FRET on FV500

[js1719]

Hello,
  I have a user that is going to do FLIM-FRET on our Olympus FV500  
system soon and I was wondering if any other people on the list serve  
have an established (written) protocol for doing either de-
constructive acceptor photo-bleaching (using the protocol processor  
method) or synthesized emission. We added the FRET module a few years  
ago but no one here ever used it. He is set up to use CFP/YFP. I have  
told him to do CFP and YFP alone as controls and I think I will teach  
him the de-constructive method first since as I remember it being  
easier. I also wondered what analysis program people use for  
calculating FRET these days- we tend to use ImageJ a lot for analysis  
so if you have a plugin that works well can you let me know? I  
appreciate all the advice you can give me, this is user is completely  
new to microscopes so the more I can provide him with the better.
Best,
  Julia

[Periasamy, Ammasi (ap3t)]

Hi Julia
The FRET data analysis software in the Olympus system is based on our algorithm. I think they did a good job in integrating the algorithm in their system.
As you know we run a annual FRET workshop during March to train the participants on FRET data acquisition and processing using various microscope systems including wide-field, spinning disk confocal, laser scanning confocal, Spectral imaging, 2photon, lifetime imaging, acceptor photobleaching, etc.
You should consider attending the course so that you can train others and the software is free for the participants and it works with imageJ. We also added many functions...it can handle 8-bit, 10-bit, 12-bit, 14-bit, 16-bit images, time lapse and 3D FRET, etc. We can also add functions depending on the workshop participants request.
There is no fee for registration and you can register to keep your spot. You can visit the web site to see the info
http://www.kcci.virginia.edu/workshop/index.php
Hope this helps
ammasi


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of js1719
Sent: Wednesday, October 15, 2008 12:56 PM
To: [hidden email]
Subject: FRET on FV500

Hello,
  I have a user that is going to do FLIM-FRET on our Olympus FV500
system soon and I was wondering if any other people on the list serve
have an established (written) protocol for doing either de-
constructive acceptor photo-bleaching (using the protocol processor
method) or synthesized emission. We added the FRET module a few years
ago but no one here ever used it. He is set up to use CFP/YFP. I have
told him to do CFP and YFP alone as controls and I think I will teach
him the de-constructive method first since as I remember it being
easier. I also wondered what analysis program people use for
calculating FRET these days- we tend to use ImageJ a lot for analysis
so if you have a plugin that works well can you let me know? I
appreciate all the advice you can give me, this is user is completely
new to microscopes so the more I can provide him with the better.
Best,
  Julia

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:[phone]; Email:[hidden email]
http//:www.kcci.virginia.edu
************************
Workshop on FRET Microscopy, March 3-7, 2009
http://www.kcci.virginia.edu/workshop/workshop2009/index.php
 *************************

[Zoltan]

Hi Julia,

We use PixFRET very successfully, see this reference:

MICROSCOPY RESEARCH AND TECHNIQUE 68:51–58 (2005)

PixFRET, an ImageJ Plug-in for FRET Calculation That Can 

Accommodate Variations in Spectral Bleed-throughs 

JE´ ROˆ ME N. FEIGE,1 DANIEL SAGE,2 WALTER WAHLI,1 BE´ ATRICE DESVERGNE,1 AND LAURENT GELMAN1* 

and AccPbFRET for acceptor photobleaching:

http://www.biophys.dote.hu/accpbfret/

http://www.biomedcentral.com/content/pdf/1471-2105-9-346.pdf

I hope this help,

Zoltan

On Wed, Oct 15, 2008 at 5:56 PM, js1719 <[hidden email]> wrote:

Hello,
 I have a user that is going to do FLIM-FRET on our Olympus FV500 system soon and I was wondering if any other people on the list serve have an established (written) protocol for doing either de-constructive acceptor photo-bleaching (using the protocol processor method) or synthesized emission. We added the FRET module a few years ago but no one here ever used it. He is set up to use CFP/YFP. I have told him to do CFP and YFP alone as controls and I think I will teach him the de-constructive method first since as I remember it being easier. I also wondered what analysis program people use for calculating FRET these days- we tend to use ImageJ a lot for analysis so if you have a plugin that works well can you let me know? I appreciate all the advice you can give me, this is user is completely new to microscopes so the more I can provide him with the better.
Best,
 Julia

--

Zoltan Cseresnyes
Facility manager, Imaging Suite

University of Cambridge, UK

[Jerry Sedgewick-2]

Hello All,

I know that some problems have accompanied the transfer of the confocal
listserv from the Buffalo location to the University of Minnesota
server.  Among these are messages sent back to those who are posting
regarding duplicate messages, and some messages notify the sender that
their message didn't post at all (I'm getting the latter myself).  In
both cases the messages post, but it does lead to confusion.  Just to
let you know, I am trying to get some answers from our listserv
administrator, along with a solution.  Until then, our U of M server is
garrulous (to anthropomorphize a computer).  Please have patience.

Thank you!

Jerry Sedgewick


--- Get FREE High Speed Internet from USFamily.Net! -- http://www.usfamily.net/mkt-freepromo.html ---

[Sam's Mail]

Imaging Scientist - St. Jude Children's Research Hospital.

Currently, St. Jude Children's Research Hospital has an opening for an Imaging Scientist (Job Number 16704) in the Cellular Imaging Department.

The successful candidate would be responsible for assisting faculty, postdoctoral researchers, staff, and collaborators in advance microscopy techniques and methodologies, including laser scanning and spinning disk confocal microscopy, wide-field microscopy, live cell imaging, FRET, FLIM, FCS, FRAP and TIRF, as well as routine maintenance of the instrumentation infrastructure.

Requirements:

A Bachelor's degree in an appropriate scientific field plus a minimum of ten (10) years (post-degree) of relevant and productive work experience is required OR,

A Master's degree in an appropriate scientific field plus a minimum of nine (9) years (post-degree) of relevant and productive work experience is required OR,

A PhD in an appropriate scientific field plus five (5) years (post-degree) of relevant and productive work experience is required.

To apply please visit our Web site
http://www.stjude.org/jobs

St. Jude Children's Research Hospital is an Equal Opportunity Employer.


--
Samuel A. Connell
Director of Light Microscopy
Cell & Tissue Imaging Center
St. Jude Children's Research Hospital
262 Danny Thomas Place
Memphis, TN 38105-3678
Office [phone]
Cell [phone]
[hidden email]

[Christian-103]

I have always received that error that emails were duplicates or not going through, and then they'd post just a few minutes later.  This was BEFORE the move, so could it just be a list-serv software error based on where the emails are coming from (Yahoo in this case)?

[Zoltan]

I've always had the same problem on the old server as well; it's not really a problem once you realise that you just have to wait a bit and ignore the rejection message.  I'm sending my messages from gmail.

On Thu, Oct 16, 2008 at 1:38 PM, Christian <[hidden email]> wrote:

I have always received that error that emails were duplicates or not going through, and then they'd post just a few minutes later.  This was BEFORE the move, so could it just be a list-serv software error based on where the emails are coming from (Yahoo in this case)?

--

Zoltan Cseresnyes
Facility manager, Imaging Suite

University of Cambridge, UK

[Tino Jäger]

Hi,

does anyone have experience with the high content screening system OPERA
prom Perkin Elmer or scan^r from Olympus?

Is there any other HCS system you can suggest (high end objective & cam
are necessary)?

Thanks for your help,

Tino

[Mike Mathews]

Molecular Devices Image Express Micro and Ultra have nice features, Meta runs and performs data analysis.  Nice application modules.

 

 

On Thu, Oct 16, 2008 at 11:04 AM, Jäger Tino <[hidden email]> wrote:

Hi,

does anyone have experience with the high content screening system OPERA
prom Perkin Elmer or scan^r from Olympus?

Is there any other HCS system you can suggest (high end objective & cam
are necessary)?

Thanks for your help,

Tino

--
Mike Mathews
Spectrofuge Corporation
919-361-2197  Office
919-423-1131  Cell
[hidden email]
www.Spectrofuge.com

[Julio Vazquez]

-

Tino, 

There are many HCA/HCS systems with different capabilities and very different price tags; you can find a review here:

http://clients.parabolasoft.co.uk/HTStec_RSS/documents/DDW%20High%20Content%20Analysis%20Tools%20Final.pdf

John Comley also published a market survey of HCS instruments. I can't find the URL, but I will send a pdf to your personal email address.

Also, on volume of Methods in Molecular Biology (Vol 356), and one of Methods in Enzymology (Vol 14, 2006) are devoted to HCS. Might be worth having a look at them. This might give you some ideas of teh different factors to consider, depending on your own needs...

http://www.amazon.com/Content-Screening-Methods-Molecular-Biology/dp/1588297314

Julio.

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org/

==

On Oct 16, 2008, at 8:04 AM, Jäger Tino wrote:

Hi,

does anyone have experience with the high content screening system OPERA

prom Perkin Elmer or scan^r from Olympus?

Is there any other HCS system you can suggest (high end objective & cam

are necessary)?

Thanks for your help,

Tino

[Bowman, Doug]

Dear colleagues,

 

We have an opening for an Imaging Scientist position to support our drug discovery efforts at Millennium Pharmaceuticals in Cambridge, Massachusetts.  The job description is posted below and can be found in the career section on the Millennium www site:

 

http://www.millennium.com/careers/index.asp

 

and go to SEARCH FOR JOBS.  The title and requisition number is listed below.

 

 

Regards,

Doug

 

Douglas Bowman

Millennium Pharmaceuticals

 

 

 

JOB TITLE	Research Investigator/Scientist I
Requisition Number	5581BR
Department Name	Molecular & Cellular Oncology
Location	Cambridge, MA
POSITION SUMMARY	Millennium Pharmaceuticals Inc, a leading fully integrated biopharmaceutical company, is searching for an Image Analyst to support our image-based assays throughout the Oncology Discovery organization. The candidate will work with image acquisition and analysis technologies that include automated high content screening, histopathology, timelapse microscopy, and cellular informatics software.
DUTIES & RESPONSIBILITIES	Duties and responsibilities will include Development of algorithms and solutions for high-content image-based assays utilizing existing and new imaging technologies that include Metamorph and Definiens image analysis software. The individual will be responsible for testing and validation of specific application solutions. This will require working in a multi-disciplinary environment with biologists, statisticians, and engineers. Consultation and training of users working in multiple functional areas. The individual will provide support for both developed solutions as well as existing hardware and software platforms that include automated microscopes, live-cell imaging systems, and associated image acquisition and analysis software. Maintenance of core technologies, e.g. microscope and software maintenance. The individual will share responsibility for maintenance of hardware and software platforms. Documentation of developed solutions. The individual will create and maintain documentation and operating procedures for use of developed solutions.
QUALIFICATIONS	Knowledge of fluorescence imaging hardware and applications for cell biology research and screening is required. Experience with any of the following technologies is desired: Metamorph software, Definiens software, high content microscopy, confocal microscopy, timelapse microscopy and image analysis. Must have excellent analytical, organizational, and communication skills both verbal and written. Basic Qualifications: PhD in Engineering , Bioengineering or Biology with 0-3 years related experience. Or, MS in Engineering, Bioengineering or Biology with 3-5 years related experience. Must have knowledge of microscopy and/or image analysis.

 

This e-mail, including any attachments, is a confidential business communication, and may contain information that is confidential, proprietary and/or privileged.  This e-mail is intended only for the individual(s) to whom it is addressed, and may not be saved, copied, printed, disclosed or used by anyone else.  If you are not the(an) intended recipient, please immediately delete this e-mail from your computer system and notify the sender.  Thank you.

[Csúcs Gábor]

Dear Tino,

I think the idea of Julio Vazquez about forming a clear idea about your
needs is excellent.  Nevertheless as we have tested (and  use) a number
of screening microscopes I can still give you some hints.
There are 2 basic questions: What does high NA means for you? (Many
systems work with a 0.95 - or similar air objective). The camera
question is "easier" - almost all systems use a camera equipped with the
same Sony interline chip (same QE). What type of screens do you want to
perform: really high throughput (you need a plate loader), or just
single plates? In this second case you don't really need a specific
screening system - you can easily do it also with a "normal" motorized
microscope equipped with a motorized X-Y stage (and the necessary
software).
But to give also a more useful answer, I'm aware of the following
companies offering a screening microscope with a high NA air objective
(of course in many cases you can use them also with an oil-objective).

BD
Olympus Biosystems
PerkinElmer (= Evotec Opera)
Molecular Devices
GE Healthcare

I've never seen a system using an "automated" oil-immersion system. The
Opera has a nice water-immersion system, and as far as I know, Olympus
is also wroking on something similar. Leica has also developed a water
immersion system for their Matrix software - with which you make a
screening microscope from your Leca confocal (SP5).

Cheers    Gabor

--
Gabor Csucs
Light Microscopy Centre, ETH Zurich
Schafmattstrasse 18, HPM F16
CH-8093, Zurich, Switzerland

Web: www.lmc.ethz.ch
Phone: [phone]
Fax: [phone]
e-mail: [hidden email]

[Tony Collins-4]

Hi Tino,

The Opera is a very nice system. The high NA not only gives you higher resolution but also shorter exposures which is not to be underestimated for HCS. One of our screens quantified changes in yeast organelle morphology.

We've a couple of images from our Opera on our website:
http://www.macbiophotonics.ca/opera2.htm

Feel free to email me off list for more details.

Regards,

Tony

Tony J. Collins, Ph.D.
McMaster Biophotonics Facility
Dept. Biochemistry and Biomedical Sciences HSC 4H21A
McMaster University, Hamilton, ON, L8N 3Z5
[hidden email]     www.macbiophotonics.ca

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Jäger Tino
> Sent: Thursday, October 16, 2008 11:05 AM
> To: [hidden email]
> Subject: [CONFOCALMICROSCOPY] High Content Screening
>
> Hi,
>
> does anyone have experience with the high content screening system
> OPERA
> prom Perkin Elmer or scan^r from Olympus?
>
> Is there any other HCS system you can suggest (high end objective & cam
> are necessary)?
>
> Thanks for your help,
>
> Tino