Re: tdTomato and LSM confocal

Posted by Mark Cannell on
URL: http://confocal-microscopy-list.275.s1.nabble.com/More-power-and-less-exposure-or-vice-versa-use-of-100-laser-tp3246685p3947364.html

Are they using an index-matched mounting medium?

Do they understand that a wide field "like what they see" is quite
different to a confocal section?

Cheers

>> 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.
>>    
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>  Kathy Spencer
>