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April 2011

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Subject:
Re: Live cell imaging with multiphoton confocal
From:
George McNamara <[log in to unmask]>
Reply To:
Confocal Microscopy List <[log in to unmask]>
Date:
Sun, 24 Apr 2011 08:29:32 -0400
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*****
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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

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