Live cell imaging with multiphoton confocal

[Kate Luby-Phelps]

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We have used 2P excitation successfully to selectively uncage in cells growing on
a feeder layer without uncaging in the feeder cells. Also, in single cells in a
cluster of pancreatic acinar cells. Inherent confocality of the focal volume in 2P
is an advantage in cases like that. I don't think this would have been possible
with 1P.

[Armstrong, Brian]

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Hi Kate, when you do these experiments are you using two lasers; uncaging with one wavelength, and imaging with another wavelength?

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 Kate Luby-Phelps
Sent: Friday, April 29, 2011 5:58 AM
To: [hidden email]
Subject: Re: Live cell imaging with multiphoton confocal

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We have used 2P excitation successfully to selectively uncage in cells growing on
a feeder layer without uncaging in the feeder cells. Also, in single cells in a
cluster of pancreatic acinar cells. Inherent confocality of the focal volume in 2P
is an advantage in cases like that. I don't think this would have been possible
with 1P.


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[Kate Luby-Phelps]

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So far we have been using one laser, which means we miss the beginning of the
timecourse after photoactivation because it takes several seconds for the Ti:S to
tune and mode-lock.  So the experiments where 2P photoactivation has been
useful involve diffusion through gap junctions between cells in a cluster or chain
because the spread of the photoactivated dye between cells is relatively slow.
However, in June we will take delivery on a new 2P system with two Ti:S lasers
so we can do it simultaneously at two different wavelengths.

Kate

[Mark Cannell]

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Hi Kate

Do you really need/want two Tl:S lasers for this?  It may be better to  
use a visible and a Ti:S as I can foresee problems with continued  
photolysis of the cage with the probe beam if it's 2P... In our work  
measuring diffusion via 2P  we used an Ar-ion with a 2P photolysis  
spot that could be either stationary or moved with the probe using the  
same scanning system. For analysis the stationary spot (produced by  
our own custom optics coupling the Ti:S bean to the rear aperture  
independent of the scanner) was the best way to go. The trouble is not  
all microscope bodies can give you the room to a fit in a short pass  
dichroic to do this. In our  old Axiovert, we had to machine up a new  
coupling component to take a small dichroic on a slider. A  diagram of  
the layout is here:

Soeller, C. & Cannell, M.B. (1999) Two-photon microscopy: Imaging in  
scattering samples and three-dimensionally resolved flash photolysis.  
Microsc. Res. Tech. 47:182-195.

Cheers Mark


On 1/05/2011, at 1:06 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
> *****
>
> So far we have been using one laser, which means we miss the  
> beginning of the
> timecourse after photoactivation because it takes several seconds  
> for the Ti:S to
> tune and mode-lock.  So the experiments where 2P photoactivation has  
> been
> useful involve diffusion through gap junctions between cells in a  
> cluster or chain
> because the spread of the photoactivated dye between cells is  
> relatively slow.
> However, in June we will take delivery on a new 2P system with two  
> Ti:S lasers
> so we can do it simultaneously at two different wavelengths.
>
> Kate

[Kate Luby-Phelps]

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Hi Mark,

Certainly for cultured cells and not too thick specimens, your way is a good
choice and I appreciate you bringing it to my attention. For thick or turbid
tissues or live animals, I think we will need 2P to image the probe after
photoactivation. We have a user who designs 2P photoactivatable probes and we
don't observe significant additional photolysis at the imaging wavelength.

Kate

[Armstrong, Brian]

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Hello Kate, would you be willing to share what 2P system, and what lasers, you chose?
What factors determined your decisions?

Thanks in advance,

We currently have a Prairie Ultima that we ordered with the second beam path. However, we have yet to add the second laser.

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 Kate Luby-Phelps
Sent: Saturday, April 30, 2011 6:06 AM
To: [hidden email]
Subject: Re: Live cell imaging with multiphoton confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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So far we have been using one laser, which means we miss the beginning of the
timecourse after photoactivation because it takes several seconds for the Ti:S to
tune and mode-lock.  So the experiments where 2P photoactivation has been
useful involve diffusion through gap junctions between cells in a cluster or chain
because the spread of the photoactivated dye between cells is relatively slow.
However, in June we will take delivery on a new 2P system with two Ti:S lasers
so we can do it simultaneously at two different wavelengths.

Kate


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