Evaluating our fluorometers

Yesterday Katie and I did some tests to evaluate the performance of our new Fluorolog.  This involved some "easy" number crunching.  Our simple number crunching required extra attention to how we organized our data, mainly due to the different ways we can analyze sample fluorescence on the new instrument, depending on geometry or detector type (for simplicity).  We analyzed the emission spectrum of quinine sulfate, a well characterized fluorophore (molecule that emits excess energy as light from its excited state).  We compared our quinine sulfate emission spectrum to the emission spectrum of quinine sulfate as presented by the National Institute of Standards & Technology (NIST; specifically Velapoldi & Mielenz, 1980).  The goal of this comparison is to see if our quinine sulfate spectrum overlaps perfectly with the NIST reference spectrum, generally following guidelines and recommendations in Cory et al. (L&O Methods, In Press).  Typically, emission spectra collected on different instruments contain signal (intensity) that is a function of (1) the fluorophore studied (how it emits light as a function of wavelength)  and (2) the response of the instrument (specific to each instrument and its components).  As you can see from my illustration above, our quinine sulfate spectrum did not overlap well with the NIST reference spectrum until we removed the instrument-specific response.
Why is this important? Because we need to be able to compare our data to data collected on other instruments in different labs now and in the future.  Removing instrument-specific response means that ideally, all signal is due only to the molecule(s) studied.  If all labs remove instrument response, we can all confidently compare trends among labs and over time.