Pump probe cross correlation fluorescence frequency domain microscope and microscopy

A scanning fluorescence lifetime microscope measures modulation and phase in fluorescence emission stimulated by spatially overlapped pump and probe beams operating at different frequencies. A pump laser modulated at a first frequency is focused onto a diffraction limited spot to excite a fluorescent sample under study. Simultaneously, a probe laser modulated at a second frequency is focused onto the same spot to induce a stimulated fluorescence emission in response to the optically combined output of the pump and probe laser light. Fluorescence emitted from the sample produces a cross-correlation signal which is dependent upon the spatial overlapping of the pump and probe beams at the focal point thereby producing a beneficial axial sectioning effect. Choosing a small differency frequency between the modulation of the first and second laser sources produces a low frequency cross correlation signal even when the modulation frequencies of the pump and probe lasers are very high. A signal processor obtains modulation and phase information from the low frequency cross correlation fluorescence signal allowing monitoring of even ultrafast fluorescence phenomena induced by high frequency modulation of the pump and probe lasers.