Re: time-domain FLIM-FRET with fixed samples

Posted by Adrian Smith-6 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Re-3D-Eroded-Object-Masks-commercial-response-tp3272647p3299027.html

Hi Guy,

What sort of speeds/specifications are you talking about for the Nikon  
Limo system?

I've been looking at LaVision Biotec's new high-speed TCSPC FLIM  
detector but I'm not clear how it compares in principle with systems  
like the Nikon (or the Becker & Hickl).

http://www.lavisionbiotec.com/en/microscopy-products/fluorescence-microscopy/

Regards,

Adrian Smith
Centenary Institute, Sydney, Australia



On 21/07/2009, at 10:29 PM, Guy Cox wrote:

> You should consider that there are much faster FLIM systems than the  
> Becker & Hickl.  Of course there is always a tradeoff, and typically  
> you will trade some degree of lifetime resolution for the extra  
> speed.  In the time-domain realm the Nikon (Europe) Limo system is  
> much faster than the B&H (but collects into only 4 gates rather than  
> 256).  It has a much higher photon efficiency and so can give good  
> results at speeds compatible with live cell imaging, and our Limo  
> system is routinely used on live cells.  (No connection other than  
> as a customer).
>
> You can probably get even higher speeds with wide-field frequency-
> domain systems such as the Lambert LIFA.
>
>                                          Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>    http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Electron Microscope Unit, Madsen Building F09,
> University of Sydney, NSW 2006
> ______________________________________________
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> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of Periasamy, Ammasi (ap3t)
> Sent: Tuesday, 21 July 2009 7:45 AM
> To: [hidden email]
> Subject: Re: time-domain FLIM-FRET with fixed samples
>
> Hi Alexey
> Yes, Dr. Wolfgang Becker is right and the fixative produce  
> additional problems in the lifetime measurements.
> Unfortunately for some of the biological experiment it is difficult  
> to use the live samples. SO, currently we are working on it...how to  
> overcome or correct the issues involved in lifetime measurement  
> using the fixed samples versus live.
> We will post the results soon.
> Best,
> Ammasi
>
>
> Ammasi Periasamy, Ph.D.
> Director, Keck Center for Cellular Imaging (KCCI) Professor of  
> Biology and Biomedical Engineering Biology, Gilmer Hall (064),  
> McCormick Rd University of Virginia Charlottesville, VA 22904
> Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:[phone]; Email:[hidden email]
>  http//:www.kcci.virginia.edu
> ************************
> Workshop on FRET Microscopy, March 9-13, 2010 http://www.kcci.virginia.edu/workshop/workshop2010/index.php
> *************************
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of Kozlenkov, Alexey
> Sent: Monday, July 20, 2009 8:03 AM
> To: [hidden email]
> Subject: time-domain FLIM-FRET with fixed samples
>
> Dear all,
>
>
> here is a question from a newbie venturing into the field of 2photon  
> FLIM-FRET measurements.
>
> I'm considering using the Becker-Hickl time-domain FLIM / LSM NLO  
> system to measure FRET between some membrane proteins, fused to CFP  
> and YFP.
> The FLIM-based approach looked like an attractive option since it  
> should allow for analysing cells with not too highly expressed  
> proteins of interest, thus reducing the risk of obtaining FRET due  
> only to membrane overcrowding.
> However, since my proteins are partially present in a highly motile  
> pool of vesicles, I intended to use fixed cell samples (as FLIM  
> would require some tens of seconds for one measurement).
>
> Now, to my question:
> The Becker-Hickl TCSPC handbook by Wolfgang Becker makes a strong  
> point of NOT using fixed samples for FLIM-FRET, due to changes in  
> lifetimes and strongly double-exponential decay profiles. However,  
> other publications, such as a protocol in the Molecular Cloning  
> "Bible", do use fixed samples for FLIM-FRET. Thus, I would welcome  
> any comments or advice from the community about this matter. Is  
> fixed sample FLIM-FRET really not recommended, and if it is not  
> true, what would be the best methodology to use (and pitfalls to  
> avoid). How important would be the choice of particular fluorescent  
> protein, fixation methods and mounting media? Obviously, I would  
> also be grateful for links to good reviews and experimental  
> publications that I might have missed.
>
>
> Thanks in advance,
>
> Alex
>
> =============================
>
> Alexey Kozlenkov, PhD
> Molecular Physiology of Somatic Sensation Max-Delbruck Centrum for  
> Molecular Medicine
> 13125 Berlin
> Germany
> [phone]
>
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