Correction of emission spectra in microspectrofluorimetry using a reference lamp: computations

Area of Science:

• Spectroscopy
• Optical Physics
• Analytical Chemistry

Background:

• Accurate emission spectra correction in microspectrofluorimetry is crucial.
• Tungsten emissivity correction necessitates knowing the true lamp filament temperature.
• Photomultipliers respond to quanta, requiring conversion from energetic units.

Purpose of the Study:

• To provide a method for determining the true temperature of a reference lamp filament from its color temperature.
• To outline procedures for converting spectral radiance units for photomultiplier detection.
• To enable accurate spectral correction, especially for fluorescence spectra extending into the far-red region.

Main Methods:

• Calculating true filament temperature from color temperature.
• Converting spectral radiance from energetic units to quanta.
• Utilizing non-linear regression on PC with curve fitting for temperature adjustment at lower voltages.
• Calibrating halogen lamps against certified ribbon filament lamps for high accuracy.

Main Results:

• A method is presented to derive true lamp temperature from color temperature.
• Procedures for unit conversion ensure compatibility with photomultiplier detectors.
• Non-linear regression allows accurate determination of spectral distribution at adjusted lamp temperatures.
• Tungsten halogen lamps are suitable references due to size and stability.

Conclusions:

• Accurate microspectrofluorimetry relies on precise calibration of reference lamps.
• The described methods facilitate accurate spectral correction by accounting for lamp emissivity and detector response.
• Adjusting lamp temperature and using appropriate unit conversions enhance spectral analysis accuracy.