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So if it is linear with intensity (1P), should it roll off to 1/4 in terms
of distance from the ideal focal point?  Intensity drops to 1/4 at 1 unit
distance from a point source, so I'm approximating the center of the focal
point here.  Am I right in my thinking?

Craig



On Wed, Apr 27, 2011 at 8:38 PM, Mark Cannell <[log in to unmask]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> At low powers the uncaging is linear in intensity so there should be no
> threshold.  So, the uncaging volume at low powers is the same as the PSF and
> for 2P its the 2P PSF. If ground state depletion develops the FWHM of the
> focal uncaging spot grows (of course).
>
> Mark
>
>
> On 28/04/2011, at 2:18 PM, Craig Brideau wrote:
>
>  *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> How much uncaging occurs outside the focal region in single photon?  If
>> you
>> don't have your laser turned up to much the energy density should only
>> exceed the uncaging threshold near the focal point, yes?
>>
>> Craig
>>
>>
>> On Wed, Apr 27, 2011 at 6:48 PM, Rosemary White <[log in to unmask]
>> >wrote:
>>
>>  *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> 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 [log in to unmask]
>>>
>>>
>>>
>>> On 28/04/11 10:10 AM, "Craig Brideau" <[log in to unmask]> wrote:
>>>
>>>  *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> 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
>>>> <[log in to unmask]>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
>>>>>
>>>>>
>>>