Re: Live cell imaging with multiphoton confocal

Posted by Mark Cannell on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Live-cell-imaging-with-multiphoton-confocal-tp6297623p6311871.html

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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:

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>
> 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 <[hidden email]
> >wrote:
>
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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:
>>>>
>>>>> *****
>>>>> 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:
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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
>>>>
>>