Emerging applications of fluorescence spectroscopy to cellular imaging: lifetime imaging, metal-ligand probes, multi-photon excitation and light quenching.

Advances in time-resolved fluorescence spectroscopy can be applied to cellular imaging. Fluorescence lifetime imaging microscopy (FLIM) creates image contrast based on the decay time of sensing probes at each point in a two-dimensional image. FLIM allows imaging of Ca2+ and other ions without the need for wavelength-ratiometric probes. Ca2+ imaging can be performed by FLIM with visible wavelength excitation. Instrumentation for FLIM is potentially simple enough to be present in most research laboratories. Applications of fluorescence are often limited by the lack of suitable fluorophores. New, highly photostable probes allow off-gating of the prompt autofluorescence, and measurement of rotational motion of large macromolecules. These luminescent metal-ligand complexes will become widely utilized. Modern pulse lasers allow new experiments based on non-linear phenomena. With picosecond and femtosecond lasers fluorophores can be excited by simultaneous absorption of two or three photons. Hence, Ca2+ probes, membrane probes, and even intrinsic protein fluorescence can be excited with red or near infrared wavelengths, without ultraviolet lasers or optics. Finally, light itself can be used to control the excited state population. By using light pulses whose wavelength overlaps the emission spectrum of a fluorophore one can modify the excited state population and orientation. This use of non-absorbed light to modify emission can have wide reaching applications in cellular imaging.