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Hi Steve,

I think you have the only one option  - 2x2 binning with Orca-AG (ER, Orca II). 
I haven't compared -AG and -ER side-by-side but AG might be slightly better. 
OrcaII will be cleraly better especially at 500 msec exposures or longer.
 
mRFP1 is a good choice in relation to SNR, having mCherry would cause less 
aggregation. CFP, GFP channels are very noisy. YFP is OK. 

EM-CCDs with the gigantic pixels and 40x lens will cause huge loss of signal.
Fom my experience, as 60x NA 1.4 is the brightest lens, and Orca ER (AG or II)
at 2x2 binning is the best compromise. Nikon 100x NA 1.45 TIRF lens would give 
a signal of only slightly lower intensity than 60x NA 1.4 likely due to lower 
undersampling.    

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.

Good luck,

Vitaly
NCI-Frederick
301-846-6575

Quoting Stephen Bunnell <[log in to unmask]>:


Quoting Stephen Bunnell <[log in to unmask]>:

> 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
> Jaharis Bldg., Room 512
> 150 Harrison Ave.
> Boston, MA 02111
> 
> Phone: (617) 636-2174
> Fax:   (617) 636-2990
> Email: [log in to unmask]
> 




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