Fluorescence spectroscopy, also known as fluorometry or spectrofluorometry, is a type of electromagnetic spectroscopy, which analyzes fluorescence from a sample. It involves using a beam of light that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. It is a useful technique in many biological (chlorophyll and carotenoid), biochemical (diagnosis of malignancies) and environmental applications.
For most fluorescence applications the amount of fluorescent energy emitted is only 3% of the amount of excitation light energy. Fluorescent light has a lower energy (higher wavelength) than the excitation energy and is usually scattered light. This means it emits energy in all directions.
For optimal performance, assuming the time acquisition window is not limited, Avantes recommends our AvaSpec-ULS2048LTEC spectrometer for this application, since it can support long integration times often exceeding 5 seconds. When higher-speed acquisition is required, Avantes recommends the AvaSpec-ULS2048XL back-thinned CCD spectrometer. For maximal sensitivity the top model of the SensLine, our AvaSpec-HERO spectrometer is recommended.
Why Avantes for your fluorescence application?
- High sensitivity for low fluorescent emission
- Long integration time capabilities (exceeding 5 seconds)
- High-speed acquisition
- Light sources for the most challenging fluorescence applications
Configuring a Setup
When configuring a measurement setup for fluorescence applications, preventing excitation light from entering the spectrometer is an important issue. Possible methods to accomplish this, where one does not exclude the other, include:
- Make use of an AvaLight-LED or AvaLight-HPLED light source for more challenging fluorescence applications. Our AvaLight-LED light sources typically have a narrow bandwidth, enabling the limitation of excitation to shorter wavelengths that are not part of the emission spectrum.
- Use a broadband light source such as the AvaLight-HAL-Mini for high output in combination with an (interference) band-pass or low-pass filter.
- Make sure the optical path for excitation light and fluorescence are perpendicular. This means the excitation light will not enter the receiving fiber (use the CUV-FL-UV/VIS or the CUV-DA).
- Use the decay time to separate excitation energy from the integration time start pulse. Use a pulsed light source to accomplish this (pulsed laser or AvaLight-XE or AvaLight-XE-HP Xenon flash light sources).
Avantes has developed a bundle especially for fluorescence measurements. This bundle is suited for various fluorescence applications.
These are some of the products mentioned above. To find the perfect setup for your specific application, contact one of our sales engineers for advice.
Below are some detailed application notes on various uses of fluorescence spectroscopy in different applications and industries.