Re: Live cell imaging with multiphoton confocal

Posted by Rosemary.White on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Live-cell-imaging-with-multiphoton-confocal-tp6297623p6311663.html

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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:

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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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>>>
>>> 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
>>