Posted by
Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Re-3D-Eroded-Object-Masks-commercial-response-tp3272647p3299080.html
I have access to Becker & Hickl and Nikon LIMO systems. I have used both on comparable samples.
See: Guy Cox, Mikhail Matz and Anya Salih, 2007. Fluorescence lifetime imaging of coral fluorescent proteins. Microscopy Research & Technique 70, 243-251.
The LIMO system will give useful data with very much shorter acquisition times than the B&H - but the downside is that you cannot isolate the different exponential components as you can in the B&H. So both have their place.
As you say, the number of photons collected is critical - and Wolfgang Becker himself admitted to me that the LIMO is about 10 times as photon efficient as the B&H system. (Obviously the exact figure will depend on the hardware, since both have many options.)
My views on the Lambert LIFA were based only on first principles and a quick play. So I'm sorry if I misled people and the expected speed is not in fact available. But I do think that if you are looking for a bi-exponential fit this isn't the correct tool for the job. Horses for courses.
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:
[hidden email]] On Behalf Of Colin Rickman
Sent: Tuesday, 21 July 2009 10:52 PM
To:
[hidden email]
Subject: Re: time-domain FLIM-FRET with fixed samples
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:
[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:434-982-5210;
> 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
> +49 (0)30 9406 3212
>
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