Fluorescence lifetime imaging in confocal microscopy: signal-to-noise considerations

By utilizing lifetime information, in addition to spectral properties, new and interesting possibilities can be realized in confocal fluorescence microscopy. In combination with the previously described Intensity-modulated Multiple-beam Scanning (IMS) technique it is possible to strongly increase the number of fluorophores that can be simultaneously and independently recorded. Another possibility is to detect, simultaneously, changes in the lifetimes of multiple fluorophores caused by changes in the chemical environment such as pH or ion concentration. A factor that critically determines the usefulness of lifetime information is the signal-to-noise ratio (SNR) that can be attained. This paper presents a theoretical investigation of the SNR when using the IMS in combination lifetime measurements. It is found that when increasing the number of simultaneously detected fluorophores, it is important to select fluorophores with widely different lifetimes. For lifetime measurements the precision can be expressed in terms of the number of detected photons.