Dear list members,
I'm seeking opinion from users of PTI TimeMaster for time domain FLIM in wide field microscopy, namely its performance, speed of acquisition, bleaching of specimen & phototoxicity for living cells, software user-friendliness, maintenance cost, etc More generally, how does time domain FLIM in wide field microscopy compare with FLIM in 2P LSM (besides the confocal optical sectioning feature). I'm considering the purchase of a FLIM system for our core imaging facility, which is already equipped with a LSM510NLO & MaiTai laser. Costs (direct & indirect), user-friendliness and occupancy of the LSM are among my concerns. So, wide field or confocal FLIM?, that is my question THANKS for your input. Cheers. Jean-Marie. Jean-Marie Vanderwinden, M.D., Ph.D. Research Director, National Fund for Scientific Research (Belgium) Laboratoire de Neurophysiologie (Prof. S.N. Schiffmann), Faculté de Médecine, Campus Erasme, CP 601, Université Libre de Bruxelles, 808 route de Lennik, B-1070 Brussels, Belgium. phone: Secretary: +(32).2.555.42.30, Direct: +(32).2.555.69.88 fax: +(32).2.555.41.21, email: [hidden email] http://www.ulb.ac.be/medecine/neurophy/ |
Alessandro Esposito |
Dear Jean-Marie,
unfortunately I do not remember what is a PTI TimeMasterT and PTI does not provide enough information on their website. I am not sure that is a wide- field FLIM, but do correct me if I am wrong. WF vs LSM: as for other techniques the answer relies in the applications you wish to pursue. If speed (and perhaps costs) is an issue, wide field FLIMs are usually very useful (Lambert Instruments, LaVision / LaVision biotek, ...). Although there is not yet a single-shot wide-field FLIM commercially available, you can get images in 1-5s typically. Speeds up to 100Hz have been demonstrated, but I would say that commercial systems will be not able to achieve that. This will soon change, but currently this is the situation. There is a WF TCSPC available from EuroPhoton GmbH, perfect for precise measurements, but only at low count rates. LaVision Biotek has also a good option with their TriM Scope, a multi foci TPM that can be coupled with wide-field FLIM detectors. Finally, laser scanning systems. Often they offer data of better quality, resolution and contrast, but you get images with acquisition times >10s, typically in the 1-5min range and sometimes longer if you purchase a MCP-PMT. Personally, I have been very happy with either wide-field or laser scanning FLIM systems. Which are the typical experiments you wished to implement? Regarding the upgrade of the LSM510NLO/MaiTai to FLIM, that is a perfectly reasonable/feasible solution. It is cost-effective (it would not be if you had to purchase the confocal and Ti:Sapphire laser!) and you have commercial solutions available (B&H, picoQuant, ISS and Nikon, ...). No commercial interest in the systems mentioned, and I have certainly forgotten some companies! Cheers Alessandro www.quantitative-microscopy.org www.wikiscope.org www.hutchison-mrc.cam.ac.uk |
George McNamara |
In reply to this post by jmvdwin
Hi Jean-Marie,
With respect to more generally ... Another post already mentioned major players. As a customer of both Becker&Hickl (TCSPC FLIM, the SPC-830 board is capable of FCS/FCCS though we have never done it) and ISS (we have FCS/FCCS - ISS also has FLIM and more, see www.iss.com) on a Leica MP/SP5, I can say that you should check them out. Wolfgang Becker of Becker&Hickl told me that excellent confocal/multiphoton TCSPC FLIM can be achieved in 2 seconds (contrary to the other post!!!) on his company's current system(s) - both their own rigs and as add on to a Zeiss LSM510NLO. For the latter, you will probably want to use the NDD port with Becker&Hickl selected detectors. Besides Wolfgang's book - or TCSPC manual online at the company website, see PubMed search: becker w fluorescence lifetime. The Becker&Hickl SPCm output .SDT files can be read by ImageJ/Bio-Formats (note to the Bio-Formats developers - please email Wolfgang about current format and more details about .SDT) and SPCimage can export in the "Matrix" section the photon counts per pixel, and each component as ASCII files (space delimited, i.e. 128 rows x 128 columns for a 128x128 pixel image). Since photons/pixel can be very high, that is, much greater than the 65,535 limit of a 16-bit file format, this is a reasonable approach to getting the FLIM data out (by the way, I am not claiming that you will get >65K counts in each pixel in 2 seconds). I copied Wolfgang on this message and will mention your post to Ben Barbieri of ISS - both of whom are speaking here in Miami on Monday. As to your question: So, wide field or confocal FLIM?, that is my question I have no experience with widefield FLIM, but the advantages of multiphoton fluorescence excitation (and SHG, THG) for live cell imaging, the fact that TCSPC can be fast, and that you already own an LSM510NLO that can be upgraded, strongly favor multiphoton. You can also discuss with vendor(s) about using an SHG crystal to 'up-convert" the MP laser output to pulsed single photon excitation. As for efficiency, I suggest that the combination of FLIM and "micropatterned adhesive substrates", for example from www.cytoo.com (another talk in Miami on Monday) and the type of analysis done by Schauer et al 2010 (who used conventional 3D images) will keep instrument throughput high. Schauer K, Duong T, Bleakley K, Bardin S, Bornens M, Goud B. Probabilistic density maps to study global endomembrane organization. Nat Methods. 2010 Jul;7(7):560-6. PMID: 20512144 Sincerely, George At 04:27 AM 8/25/2010, you wrote: Dear list members, George McNamara, Ph.D. Image Core Manager Analytical Imaging Core Facility University of Miami, Miller School of Medicine Miami, FL 33136 [hidden email] [hidden email] 305-243-8436 office http://www.sylvester.org/AICF (Analytical Imaging Core Facility) http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file) http://home.earthlink.net/~geomcnamara |
Periasamy, Ammasi (ap3t) |
Wide-field or confocal FLIM…………. You can choose the FLIM system depending on your application and
your experience in lifetime imaging and data analysis and interpretation of the
data collected from the biological systems. You may need a confocal or 2photon
based system if you have to measure the lifetime distribution inside the cell
or tissue. Lifetime imaging is carried out in two ways, Frequency domain and
time domain and both can be implemented either in wide-field or confocal
(2photon based system too). Plus and minuses of these two FLIM methodologies (wide-field, Confocal,
and two-photon based FLIM systems) for various biological applications are
widely discussed in our book on “FLIM Microscopy in Biology and Medicine”
(Eds: Periasamy & Clegg; Taylor & Francis Group, CRC Press, 2010). The chapters in
this book are written to cover the beginner to FLIM as well as the experienced scientists
in lifetime imaging. We discuss widely both theory and practical training on the
lifetime imaging methodology in our upcoming 10th annual hands on
training workshop on FRET microscopy http://www.kcci.virginia.edu/workshop/workshop2011/index.php
The listed companies below bring their instruments to train the
workshop participants on FLIM data acquisition, processing and interpretation of
the data. Established investigators in FLIM area closely interact with the participants
during the workshop. Becker & Hickl (Boston Electronics) http://www.becker-hickl.de/ Intelligent Imaging Innovations http://www.intelligent-imaging.com/ ISS www.iss.com Lambert Instruments http://www.lambert-instruments.com/ Picoquant http://www.picoquant.com/ Hope this helps. If you need any particular explanation contact
me through my personal email <[hidden email]>. My contact information
is given below Prof. Ammasi Periasamy Director, Keck Center for Cellular Imaging (KCCI) Professor of Biology and Biomedical Engineering Biology, Gilmer Hall (064), 485 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/Contact/peri.php ************************ 10th Annual Workshop on FRET Microscopy, March 8-13, 2011 http://www.kcci.virginia.edu/workshop/workshop2011/ One Day Symposium to honor Prof. Theodor Förster's 100th Birth
Day, March 10, 2011 http://www.kcci.virginia.edu/workshop/workshop2011/SymposiumBrochure.pdf ************************* |
Periasamy, Ammasi (ap3t) |
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear All Happy New Year 2011! I am posting for Dr. Margarida Barroso. Please contact her [hidden email]<mailto:[hidden email]> Best wishes, Ammasi POST-DOCTORAL POSITION Center for Cardiovascular Sciences Albany Medical College Albany, NY A three year postdoctoral fellowship position is available to study iron transport mechanisms and membrane trafficking in polarized epithelial cells using advanced imaging approaches. For more information on our research, check Margarida Barroso in Linkedin.com The optimal applicants should have hands on experience in molecular biology techniques (ex. Molecular cloning, transfection or RNAi techniques) and an interest in learning advanced imaging techniques. In our lab, candidates will have the opportunity of using Confocal and TIRF microscopy to perform quantitative imaging techniques, such as FRET, FRAP and others. The work is highly analytical and requires good technical expertise and computer skills. Preferred candidates are individuals with a recent PhD degree with a track record of productivity as evidenced by publications and good command of English. The successful candidates will be offered competitive salary and health coverage. Please email a cover letter describing your past research experience, a CV and the names and contact information of 2 references to: Margarida Barroso, Ph.D. Assistant Professor Center for Cardiovascular Sciences Albany Medical College 47 New Scotland Av Albany, NY 12208 [hidden email]<mailto:[hidden email]> Prof. Ammasi Periasamy Director, Keck Center for Cellular Imaging (KCCI) Biology, Gilmer Hall (064), 485 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/Contact/peri.php ************************ 10th Annual Workshop on FRET Microscopy, March 8-13, 2011 http://www.kcci.virginia.edu/workshop/workshop2011/ One Day Symposium to honor Prof. Theodor Förster's 100th Birth Day, March 10, 2011 http://www.kcci.virginia.edu/symposium/ ************************* |
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