For the various optical measurements three different spectra are necessary:

a) Measurement spectrum: the spectrum that is acquired from the spectrometer during the experimental procedure i.e. the spectrum that is transformed due the interaction with the sample under measurement.

b) Reference spectrum: the spectrum that is acquired from the spectrometer with experimental set-up, but at the absence of the sample to be characterized i.e. the spectrum that is emitted from the light source + the efficiency of the used fibers + the response vs. wavelength of the spectrometer. The reference spectrum is used for the normalization of the measurement spectrum due: i) the non linear intensity of light vs. the wavelength that emitted from the light source ii) the light propagation efficiency of the optical fibers and iii) the non linear spectrometer's response vs. wavelength. E.g. figure 1 shows the reference, measurement spectrum (red and yellow line) plus the normalized measurement spectrum (measurement / reference) that take into account the non linearity of the light source and the spectrometer efficiency.

c) Dark spectrum: the spectrum that is acquired from the spectrometer  at the absence of the emitting light from light source (normally by using a shutter). The dark spectrum is subtracted from both measurement and reference spectra to correct: i) the background electronic noise from the liner CCD of spectrometer detector and ii) the influence of the ambient light.

The reference and dark spectra are acquired and stored at the beginning of the experiment once. These spectra remain unchanged if the experimental set-up (including light source, optical fibers and spectrometer configuration) is the same. The measurement spectrum can be acquired dynamically or statically, following the particular experimental needs.