Time domain FLIM in wide field microscopy

classic Classic list List threaded Threaded
5 messages Options
jmvdwin jmvdwin
Reply | Threaded
Open this post in threaded view
|

Time domain FLIM in wide field microscopy

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 Alessandro Esposito
Reply | Threaded
Open this post in threaded view
|

Re: Time domain FLIM in wide field microscopy

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 George McNamara
Reply | Threaded
Open this post in threaded view
|

Re: Time domain FLIM in wide field microscopy

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,

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/







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) Periasamy, Ammasi (ap3t)
Reply | Threaded
Open this post in threaded view
|

Re: Time domain FLIM in wide field microscopy

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) Periasamy, Ammasi (ap3t)
Reply | Threaded
Open this post in threaded view
|

Postdoctoral position

*****
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/
*************************