Implementation of intensity-modulated laser diodes in time-resolved, pump–probe fluorescence microscopy

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Abstract
We present the implementation of intensity-modulated laser diodes for applications in frequency-domain pump–probe fluorescence microscopy. Our technique, which is based on the stimulated-emission approach, uses two sinusoidally modulated laser diodes. One laser (635 nm) excites the chromophores under study, and the other laser (680 nm) is responsible for inducing stimulated emission from excited-state molecules. Both light sources are modulated in the 80-MHz range but with an offset of 5 kHz between them. The result of the interaction of the pump and the probe beams is that a cross-correlation fluorescence signal at 5 kHz is generated primarily at the focal volume. Microscope imaging at the cross-correlation signal results in images with high contrast, and time-resolved high-frequency information can be acquired without high-speed detection. A detailed experimental arrangement of our methodology is presented along with images acquired from a 4.0-µm-diameter fluorescent sphere and TOTO-3–labeled mouse STO cells. (TOTO-3 is a nucleic acid stain.) Our results demonstrate the feasibility of using sinusoidally modulated laser diodes for pump–probe imaging, creating the exciting possibility of high-contrast time-resolved imaging with low-cost laser-diode systems.