Re: Live cell imaging with multiphoton confocal

Posted by Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Live-cell-imaging-with-multiphoton-confocal-tp6297623p6311609.html

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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:

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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:
>
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>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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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
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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
>>>
>>>
>> 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
>