It is important to be careful when comparing different systems and their
relative speed of acquisition. Few groups have more than one system at
any one time to compare, using the same sample, and reported speeds and
accuracies are rarely from the sample (high speed and high accuracy are
normally achieved through differing acquisition settings).
We routinely use a Becker & Hickl system for mono and bi-exponential
FLIM recordings (both from live cells and fixed samples). Recently we
trialled a Lambert LIFA system for our wide-field microscope (we were
hoping to use it under TIRF illumination). On the face of it this system
should be able to acquire lifetime data far faster. With low temporal
accuracy and a single exponential fit the system is faster using
widefield illumination than our Becker and Hickl system (normally around
30s). However, with comparable accuracy requirements and a
bi-exponential fit the acquisition time was far longer on the Lambert
system. The problem turned out to be the way the system works - by
modulating the voltage on the MCP. To achieve a modulation the voltage
oscillates around 0V (ie off) meaning the MCP is very insensitive.
The main limitation for all FLIM recordings is the number of detected
photons used for the fitting. More photons = longer acquisition = higher
temporal accuracy and vice versa. Depends what you want to measure.
Colin
--
Dr Colin Rickman
Centre for Integrative Physiology
School of Biomedical Sciences
University of Edinburgh
Hugh Robson Building
George Square
Edinburgh
EH8 9XD
Tel: +44 131 6511512
Fax: +44 131 6503128
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
> ______________________________________________
> Phone +61 2 9351 3176 Fax +61 2 9351 7682
> Mobile 0413 281 861
> ______________________________________________
> http://www.guycox.net
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Periasamy, Ammasi (ap3t)
> Sent: Tuesday, 21 July 2009 7:45 AM
> To: [log in to unmask]
> 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:434-982-5210; Email:[log in to unmask] 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:[log in to unmask]] On Behalf Of Kozlenkov, Alexey
> Sent: Monday, July 20, 2009 8:03 AM
> To: [log in to unmask]
> 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
> +49 (0)30 9406 3212
>
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