Technology

laser-induced fluorescence spectroscopy

Due to a very high sensitivity and specifity the detection of fluorescence signals has an increasing importance in biotechnological and medical analytics. Commonly and most often used are measurements of fluorescence intensities, fluorescence spectra and fluorescence polarisations. These information can be used for the sensitive detection of low analyte concentrations, of binding reactions (e.g. between antigen and antibody) or the investigation of chemical reaction kinetics. A less common quantity to be measured is the value of the fluorescence lifetime. This value is a physical constant specific for each type of fluorescing molecules (specific for each fluorescence label) and is determined by its concrete physical and chemical state. Fluorescence lifetimes are potentially influenced by the local micro-environment of the molecule, by the solvent, pH, binding state, temperature, ...

Fluorescence lifetimes of most fluorescing organic chromophores are to be found in the range of a few nanoseconds. Usually short laser pulses are required to excite the sample while the fast fluorescence decay is detected with a high temporal resolution. The microplate reader LF 500 NanoScan FLT uses for the sample excitation a short pulsed combination of a Nitrogen laser with a dye laser. It is the basis for a huge dynamic range of more than 6 decades regarding resolvable lifetimes between 1 nanosecond and several milliseconds. The instrument LF 402 Metabolic also uses the laser-induced and time-resolving method of fluroescence detection in order to achieve an improved selectivity and sensitivity for metabolism-related auto fluorescence of cells and tissues. Here the "tool" for the user is a fibre-optic probe which eliminates the constraint of having samples inside of cuvettes.

What is fluorescence lifetime?

The decay of fluorescence signals after excitation with a short (laser) pulse follows an exponential function: