>>>> 860-486-4331 (fax)
>>>>>>>
>>>>>>>
>>>>>>>
>>>>>>>
>>>>>>
>>>>>> --
>>>>>>
>>>>>>
>>>>>> George McNamara, PhD
>>>>>> Analytical Imaging Core Facility
>>>>>> University of Miami
>>>>>>
>>>>>>
>>>>

[Craig Brideau]

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

Ah, that makes sense to me now.  Less intensity, but
more fluorophores exposed to that intensity as the area increases, leading
to a net linear response.

Thanks!

Craig


On Thu, Apr 28, 2011 at 2:03 PM, Mark Cannell <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Yes but the area is increased by a similar amount so the total released
> does not fall with distance (assuming no inner filtering effects etc)
>
> Cheers
>
> On 29/04/2011, at 7:41 AM, Craig Brideau wrote:
>
>  *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> So if it is linear with intensity (1P), should it roll off to 1/4 in terms
>> of distance from the ideal focal point?  Intensity drops to 1/4 at 1 unit
>> distance from a point source, so I'm approximating the center of the focal
>> point here.  Am I right in my thinking?
>>
>> Craig
>>
>>
>>
>> On Wed, Apr 27, 2011 at 8:38 PM, Mark Cannell <[hidden email]
>> >wrote:
>>
>>  *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> At low powers the uncaging is linear in intensity so there should be no
>>> threshold.  So, the uncaging volume at low powers is the same as the PSF
>>> and
>>> for 2P its the 2P PSF. If ground state depletion develops the FWHM of the
>>> focal uncaging spot grows (of course).
>>>
>>> Mark
>>>
>>>
>>> On 28/04/2011, at 2:18 PM, Craig Brideau wrote:
>>>
>>> *****
>>>
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> How much uncaging occurs outside the focal region in single photon?  If
>>>> you
>>>> don't have your laser turned up to much the energy density should only
>>>> exceed the uncaging threshold near the focal point, yes?
>>>>
>>>> Craig
>>>>
>>>>
>>>> On Wed, Apr 27, 2011 at 6:48 PM, Rosemary White <[hidden email]
>>>>
>>>>> wrote:
>>>>>
>>>>
>>>> *****
>>>>
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> Dear all,
>>>>>
>>>>> Haven't followed this discussion closely, but would one advantage of 2P
>>>>> lie
>>>>> in the ability to uncage caged compounds - fluorescent tracers, for
>>>>> example,
>>>>> within a single cell?  We've used UV uncaging and while you get most
>>>>> uncaging in the target cell, you also get cones of uncaging above and
>>>>> below
>>>>> the plane of focus.  Seems that 2P would overcome this problem.
>>>>>
>>>>> Rosemary White
>>>>>
>>>>> Dr Rosemary White
>>>>> CSIRO Plant Industry
>>>>> GPO Box 1600
>>>>> Canberra, ACT 2601
>>>>> Australia
>>>>>
>>>>> T 61 2 6246 5475
>>>>> F 61 2 6246 5334
>>>>> E [hidden email]
>>>>>
>>>>>
>>>>>
>>>>> On 28/04/11 10:10 AM, "Craig Brideau" <[hidden email]> wrote:
>>>>>
>>>>> *****
>>>>>
>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>> *****
>>>>>>
>>>>>> I second the previous lister's opinions.  At the end of the day MP is
>>>>>>
>>>>>>  really
>>>>>
>>>>>  only good for thick tissue sections.  If you are looking at cell
>>>>>> layers
>>>>>>
>>>>>>  just
>>>>>
>>>>>  use a conventional 1P confocal.  The key advantage of 2P is
>>>>>> penetration
>>>>>> depth, which is moot when you are looking at cells.  That said, John's
>>>>>> comments about shorter UV imaging are still valid; it's easier to get
>>>>>> a
>>>>>>
>>>>>>  NIR
>>>>>
>>>>>  Ti:Saph beam through conventional optics than a very UV beam.  This
>>>>>> only
>>>>>> applies if you are using dyes that need very UV excitation of course.
>>>>>>
>>>>>> Craig
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> On Sun, Apr 24, 2011 at 6:29 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
>>>>>>> *****
>>>>>>>
>>>>>>> Hi David,
>>>>>>>
>>>>>>> The tuning range of Ti:Sapphire (ex. 690-1040 nm) used to be
>>>>>>> problematic
>>>>>>> for exciting far red and infrared fluorophores. There were some
>>>>>>>
>>>>>>>  exceptions,
>>>>>>
>>>>>
>>>>>  such as Alexa Fluor 488m 568, 594, and 633 all exciting well around
>>>>>> 755
>>>>>>
>>>>>>>
>>>>>>>  nm
>>>>>>
>>>>>
>>>>>  (Fig 5D of Dickinson et al 2003 J Biomed Optics 8: 329-338), but since
>>>>>>
>>>>>>>
>>>>>>>  you
>>>>>>
>>>>>
>>>>>  are ok with cost and complexity, adding an OPO(s) and/or multiple MP
>>>>>>
>>>>>>>
>>>>>>>  lasers,
>>>>>>
>>>>>
>>>>>  gives you full range (see
>>>>>>
>>>>>>> http://www.coherent.com/products/?1570/Chameleon-OPO-Tuning-Rangefor
>>>>>>> OPO).
>>>>>>>
>>>>>>> Depending on cell type and physiological needs, you may be able to
>>>>>>>
>>>>>>>  suppress
>>>>>>
>>>>>
>>>>>  phototoxicity by lowering O2 - cell culture at ~18% O2 is something of
>>>>>>
>>>>>>>
>>>>>>>  an
>>>>>>
>>>>>
>>>>>  artifact for many cell types - by using catalase/glucose
>>>>>> oxidase/glucose
>>>>>>
>>>>>>>
>>>>>>>  or
>>>>>>
>>>>>
>>>>>  Oxyrase (for the latter, www.oxyrase.com, see Waterman-Storer 1993
>>>>>> and
>>>>>>
>>>>>>> Mikhailov 1995 Cell Motil Cytokseleton).
>>>>>>>
>>>>>>> Enjoy,
>>>>>>>
>>>>>>> George
>>>>>>>
>>>>>>>
>>>>>>> On 4/23/2011 6:03 PM, David Knecht charter wrote:
>>>>>>>
>>>>>>> *****
>>>>>>>
>>>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>>>> *****
>>>>>>>>
>>>>>>>> Is there any disadvantage to multiphoton for cultured cells (besides
>>>>>>>>
>>>>>>>>  cost
>>>>>>>
>>>>>>
>>>>>  and complexity)? Cell viability?  Phototoxicity?  Dave
>>>>>>
>>>>>>>
>>>>>>>> On Apr 23, 2011, at 11:21 AM, Kate Luby-Phelps wrote:
>>>>>>>>
>>>>>>>>
>>>>>>>>
>>>>>>>> *****
>>>>>>>>
>>>>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>>>>> *****
>>>>>>>>>
>>>>>>>>> Multiphoton has no clear advantage for cells in culture. For cell
>>>>>>>>>
>>>>>>>>>  culture
>>>>>>>>
>>>>>>>
>>>>>  samples,
>>>>>>
>>>>>>> we use two photon  only to excite DAPI or other UV and near-UV fluors
>>>>>>>>> since we
>>>>>>>>> had to make a choice between the Ti:S and a 405 laser on our scope.
>>>>>>>>>
>>>>>>>>> Kate Luby-Phelps
>>>>>>>>>
>>>>>>>>>
>>>>>>>>> Dr. David Knecht
>>>>>>>>>
>>>>>>>> Department of Molecular and Cell Biology
>>>>>>>> Co-head Flow Cytometry and Confocal Microscopy Facility
>>>>>>>> U-3125
>>>>>>>> 91 N. Eagleville Rd.
>>>>>>>> University of Connecticut
>>>>>>>> Storrs, CT 06269
>>>>>>>> 860-486-2200
>>>>>>>> 860-486-4331 (fax)
>>>>>>>>
>>>>>>>>
>>>>>>>>
>>>>>>>>
>>>>>>>>
>>>>>>> --
>>>>>>>
>>>>>>>
>>>>>>> George McNamara, PhD
>>>>>>> Analytical Imaging Core Facility
>>>>>>> University of Miami
>>>>>>>
>>>>>>>
>>>>>>>
>>>>>