An internal standardization procedure for spectrally resolved fluorescence lifetime imaging

An internal standardization procedure, provided by comparing the modulation depth and phase shift of the laser light to that of a standard, for spectrally resolved fluorescence lifetime imaging (FLIM) was discussed. The internal standardization can be implemented on sFLIM using a scattering surface and a holographic notch filter. It was indicated that a relatively simple and desirable introduction of a holographic notch filter into the optical arrangement of a spectrally resolved FLIM system results in a stabilization of the lifetime measurements over time. The notch filter blocks most of the excitation light and allows the widest spectral range for the emitted fluorescence, and hence, gives the greatest versatility.