Posted by
Rietdorf, Jens on
URL: http://confocal-microscopy-list.275.s1.nabble.com/More-power-and-less-exposure-or-vice-versa-use-of-100-laser-tp3246685p3950640.html
Dear Kathy,
your mercury arc has a nice bright peak at that ex and the HeNe 543 is
the weakest laser on the list. Likewise the PMT has a QE below 10% in
that range of the em spectrum, your camera may have up to 90%.
What happens if you use the arc & a camera on the FV500?
Do they use lenses with similar NA on both systems?
Is the pixel size x,y similar?
Is the pinhole aligned?
Is the PSF of the confocal ok?
Just a few points to check. Good luck, jens
-----Original Message-----
From: Confocal Microscopy List [mailto:
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On Behalf Of Kathryn Spencer
Sent: Wednesday, November 04, 2009 6:57 PM
To:
[hidden email]
Subject: tdTomato and LSM confocal
Hello all;
>
> I have a colleague who is unhappy with the images of td-Tomato
> AI9 mice on our Fluoview 500. They first view their brain sections
> on our AX70, with mercury lamp illumination, a TRITC 535/50 EX filter,
> 610/75 EM filter, and like what they see. On our FV500 with 543 laser
> illumination (HeNe-Green 50% default AOTF increased to 90%), we have
> switched to a 560LP filter from the 560-600EM filter. This improved
> their results, but they still are disappointed in signal intensity. I
> recently had our system checked over by Olympus service, and we
> measured the laser output as 0.45mW out of the fiber (non-wavelength
> specific power meter), which is just off the best it's ever been. I
> have suggested opening the pinhole and sacrificing Z resolution for
> brightness.
>
> Would you have any other suggestions for increasing the intensity of
> their signal? I have explained filters and Z-thickness versus
> brightness. I don't know what to tell them, and they are faulting the
> system. From all spectra I can find, our system should be great as
seeing this construct.
Kathy Spencer