Correction of emission spectra in microspectrofluorimetry using a reference lamp: computations.
Accurate correction of emission spectra in microspectrofluorimetry, using a reference lamp, may require that a factor for the emissivity of tungsten be introduced. This is only possible provided that the true temperature of the lamp filament is known. A method is given for obtaining the true temperature from the knowledge of the colour temperature. Also, the values of the spectral concentration of the radiance of the black body, either computed according to Planck's equation or taken from available published tables, have to be converted from energetic units to units of quanta since the photomultiplier is linear not to absorbed power but to units of quanta. When the fluorescence spectra to be corrected extend into the far red it is preferable to use a lower temperature (by lowering the supply voltage) than that for which the lamp is certified. It is possible to determine the new temperature (and then the corresponding spectral distribution) by taking a few pairs of measurements at different wavelengths both at the lower voltage and at the voltage for which the lamp is certified and then introducing these values in a non-linear regression soluble on a PC with a curve fitting program. The microscope tungsten halogen lamp can conveniently be used as a reference, thanks to its small size and its steady spectral characteristics. When high accuracy is required, however, the halogen lamp should be calibrated against a certified ribbon filament lamp.