Dear List,
Thank you Guy for mentioning the Lambert LIFA system.
I would really appreciate your feedback on the Lambert LIFA system including
options/flexibility/upgrades along the long-term use.
Best,
Vitaly
NCI-Frederick,
301-846-6575
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Guy Cox
Sent: Tuesday, July 21, 2009 7:29 AM
To: [log in to unmask]
Subject: Re: time-domain FLIM-FRET with fixed samples
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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