http://confocal-microscopy-list.275.s1.nabble.com/Re-3D-Eroded-Object-Masks-commercial-response-tp3272647p3299097.html
I've no experience with the LaVision system - I know their Trimscope but not their FLIM detector. It certainly sounds interesting.
The LIMO will typically give robust lifetime images in 1 - 4s acquisition time (but I do work with rather bright samples). You can always (Adrian, that is, not the List) walk over and have a play!
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).
> 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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