[FDS] FDS sensitivity

[Tom Laue]

Hi Bo, et al.
I agree that a systematic study of sensitivities is a good idea. Be 
aware that a dye like SBYR Green changes its quantum yield dramatically 
(which is why it is used in PCR to detect dsDNA). The problem of 
changing quantum yield is general to fluorescence, and not FDS specific. 
Jack Correia is working on a paper about the issues that need to be 
addressed if one wants to calculate a concentration from a fluorescence 
signal. By the way, would you add Jack and Rachel to the FDS list (if 
not already there)? They have as much experience (or more) with the FDS 
than anyone.
I will have to see how much GFP we have. Unbound GFP is a good choice 
since it does not quench readily. On the other hand, it may change its 
quantum yield when bound to another molecule due to changes in the local 
dipole moment.
Best wishes,
Tom

Borries Demeler wrote:
> Tom and Steve,
>
> Once we get our machine working I would like to do a systematic study on
> sensitivity responses and the different gain settings so we can generate
> some instrument-specific calbration curves that can be compared between
> instruments. We probably need to do this for each dye and check a large
> concentration range of dye to precisely predict what kind of response
> can be expected. Besides the "order of magnitude" degassing effect on 
> intensity, I would also like to carefully measure the effect of photobleaching,
> especially for dyes like sybrgreen in DNA. I have a number of experiments
> to do with low concentration DNA and virus particles from Phil Serwer's 
> samples, and low concentration DNA is absolutely essential because of
> the superlarge size of these molecules to avoid hydrodynamic nonideality.
> Steve, I would suspect you chromatin people are in the same boat, and
> sybrgreen could potentially work very well for that - I just don't know.
> Just something I would love to try out once we get our machine working.
>
> Basically, I would like to take up Tom's offer to test our machine on
> the GFP samples for which calibration standards exist from his machine
> and then compare that sample among our machines to see the variation in
> intensity observed between machines. So I would suggest we send it around
> and see what everyone can measure with it.
>
> -Borries
>
>
>   
>> Tom, is the radial and focus calibration procedure accurately described in the 
>> AOS manual? Or is there a document describing this I can access? Can this 
>> become a regular part of instrument upkeep, ie performing this procedure 
>> monthly?
>> Obviously, we would all like to get the most sensitivity our of our machines. 
>> My only real check of signal/sensitivity is the calibration centerpiece I 
>> loaded with free fluorosceinin january, and I wonder if this may not 
>> accurately represent the anticipated signal strength anymore. 
>> thanks, 
>> steve
>>     
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-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
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E-mail: Tom.Laue at unh.edu
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