Fluorescence Multi-parametric Microscopy Study

Fluorescence spectroscopy and microscopy have evolved into powerful tools in biomedicine. The combination of the two technologies provides not only high molecular specificity and multiple microenvironmental parameters, but also high spatial resolution. One demand for the detection and imaging systems in biomedicine is to obtain multi-functional information with high sensitivity and specificity and in one measurement. Fluorescence spectrum measurement can be used for distinguishing different molecular species in a sample and contrasting different fluorophores. Like fluorescence spectrum, fluorescence lifetime provides another unique contrast mechanism in biomedical imaging, particularly for the identification of fluorophores with overlapping spectra. In addition, lifetime measurement is very sensitive to the microenvironment of the fluorophores. Therefore a lot of physiological parameters including pH, Ca²⁺, Na⁺ and pO₂ can be quantified from fluorescence lifetime. The simultaneous measurement of fluorescence spectrum and lifetime provides distinct contrast mechanisms and complementary functional information in fluorescence imaging, which attracted more attention in biomedical community. Another demand for the detection and imaging systems is to be able to monitor the dynamic processes of living cells non-invasively and in real time. Multifocal multiphoton microscopy (MMM) can acquire three-dimensional fluorescence microscopic images by multiphoton excitation with the advantages of high speed, reduced photobleaching and photodamage, enhanced penetration depth and high signal to noise ratio. Fluorescence lifetime imaging has also been demonstrated in MMM by several groups, but the presented schemes do not have high temporal resolution, and more severely, no...