Fluorescence Lifetime Sensing

A cornerstone of Ciencia's technology base is patented instrumentation and methods for fluorescence lifetime sensing. A general-purpose research instrument based on this technology, LifeSense™, is being marketed by Oriel Instruments under a license agreement.

Fluorescence is the process whereby certain materials (fluorophores) emit light at certain wavelengths after absrobing light of a shorter wavelength. Fluorescence emission can be characterized by several parameters which include intensity, wavelength (excitation and emission spectra), quantum yield, polarization, and lifetime. These parameters may be sensitive to the microenvironment of the fluorophore; thus in sensing and bioanalytical applications, extrinsic fluorescent probes are often conjugated to biomolecules, as signal transduction elements.

Fluorescence intensity is the fluorescence parameter most widely used for signal transduction in sensing and analytical applications. However, fluorescence intensity is a relative measurement which depends on instrument characteristics. This makes comparison of data taken with different instruments or even with the same instrument at different times difficult. Intensity measurements are also affected by photobleaching and leaching of fluorophore from the sensing matrix. This creates troublesome calibration issues. Additionally, turbidity or the presence of colored compounds in the sample further complicate the interpretation of the measurements.

Some of the drawbacks of intensity measurements can be circumvented with the use of ratiometric dyes, which undergo a change in excitation or emission spectral characteristics in reponse to the event of analytical interest (e.g., binding to the analyte). In principle, obtaining the ratio of individually measured intensities at two emission or excitation wavelenghts may compensate for the spurious factors that may affect the measurement of intensity alone. However, the number of useful ratiometric dyes is quite limited preventing this technique from offering a universal approach. Moreover, ratiometric measurements require an increase in instrument complexity and are instrument-dependent making results obtained with different instruments not directly comparable without careful calibration.

In contrast, fluorescence lifetime measurements yield an absolute quantity whose value is independent of the measurement platform. Furthermore, its value is independent of the measured intensity, and thus it is highly immune to photobleaching and changes in fluorophore concentration, turbidity in the sample, optical misalignment and other variables that affect fluorescence intensity measurements. Lifetime sensing also permits simultaneous quantitative multi-label detection and reliable subtraction of non-specific background fluorescence. The robustness of fluorescence lifetime sensing makes this approach an ideal tool for measurements on difficult-to-control "real world" samples, as are found in environmental, biomedical, and industrial applications.