CONFOCALMICROSCOPY Archives

April 2011

CONFOCALMICROSCOPY@LISTS.UMN.EDU
Options: Use Monospaced Font
Show Text Part by Default
Condense Mail Headers

Message: [<< First] [< Prev] [Next >] [Last >>]
Topic: [<< First] [< Prev] [Next >] [Last >>]
Author: [<< First] [< Prev] [Next >] [Last >>]

Print Reply
Mime-Version:
1.0 (Apple Message framework v936)
Content-Type:
text/plain; charset=US-ASCII; format=flowed; delsp=yes
Date:
Fri, 29 Apr 2011 08:03:32 +1200
Reply-To:
Confocal Microscopy List <[log in to unmask]>
Subject:
Re: Live cell imaging with multiphoton confocal
From:
Mark Cannell <[log in to unmask]>
In-Reply-To:
<[log in to unmask]>
Content-Transfer-Encoding:
7bit
Sender:
Confocal Microscopy List <[log in to unmask]>
Parts/Attachments:
text/plain (262 lines) 
*****
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- 
>>>>>> 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
>>>>>>
>>>>>>
>>>>

ATOM RSS1 RSS2