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Frequency FLIM system

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sprag

Frequency FLIM system

Dear all,

I am curious to know what off-the-shelf systems are available for measuring FLIM in the frequency domain.

The only one I am know is the LIFA from Lambert Instruments.

Does anyone have any experience using this system, or any other similar?

Vendors are also encourage to reply, either here or off the list.

Thanks in advance for your advice,

Regards,

Soren Prag

Instituto de Medicina Molecular
Portugal

e-mail: [hidden email]

Kelly Lundsten

Re: Frequency FLIM system

I don’t know if they do business in Portugal, but I know Intelligent Imaging Innovations (3i) offers frequency domain systems. Their website is www.intelligent-imaging.com. They demo it at the AQLM course every year.

Kelly Lundsten

Sr. Field Application Scientist

ART, Inc.

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Soren Prag
Sent: Thursday, December 11, 2008 9:50 AM
To: [hidden email]
Subject: Frequency FLIM system

Dear all,

I am curious to know what off-the-shelf systems are available for measuring FLIM in the frequency domain.

The only one I am know is the LIFA from Lambert Instruments.

Does anyone have any experience using this system, or any other similar?

Vendors are also encourage to reply, either here or off the list.

Thanks in advance for your advice,

Regards,

Soren Prag

Instituto de Medicina Molecular
Portugal

e-mail: [hidden email]

Asson-Batres, Mary Ann

Microcosm Inc.

Does anyone know how to contact Microcosm Inc – an imaging company that was in Columbia MD and that services confocal microscopes. We need service and they seem to have moved? In any case our contact info no longer works.

Thanks,

Mary Ann Asson-Batres

Tennessee State University

[hidden email]

Barbara Foster

Re: Microcosm Inc.

Hi, Mary Ann

Brad Bobbitt, the key person at Microcosm has formed LSM

You can reach him at 717-227-4634 or [hidden email]

Hope this is helpful.

Best regards
Barbara Foster, President and Sr. Consultant

Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: (972)924-5310 Skype: fostermme
W: www.MicroscopyEducation.com

NEWS! Visit the NEW and IMPROVED www.MicroscopyEducation.com! And don't forget: MME is now scheduling customized, on-site courses through March 2009. Call me for a free assessment and quote.

At 01:37 PM 12/11/2008, you wrote:

Does anyone know how to contact Microcosm Inc an imaging company that was in Columbia MD and that services confocal microscopes. We need service and they seem to have moved? In any case our contact info no longer works.

Thanks,
Mary Ann Asson-Batres

Tennessee State University
[hidden email]

Boswell, Carl A - (cboswell)

Re: Microcosm Inc.

In reply to this post by Asson-Batres, Mary Ann

Hi Mary Ann,

They're going through bankruptcy right now, so don't expect any new service from them. It may be difficult to actually get one's service agreement fulfilled, but I know they are trying. For those of us with ancient Nikon PCM2000 confocals, I'm not sure what the alternatives are. Any suggestions from anyone?

Good luck to us all.

Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX 520-621-3709

----- Original Message -----

From: [hidden email]

To: [hidden email]

Sent: Thursday, December 11, 2008 12:30 PM

Subject: Microcosm Inc.

Does anyone know how to contact Microcosm Inc an imaging company that was in Columbia MD and that services confocal microscopes. We need service and they seem to have moved? In any case our contact info no longer works.

Thanks,

Mary Ann Asson-Batres

Tennessee State University

[hidden email]

Peter Carroll

Re: Microcosm Inc.

In reply to this post by Asson-Batres, Mary Ann

All of us with their product need service... unless you happen to be the
only person in the world who actually got some stable hardware and
working software from them... just today our 410 catapulted a sample
well into the air when the machine just decided to randomly go into
Z-stack mode (the only time that mode has actually worked, believe it or
not) and rammed the objective up towards the stage at an alarming rate
of speed... just the latest entry in a seemingly endless list of bugs
and problems we've had with everything Microcosm, starting with them
putting in the completely wrong filters and dichroics in the initial
installation, then trying to convince us it we must be doing something
wrong to not get any sort of signal through the scope...

Methinks there's a reason behind their bankruptcy...

Asson-Batres, Mary wrote:

>
> Does anyone know how to contact Microcosm Inc – an imaging company
> that was in Columbia MD and that services confocal microscopes. We
> need service and they seem to have moved? In any case our contact info
> no longer works.
>
> Thanks,
>
> Mary Ann Asson-Batres
>
> Tennessee State University
>
> [hidden email] <mailto:[hidden email]>
>

Asson-Batres, Mary Ann

Re: Microcosm Inc.

In reply to this post by Barbara Foster

Thanks for the suggestion. I tried the telephone number for Brad Bobbitt and it is no longer in service.

Mary Ann

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Barbara Foster
Sent: Thursday, December 11, 2008 2:13 PM
To: [hidden email]
Subject: Re: Microcosm Inc.

Vickery Trinkaus-Randall

FLASH

In reply to this post by Kelly Lundsten

hello
We are trying to use FLASH and are having trouble with getting a good signal over the noise. It appears that there is signal if you even put the FLASH in medium and examine it. Does anyone have some great ideas? We are trying to tag it to a protein and then evaluate where the protein goes in a cell.
Vickery Trinkaus-Randall

Kelly Lundsten wrote:

I don’t know if they do business in Portugal, but I know Intelligent Imaging Innovations (3i) offers frequency domain systems. Their website is www.intelligent-imaging.com. They demo it at the AQLM course every year.

Kelly Lundsten

Sr. Field Application Scientist

ART, Inc.

From: Confocal Microscopy List [[hidden email]] On Behalf Of Soren Prag
Sent: Thursday, December 11, 2008 9:50 AM
To: [hidden email]
Subject: Frequency FLIM system

Dear all,

I am curious to know what off-the-shelf systems are available for measuring FLIM in the frequency domain.

The only one I am know is the LIFA from Lambert Instruments.

Does anyone have any experience using this system, or any other similar?

Vendors are also encourage to reply, either here or off the list.

Thanks in advance for your advice,

Regards,

Soren Prag

Instituto de Medicina Molecular
Portugal

e-mail: [hidden email]

Stephen Cody-2

Re: Frequency FLIM system

In reply to this post by Kelly Lundsten

Dear Soren,

Lambert Instruments manufacture a frequency domain system that is incredibly easy to use.

http://www.lambert-instruments.com/

No commercial affiliation with Lambert, I'm a satisfied customer.

Stephen Cody

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Soren Prag
Sent: Thursday, December 11, 2008 9:50 AM
To: [hidden email]
Subject: Frequency FLIM system

Dear all,

I am curious to know what off-the-shelf systems are available for measuring FLIM in the frequency domain.

The only one I am know is the LIFA from Lambert Instruments.

Does anyone have any experience using this system, or any other similar?

Vendors are also encourage to reply, either here or off the list.

Thanks in advance for your advice,

Regards,

Soren Prag

Instituto de Medicina Molecular
Portugal

e-mail: [hidden email]

--
Stephen Cody

John Gibas

Re: Microcosm Inc.

In reply to this post by Asson-Batres, Mary Ann

Mary Ann and Anyone Else

You can call Don. He is doing what he can as an independent contractor since Microcosm is now defunct. I'm not sure if there are any legal limitations on his end. His cell is 443-745-2087. Peter, sorry about that filter issue. I believe and according to Don, a number of those filters were ordered wrong by our administrative staff at the time. I still should have caught that, my fault. There were/are a number of systems working better than yours for some reason. Unfortunately, we didn't get the internal support from management to correct some of these issues which is why I left. I have been working at Johns Hopkins the last few of years. You can contact me there. The lab phone number is 410-614-0134.

John J Gibas
Cell 410-507-4952

--- On Thu, 12/11/08, Asson-Batres, Mary <[hidden email]> wrote:

From: Asson-Batres, Mary <[hidden email]>
Subject: Re: Microcosm Inc.
To: [hidden email]
Date: Thursday, December 11, 2008, 5:09 PM

Thanks for the suggestion. I tried the telephone number for Brad Bobbitt and it is no longer in service.

Mary Ann

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Barbara Foster
Sent: Thursday, December 11, 2008 2:13 PM
To: [hidden email]
Subject: Re: Microcosm Inc.

Hi, Mary Ann

Brad Bobbitt, the key person at Microcosm has formed LSM

You can reach him at 717-227-4634 or [hidden email]

Hope this is helpful.

Best regards
Barbara Foster, President and Sr. Consultant

Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: (972)924-5310 Skype: fostermme
W: www.MicroscopyEducation.com

NEWS! Visit the NEW and IMPROVED www.MicroscopyEducation.com! And don't forget: MME is now scheduling customized, on-site courses through March 2009. Call me for a free assessment and quote.

At 01:37 PM 12/11/2008, you wrote:

Does anyone know how to contact Microcosm Inc – an imaging company that was in Columbia MD and that services confocal microscopes. We need service and they seem to have moved? In any case our contact info no longer works.

Thanks,
Mary Ann Asson-Batres

Tennessee State University
[hidden email]

Claire Brown

Re: Frequency FLIM system

In reply to this post by sprag

ISS also has a system. I have never used it but here is the web link.

http://www.iss.com/products/albaflim/index.html

Claire

S. Brunet

Re: Microcosm Inc.

In reply to this post by Asson-Batres, Mary Ann

Hello Mary Ann:

New website for Brad: http://www.lsmtech.com/
New phone number: 717-938-4643

Bye!
Sophie
____________________________________________________
Sophie M. K. Brunet, Ph. D.
Research Officer
Optical Spectroscopy, Laser Systems and Applications
Chemistry 112 sessional lecturer
[hidden email]
306-966-1719 (office) 306-966-1702 (fax)
____________________________________________________
Saskatchewan Structural Sciences Centre
University of Saskatchewan
Thorvaldson Bldg.
110 Science Place
Saskatoon, Sk S7N 5C9
____________________________________________________

Quoting "Asson-Batres, Mary" <[hidden email]>:

> Thanks for the suggestion. I tried the telephone number for Brad Bobbitt and
> it is no longer in service.
>
> Mary Ann
>
>
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of Barbara Foster
> Sent: Thursday, December 11, 2008 2:13 PM
> To: [hidden email]
> Subject: Re: Microcosm Inc.
>
> Hi, Mary Ann
>
> Brad Bobbitt, the key person at Microcosm has formed LSM
>
> You can reach him at 717-227-4634 or [hidden email]
>
>
> Hope this is helpful.
>
> Best regards
> Barbara Foster, President and Sr. Consultant
>
> Microscopy/Microscopy Education
> 7101 Royal Glen Trail, Suite A
> McKinney TX 75070
> P: (972)924-5310 Skype: fostermme
> W: www.MicroscopyEducation.com
>
> <http://www.microscopyeducation.com/>NEWS! Visit the NEW and IMPROVED
> www.MicroscopyEducation.com<http://www.microscopyeducation.com/>! And don't
> forget: MME is now scheduling customized, on-site courses through March
> 2009. Call me for a free assessment and quote.
>
>
>
> At 01:37 PM 12/11/2008, you wrote:
>
> Does anyone know how to contact Microcosm Inc - an imaging company that was
> in Columbia MD and that services confocal microscopes. We need service and
> they seem to have moved? In any case our contact info no longer works.
>
> Thanks,
> Mary Ann Asson-Batres
>
> Tennessee State University
> [hidden email]<mailto:[hidden email]>
>
>

Knecht, David

Re: FLASH

In reply to this post by Vickery Trinkaus-Randall

Both Flash and Reash have some amount of cellular background in our hands, but it is not huge. Not sure what cells you are using, but we have found (and have a recently accepted manuscript) that in Dictyostelium, Flash does not work, but Reash works great. Dave

On Dec 11, 2008, at 5:17 PM, Vickery Trinkaus-Randall wrote:

hello
We are trying to use FLASH and are having trouble with getting a good signal over the noise. It appears that there is signal if you even put the FLASH in medium and examine it. Does anyone have some great ideas? We are trying to tag it to a protein and then evaluate where the protein goes in a cell.
Vickery Trinkaus-Randall

Kelly Lundsten wrote:
I don’t know if they do business in Portugal, but I know Intelligent Imaging Innovations (3i) offers frequency domain systems. Their website is www.intelligent-imaging.com. They demo it at the AQLM course every year.

Kelly Lundsten
Sr. Field Application Scientist
ART, Inc.

From: Confocal Microscopy List [[hidden email]] On Behalf Of Soren Prag
Sent: Thursday, December 11, 2008 9:50 AM
To: [hidden email]
Subject: Frequency FLIM system

Dear all,

I am curious to know what off-the-shelf systems are available for measuring FLIM in the frequency domain.
The only one I am know is the LIFA from Lambert Instruments.

Does anyone have any experience using this system, or any other similar?

Vendors are also encourage to reply, either here or off the list.

Thanks in advance for your advice,

Regards,

Soren Prag
Instituto de Medicina Molecular
Portugal

e-mail: [hidden email]

Dr. David Knecht

Department of Molecular and Cell Biology

Co-head Flow Cytometry and Confocal Microscopy Facility

U-3125

91 N. Eagleville Rd.

University of Connecticut

Storrs, CT 06269

860-486-2200

860-486-4331 (fax)

John Oreopoulos

detection of homoFRET with FLIM?

In reply to this post by Claire Brown

I have a question for all of the FRET and/or FLIM experts out there
in the world. I have read that FLIM can be used to spatially detect
heteroFRET between CFP and YFP labeled proteins by looking for a drop
in the donor fluorescence lifetime, but I'm wondering what happens to
the fluorescence lifetime in the case of homoFRET between two YFP
labeled proteins. We have a YFP labeled membrane protein that tends
to form dimers with itself in some areas of the cell membrane. We
imaged the polarization anisotropy of the fluorescence to confirm the
presence of homoFRET with a spatially heterogeneous distribution on
the membrane. I thought it would be great to use another sensitive
FRET microscopy method like FLIM to verify this observation. Can
anyone enlighten me on this topic or direct me to some relevant
literature references? Thank you!

John Oreopoulos

Sam's Mail

Re: detection of homoFRET with FLIM?. .

Hi John,

Using FLIM as a tool for Homo FRET is a bit of a quagmire I believe, for if the FRET is between identical FP's, then the lifetime itself shouldn't be perturbed.

This group describes what they call pseudo-homo-FRET where the transfer is taking place between blue emitting sub-populations of CFP.

Monitoring protein interactions in the living cell through the fluorescence decays of the Cyan Fluorescent Protein", Chemphyschem 7:1442-1454 (2006).

Another technique that might be useful for this is a correlation spectroscopy approach. There are a handful of approaches to visualizing multimerization and aggregation status of your FP of interest. One that may be easily applicable to your work as it has been shown to be useful even in the absence of photon counting detectors is Number and Brightness analysis from Enrico Gratton's group at UCI.

Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode Microsc Res Tech. 2008 Jan;71(1):69-81.

There are other FCS approaches that would be useful of course, such as the use of photon counting histogram (PCH) analysis or Fluorescence Intensity Distribution Anaysis (FIDA) or Fluorescence Moment Image Analysis, but require a bit more specialized equipment.

Oligomerization of the EGF Receptor Investigated by Live Cell Fluorescence Intensity Distribution Analysis Biophys. J., August 1, 2007; 93(3): 1021 - 1031

--
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 (901) 495-2536
Cell (901) 603-3162
[hidden email]

On 12/17/08 11:13 AM, "John Oreopoulos" <[hidden email]> wrote:

I have a question for all of the FRET and/or FLIM experts out there
in the world. I have read that FLIM can be used to spatially detect
heteroFRET between CFP and YFP labeled proteins by looking for a drop
in the donor fluorescence lifetime, but I'm wondering what happens to
the fluorescence lifetime in the case of homoFRET between two YFP
labeled proteins. We have a YFP labeled membrane protein that tends
to form dimers with itself in some areas of the cell membrane. We
imaged the polarization anisotropy of the fluorescence to confirm the
presence of homoFRET with a spatially heterogeneous distribution on
the membrane. I thought it would be great to use another sensitive
FRET microscopy method like FLIM to verify this observation. Can
anyone enlighten me on this topic or direct me to some relevant
literature references? Thank you!

John Oreopoulos

Rietdorf, Jens

Re: detection of homoFRET with FLIM?

In reply to this post by John Oreopoulos

Dear John,

Though I cannot provide a reference, I think it should be well possible
to measure homo-FRET using FLIM; FRET will increase the probability to
de-populate the donor exited state and thus shorten its lifetime, so a
part of the GFP should show reduced lifetimes in case of homo-FRET. The
sensitivity of the approach will be reduced compared to hetero-FRET,
because you cannot spectrally restrict your observation to the
population of the donor molecules.

Just out of curiosity: How do you distinguish between the anisotropy due
to FRET from the anisotropy resulting from the proteins being
rotationally restricted by the membrane?
Jahnig, F. (1979) Structural order of lipids and proteins in membranes:
Evaluation of fluorescence anisotropy data. Proc. Nati. Acad. Sci. USA
Vol. 76, No. 12, pp. 6361-6365.

Best, jens.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of John Oreopoulos
Sent: Wednesday, December 17, 2008 6:13 PM
To: [hidden email]
Subject: detection of homoFRET with FLIM?

I have a question for all of the FRET and/or FLIM experts out there
in the world. I have read that FLIM can be used to spatially detect
heteroFRET between CFP and YFP labeled proteins by looking for a drop
in the donor fluorescence lifetime, but I'm wondering what happens to
the fluorescence lifetime in the case of homoFRET between two YFP
labeled proteins. We have a YFP labeled membrane protein that tends
to form dimers with itself in some areas of the cell membrane. We
imaged the polarization anisotropy of the fluorescence to confirm the
presence of homoFRET with a spatially heterogeneous distribution on
the membrane. I thought it would be great to use another sensitive
FRET microscopy method like FLIM to verify this observation. Can
anyone enlighten me on this topic or direct me to some relevant
literature references? Thank you!

John Oreopoulos

John Oreopoulos

Re: detection of homoFRET with FLIM?

Thank you to those who have replied so far. Looks like I'll just have to try it out when I get a chance if a FLIM system in Toronto becomes available and see if I can observe a heterogeneous distribution of lifetimes on the membrane as well that match the spatial pattern of anisotropy.

In regards to distinguishing anisotropy changes due to homoFRET vs. rotational restriction, there was a very nice recent paper that showed you could exploit the Red-edge anisotropy effect to distinguish the two cases:

Squire, A., P.J. Verveer, O. Rocks, and P.I.H. Bastiaens, 2004. Red-edge anisotropy microscopy enables dynamic imaging of homo-fret between green fluorescent proteins in cells. J. Struct. Biol. 147 (1), 62-69.

I posted to the listserver a few months ago asking about Red-edge effects because I had never heard of them before. The responses I got were very informative and It's turned out to be a very useful tool for this project.

John Oreopoulos

On 17-Dec-08, at 3:18 PM, Rietdorf, Jens wrote:

Dear John,

Though I cannot provide a reference, I think it should be well possible
to measure homo-FRET using FLIM; FRET will increase the probability to
de-populate the donor exited state and thus shorten its lifetime, so a
part of the GFP should show reduced lifetimes in case of homo-FRET. The
sensitivity of the approach will be reduced compared to hetero-FRET,
because you cannot spectrally restrict your observation to the
population of the donor molecules.

Just out of curiosity: How do you distinguish between the anisotropy due
to FRET from the anisotropy resulting from the proteins being
rotationally restricted by the membrane?
Jahnig, F. (1979) Structural order of lipids and proteins in membranes:
Evaluation of fluorescence anisotropy data. Proc. Nati. Acad. Sci. USA
Vol. 76, No. 12, pp. 6361-6365.

Best, jens.

-----Original Message-----
From: Confocal Microscopy List [[hidden email]]
On Behalf Of John Oreopoulos
Sent: Wednesday, December 17, 2008 6:13 PM
To: [hidden email]
Subject: detection of homoFRET with FLIM?

I have a question for all of the FRET and/or FLIM experts out there
in the world. I have read that FLIM can be used to spatially detect
heteroFRET between CFP and YFP labeled proteins by looking for a drop
in the donor fluorescence lifetime, but I'm wondering what happens to
the fluorescence lifetime in the case of homoFRET between two YFP
labeled proteins. We have a YFP labeled membrane protein that tends
to form dimers with itself in some areas of the cell membrane. We
imaged the polarization anisotropy of the fluorescence to confirm the
presence of homoFRET with a spatially heterogeneous distribution on
the membrane. I thought it would be great to use another sensitive
FRET microscopy method like FLIM to verify this observation. Can
anyone enlighten me on this topic or direct me to some relevant
literature references? Thank you!

John Oreopoulos

brad-4

Re: Microcosm Inc.

In reply to this post by Asson-Batres, Mary Ann

I would like to clarify some of the information that has been posted
recently. LSM Tech is the only Zeiss authorized service provider for the
Zeiss LSM 10 (LSM210), LSM 310 and LSM 410 confocal microscope systems.
We provide service support, upgrades, and technical assistance worldwide
for these systems. Our correct contact information is:

LSM Tech
717-938-4643 office
717-938-4588 fax
www.lsmtech.com
[hidden email]

Happy Holidays!

Best regards,
Brad Bobbitt
President
[hidden email]

By the way, LSM Tech is not and has never been affiliated or associated
with Microcosm, Inc.

*************************************************************

> Thanks for the suggestion. I tried the telephone number for Brad Bobbitt
> and it is no longer in service.
>
> Mary Ann
>
>
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Barbara Foster
> Sent: Thursday, December 11, 2008 2:13 PM
> To: [hidden email]
> Subject: Re: Microcosm Inc.
>
> Hi, Mary Ann
>
> Brad Bobbitt, the key person at Microcosm has formed LSM
>
> You can reach him at 717-227-4634 or [hidden email]
>
>
> Hope this is helpful.
>
> Best regards
> Barbara Foster, President and Sr. Consultant
>
> Microscopy/Microscopy Education
> 7101 Royal Glen Trail, Suite A
> McKinney TX 75070
> P: (972)924-5310 Skype: fostermme
> W: www.MicroscopyEducation.com
>
> <http://www.microscopyeducation.com/>NEWS! Visit the NEW and IMPROVED
> www.MicroscopyEducation.com<http://www.microscopyeducation.com/>! And
> don't forget: MME is now scheduling customized, on-site courses through
> March 2009. Call me for a free assessment and quote.
>
>
>
> At 01:37 PM 12/11/2008, you wrote:
>
> Does anyone know how to contact Microcosm Inc - an imaging company that
> was in Columbia MD and that services confocal microscopes. We need
> service and they seem to have moved? In any case our contact info no
> longer works.
>
> Thanks,
> Mary Ann Asson-Batres
>
> Tennessee State University
> [hidden email]<mailto:[hidden email]>
>
>