Fluorescence Measurements

Fluorescence measurements are required in many biology (chlorophyll and carotenoid), biomedical (fluorescence diagnosis of malignancies) and environmental applications.

Fluorescence measurements typically need a high sensitivity setup (AvaSpec-2048TEC recommended for integration times > 5 seconds) as for most fluorescence applications, the amount of fluorescence energy emitted is only 3% of the amount of excitation light energy.

The fluorescence light is of a lower energy (higher wavelength) than the excitation energy and is usually scattered light (emits energy in all directions).

The most important consideration for the setup is to prevent excitation light from entering the spectrometer.

This can be achieved with different methods, where one does not exclude the other:

• Use an AvaLight-LED light source for excitation (small bandwidth), emitting no energy at fluorescence wavelength.
• Use an (interference) band pass or low pass filter in combination with an AvaLight-HAL light source for high output, small bandwidth excitation.
• Make sure that the optical path for excitation light and fluorescence are 90 degrees perpendicular. This way the 
  excitation light will not enter the receiving fibre (use of the CUV-UV/VIS-FL or the CUV-DA).
• Use the fluorescence decay time to separate excitation energy from the integration time start pulse. For this, a pulsed
  light source is required (pulsed laser or AvaLight-XE Xenon flash) and an AvaSpec-2048FT fast trigger spectrometer with 
  programmable delay time.

A typical setup for fluorescence measurements is shown in the illustration above.

Typical components used for fluorescence measurements