Re: Hardware Questions (TIRF, Cameras)

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Hardware-Questions-TIRF-Cameras-tp590221p590228.html

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Actually, I would recommend the combination of both if possible (both TIRF and an EMCCD camera). As long as your complexes are close to the coverslip surface (100 nm or less), you will see them easily with a huge gain in signal to noise ratio. With an objective based TIRF, you'll have to move to a higher NA objective (~1.45), and so your resolution and light collection will go up a bit as well. 

Of course, the only downside is that the pixels on the EMCCD will likely be bigger - a problem I've discussed in another thread on the confocal listserver when it comes to trying to match the Nyquist resolution afforded by the TIRF objective. Only way to know for sure is to try it in a real situation, however.

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging

Tel: W:416-946-5022

On 31-Aug-07, at 12:38 PM, Stephen Bunnell wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear microscopists,

    Here's the situation:

    I need to reduce my exposure times.

    I am visualizing dynamically moving complexes (~150nm/s) that are

composed of low abundance signaling molecules, and are close to the

coverslip-water interface.

    I need to visualize these structures, in multiple colors, every 3-5

seconds- preferably faster. Presently, we use a Yokogawa spinning disc to

detect CFP, YFP, and mRFP variants. We are getting by, but would like to be

able to do the work at more physiological chimera expression levels. Typical

exposures currently range from 500 ms (good) to 3000 ms (bad) per channel.

    I am achieving adequate resolution with a 40X NA1.3 oil immersion lens

(Zeiss). I am using the Hamamatsu ORCA-ER CCD. The camera has 6µm pixels,

which we use unbinned. I do not think I can tolerate any lower resolution.

    I have two options, and a question associated with each:

    (1) Move to a TIRF system. What are your opinions about how much gain in

sensitiviy this may provide? Has anyone worked with the Zeiss TIRF module

for the Axiovert 200M?

    (2) Change cameras. I have tried a few back thinned EM-CCDs, but did not

find that they offered much benefit once the pixel size was corrected for.

Is there a better option, that offers high resolution, high sensitivity, and

low background noise? Obviously, there will be compromises. What are your

opinions of intensified CCD cameras?

    Best regards,

    -Steve Bunnell

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

Stephen C. Bunnell, Ph.D.

Assistant Professor

Tufts University Medical School

Department of Pathology

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150 Harrison Ave.

Boston, MA 02111

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