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Ah, that makes sense to me now. Less intensity, but
more fluorophores exposed to that intensity as the area increases, leading
to a net linear response.
Thanks!
Craig
On Thu, Apr 28, 2011 at 2:03 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
> *****
>
> Yes but the area is increased by a similar amount so the total released
> does not fall with distance (assuming no inner filtering effects etc)
>
> Cheers
>
> On 29/04/2011, at 7:41 AM, Craig Brideau wrote:
>
> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> 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-Rangefor
>>>>>>> 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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