Re: Hardware Questions (TIRF, Cameras)

Posted by George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Hardware-Questions-TIRF-Cameras-tp590221p590225.html

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Stephen,

First, consider some wetware changes:

CFP (presumably ECFP?) -> mCerulean ... Piston lab
YFP (presumably EYFP?) -> mVenus ... Miyawaki lab
mRFP (presumably mRFP1?) -> mKate ... just published by Scherbo et al in Nature Methods (PubMed ID 17721542)


The mKate article compares all the far red fluorophores.

camera tips:
1. have you optimized (maximized) the gain setting on the ORCA? Your software default may be low gain. If you change the gain, you may also need to tweak the offset to keep the 'floor' above zero (I like having it at about 100 or 200 intensity levels). There are 10 gain settings on the ORCA-ER Firewire cameras. These are analog gains, before the digitizer, so output is continuous.
   Given your exposure times (3 seconds!) I suspect you are using low gain.
2. The ORCA-ER Firewire has an "IR mode", that is useful for far-red/near-infrared signals (on for mRFP1 or mKate or Cy5, off for visible fluorophores). It increases the QE in the red by grabbing photoelectons from deeper in the CCD (blue is absorbed first, then green, then red, then IR), at the cost of a slightly higher background (more volume of the CCD, results in also grabbing more thermal electrons, so keep it off for blue, green, yellow).
Zeiss AxioVision and MetaMorph (and I'm sure Compix) users can enable these options in macros/journals, as well as in the acquire dialogs.
My thanks to Butch Moomaw of Hamamatsu for explaining these features.

emission filters: do you have spectrally optimal emission filters? Are the filters in excellent shape (no fingerprints, no pinholes)?

illumination: Assuming you are using laser(s), are you getting optimum output from them to the objective lens? I'm guessing you are using an Argion ion laser for the CFP and YFP - is its power output (knob or slider for Amperage, not AOTF) adjusted for good power output? Too low Amperage results in instability, too high results in shortened tube lifetime (max power can result in 1/10th tube life - of course if the tube is under service contract and/or the experiments are worth it, cranking the Amperage may get you better data).

Photobleaching: if you have higher illumination, you may (should!) have more photobleaching. Oxyrase (www.oxyrase.com) or other additives have been used to decrease O2


New camera and/or new lenses:  Get demos!!!

Lenses: besides (getting demo of) the 1.45 NA TIRF lenses, operated in TIRF and non-TIRF modes, how well does the Zeiss 63x/1.2 NA water immersion lens work for your live cell experiments? If you are micrometers away from the coverglass, RI matching MIGHT be preferable to high NA.

If you want to go faster, simultaneous acquisition of the three emission channels is possible with multiple cameras or the dualview/quadview from Optical Insights/Photometrics (some other companies have side-by side devices, and see Prabhat et al 2007 PNAS for an interesting configuration for a TIRF/widefield system).


Vitaly wrote in another response: TIRF may not work as you would excite molecules which are up to 200 nm away from the glass. Moreover TIRF people often use a thicker cover glass to reduce optical artefacts.

Why would a thicker coverglass reduce optical artifacts in TIRF? Measuring the coverglass to make sure it is correct for the emission side (i.e. 170 um for a standard 0.17 coverglass corrected lens) should be what matters. The TIRF excitation is propagating inside the coverglass at the coverglass-cell culture medium interface - how would it know how thick the coverglass is to the objective lens side?



best wishes,



At 12:38 PM 8/31/2007, you 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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Boston, MA 02111

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George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
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