http://confocal-microscopy-list.275.s1.nabble.com/Live-cell-imaging-with-multiphoton-confocal-tp6297623p6311834.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]
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>
>
> On 28/04/11 10:10 AM, "Craig Brideau" <
[hidden email]> wrote:
>
> > *****
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> >
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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
> >>
>