Detecting Fluorescence Lifetimes

[Anke Bielicke]

Hello everybody,

 

I try to measure FRET with a FLIM system from Becker and Hickl containing a MCP detector from Hamamatsu (R3809U-50). This detector is very temperature sensitive (increase of dark counts with increasing temperature). Our room is air conditioned so the room temperature is constant.

 

When I measure CFP over a couple of houres (I measure live cells and allways take a fresh probe) I observe a decrease in lifetime (0.4 ns in 5 hours).

 

How can I avoid this problem??

Has anybody else observed this?

I think it could be the detector and that it is getting hot over the time. Could I cool it down somehow?

 

 

Thanks for any helpful advise.

 

Anke

 

[S. Brunet]

Hello Anke:

Try the Hamamatsu C4878 cooler (you will need th holder as well).  It keeps the
MCP-PMT at -30 celsius.  Read the intructions carefully on operating the
cooler.

No financial interest.

Regards,
Sophie
____________________________________________________
Sophie M. K. Brunet, Ph. D.
Research Officer
Optical Spectroscopy, Laser Systems and Applications
[hidden email]
306-966-1719 (office)   306-966-1702 (fax)
____________________________________________________
Saskatchewan Structural Sciences Centre
University of Saskatchewan
Thorvaldson Bldg.
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____________________________________________________


Quoting Anke Bielicke <[hidden email]>:

> Hello everybody,
>
> I try to measure FRET with a FLIM system from Becker and Hickl containing a
> MCP detector from Hamamatsu (R3809U-50). This detector is very temperature
> sensitive (increase of dark counts with increasing temperature). Our room is
> air conditioned so the room temperature is constant.
>
> When I measure CFP over a couple of houres (I measure live cells and allways
> take a fresh probe) I observe a decrease in lifetime (0.4 ns in 5 hours).
>
> How can I avoid this problem??
> Has anybody else observed this?
> I think it could be the detector and that it is getting hot over the time.
> Could I cool it down somehow?
>
>
> Thanks for any helpful advise.
>
> Anke
>
>

[Michelle Digman]

Hi Anke,
I am copying an abstract from a paper describing the photosenstivity of
CFP . Perhaps the problem is not the detector but the CFP itself. I have
also seen this decrease in lifetime with CFP. If you have Ceruleun it is
not as photosensitive as CFP.
Best,
Michelle

Research Article
Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on
FRET determination by fluorescence lifetime imaging microscopy in living
cells
Marc Tramier, Morad Zahid, Jean-Claude Mevel, Marie-Jo Masse, Maïté
Coppey-Moisan *


Abstract
Fluorescent protein-based FRET is a powerful method for visualizing
protein-protein interactions and biochemical reactions in living cells. It
can be difficult, however, to avoid photobleaching when observing
fluorescent cells under the microscope, especially those expressing CFP.
We compared the sensitivity of two protein-based FRET pairs to
light-induced fluorescence changes in the donor, on FRET determination by
fluorescence lifetime imaging microscopy (FLIM). Thanks to the very low
excitation light levels of the time- and space-correlated single photon
counting (TSCSPC) method, FLIM acquisitions were achieved without donor
photobleaching. Here, we show that photobleaching of CFP by a mercury lamp
under the microscope induced a decrease in the mean fluorescence lifetime,
which interfered with FRET determination between CFP and YFP. Importantly,
the range of light-induced variation of the mean fluorescence lifetime of
CFP was not proportional to the decrease in the steady state fluorescence
intensity and varied from cell to cell. The choice of the CFP/YFP pair
therefore requires that the cells be observed and analyzed at very low
light levels during the whole FRET experiment. In contrast, the
GFP/mCherry pair provided an accurate FRET measurement by FLIM, even if
some GFP photobleaching took place. We thus demonstrate that CFP can be an
unreliable donor for FRET determination in living cells, due to its
photosensitivity properties. We demonstrate that the GFP/mCherry pair is
better suited for FRET measurement by FLIM in living cells than the
CFP/YFP pair. Microsc. Res. Tech., 2006. © 2006 Wiley-Liss, Inc.

--
Michelle Digman, Ph.D.
Director, Optical Biology Core
University of California, Irvine
McGaugh Hall 4443
[phone] (OBC office)
[phone] (mobile)
http://dbc.bio.uci.edu/index.html
Laboratory for Fluorescence Dynamics

[Stroetz, Randolph W., R.R.T., R.C.P.]

Has anyone seen or used an adapter allowing an infinity-corrected microscope objective to be mounted on a digital SLR camera?

Thanks, Randy

[Andrew Resnick]

Leica objective threads are pretty darn close to a c-mount
specification; from there you can usually get other adaptors (i.e.
bayonet-to c-mount, etc). Not sure what the other manufacturers use.

----- Original Message -----
From: "Stroetz, Randolph W., R.R.T., R.C.P." <[hidden email]>
Date: Tuesday, July 28, 2009 4:25 pm
Subject: Odd camera adapter
To: [hidden email]

>
> Has anyone seen or used an adapter allowing an infinity-corrected
> microscope objective to be mounted on a digital SLR camera?
>
> Thanks, Randy
>

[Rosemary.White]

An adapter from one of the optical companies like Edmund Optics will
probably do this, you'll also need a tube lens in the adapter - check the
microscopy sections of these catalogues.  We've been looking into this
ourselves, and may go the route of purchasing one of their very long working
distance objectives with adapter to do time-lapse imaging of root growth.
cheers,
Rosemary

Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

ph 61 2 6246 5475
fx 61 2 6246 5334



On 29/07/09 6:31 AM, "Andy Resnick" <[hidden email]> wrote:

> Leica objective threads are pretty darn close to a c-mount
> specification; from there you can usually get other adaptors (i.e.
> bayonet-to c-mount, etc). Not sure what the other manufacturers use.
>
> ----- Original Message -----
> From: "Stroetz, Randolph W., R.R.T., R.C.P." <[hidden email]>
> Date: Tuesday, July 28, 2009 4:25 pm
> Subject: Odd camera adapter
> To: [hidden email]
>
>>
>> Has anyone seen or used an adapter allowing an infinity-corrected
>> microscope objective to be mounted on a digital SLR camera?
>>
>> Thanks, Randy
>>

[Jerry Sedgewick-2]

As Rosemary suggested, the Edmund Optics catalog has c-mount tubes with
an adapter for the objective mount, and a c-mount to t-mount that will
then attach to a Nikon or Canon mount.  In my own experience with a
non-infinity lens at 140mm from back of lens to camera chip, everything
worked well. For infinity objectives, you might be able to get away with
a 175 or 200mm focal length meniscus for the tube lens. With Edmund
Optics c-mount tubes, you may have to get adapters to include the lens
in the path (the lenses may not fit in the tube at the diameters sold).

Jerry


Rosemary White wrote:

> An adapter from one of the optical companies like Edmund Optics will
> probably do this, you'll also need a tube lens in the adapter - check the
> microscopy sections of these catalogues.  We've been looking into this
> ourselves, and may go the route of purchasing one of their very long working
> distance objectives with adapter to do time-lapse imaging of root growth.
> cheers,
> Rosemary
>
> Rosemary White
> CSIRO Plant Industry
> GPO Box 1600
> Canberra, ACT 2601
> Australia
>
> ph 61 2 6246 5475
> fx 61 2 6246 5334
>
>
>
> On 29/07/09 6:31 AM, "Andy Resnick" <[hidden email]> wrote:
>
>  
>> Leica objective threads are pretty darn close to a c-mount
>> specification; from there you can usually get other adaptors (i.e.
>> bayonet-to c-mount, etc). Not sure what the other manufacturers use.
>>
>> ----- Original Message -----
>> From: "Stroetz, Randolph W., R.R.T., R.C.P." <[hidden email]>
>> Date: Tuesday, July 28, 2009 4:25 pm
>> Subject: Odd camera adapter
>> To: [hidden email]
>>
>>    
>>> Has anyone seen or used an adapter allowing an infinity-corrected
>>> microscope objective to be mounted on a digital SLR camera?
>>>
>>> Thanks, Randy
>>>
>>>      
>
>
>  

--
Jerry (Gerald) Sedgewick
Core Facility Director, Biomedical Image Processing Lab (BIPL)
University of Minnesota, Department of Neuroscience
1-205 Hasselmo Hall
312 Church St. S.E.
Minneapolis, MN  55455
612-624-6607
[hidden email]
http://www.bipl.umn.edu
Author: "Scientific Imaging with Photoshop: Methods, Measurement and Output."

Rawlight.com (dba "Sedgewick Initiatives")
965 Cromwell Avenue
Saint Paul, MN  55114
651-788-2261
[hidden email]
http://www.rawlight.com
http://www.jerrysedgewick.com



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[Thomas Trusk]

You might want to check http://www.scopetronics.com  I use their MAXVIEW
PLUS with many digital cameras.

[Edelmann, Richard E. Dr.]

  Once upon a time Canon used to make one for their 35mm cameras (1970's-1980's).  The lens mounts for the dSLR are STANDARD lens camera manufacturer's mounts (i.e. F-mounts, Canon Mount, etc.).  I have tested a number of old (pre-digital) lenses on the Nikon dSLR's and they work well (but no auto-focus obviously).  So you could look for an old Canon microscope objective mount.


________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Stroetz, Randolph W., R.R.T., R.C.P. [[hidden email]]
Sent: Tuesday, July 28, 2009 4:25 PM
To: [hidden email]
Subject: Odd camera adapter

Has anyone seen or used an adapter allowing an infinity-corrected microscope objective to be mounted on a digital SLR camera?

Thanks, Randy

[Edelmann, Richard E. Dr.]

Quick searching:

http://www.alanwood.net/photography/olympus/objective-lens-mount.html

(Oh, and yes the older Canon made mounts were designed to be used with a macro-bellow system for proper focusing).

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Stroetz, Randolph W., R.R.T., R.C.P. [[hidden email]]
Sent: Tuesday, July 28, 2009 4:25 PM
To: [hidden email]
Subject: Odd camera adapter

Has anyone seen or used an adapter allowing an infinity-corrected microscope objective to be mounted on a digital SLR camera?

Thanks, Randy

[Armstrong, Brian]

Diagnostic Instruments also makes adapters for "unique" applications.
Not quite sure how costs would compare to Edmond's for example.
http://www.diaginc.com/accessories/coupler/

No commercial interest.

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872
http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
ing/Pages/default.aspx

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Edelmann, Richard E. Dr.
Sent: Wednesday, July 29, 2009 8:55 AM
To: [hidden email]
Subject: Re: Odd camera adapter

Quick searching:

http://www.alanwood.net/photography/olympus/objective-lens-mount.html

(Oh, and yes the older Canon made mounts were designed to be used with a
macro-bellow system for proper focusing).

________________________________________
From: Confocal Microscopy List [[hidden email]] On
Behalf Of Stroetz, Randolph W., R.R.T., R.C.P.
[[hidden email]]
Sent: Tuesday, July 28, 2009 4:25 PM
To: [hidden email]
Subject: Odd camera adapter

Has anyone seen or used an adapter allowing an infinity-corrected
microscope objective to be mounted on a digital SLR camera?

Thanks, Randy


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[Michelle Digman]

Dear List,

Announcing the 4th LFD Workshop in Advanced Fluorescence Imaging and Dynamics

October 26-30, 2009

For full details on course content, registration procedures, and much more
information, please see visit our website at
http://www.lfd.uci.edu/workshop/ .

Please note that there remain a limited number of laboratory training
section spaces available.  We assure our applicants that the computer
training covers the exact same course material, and we also maintain a
laboratory training waitlist.


Thank you for forwarding this announcement to colleagues you think may be
interested in our workshop!

***************************ATTENTION! AWARDS FOR STUDENTS!!*************
Registration Awards for Students

There will be 6 registration waivers for students. The winners will have
the opportunity to present their work in a 10 minutes presentation during
the workshop. Please submit a short abstract of your work (half page) and
a support letter from your advisor before September 26, 2009, to:


Enrico Gratton
University of California, Irvine
Biomedical Engineering Department
3210 Natural Sciences 2
Irvine, CA, 92697-2715