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

We present the implementation of intensity-modulated laser diodes for appli cations in frequency-domain pump-probe fluorescence microscopy. Our techniq ue, which is based on the stimulated-emission approach, uses two sinusoidal ly modulated laser diodes. One laser (635 nm) excites the chromophores unde r study, and the other laser (680 nm) is responsible for inducing stimulate d emission from excited-state molecules. Both light sources are modulated i n 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. M icroscope imaging at the cross-correlation signal results in images with hi gh contrast, and time-resolved high-frequency information can be acquired w ithout high-speed detection. A detailed experimental arrangement of our met hodology is presented along with images acquired from a 4.0-mum-diameter fl uorescent sphere and TOTO-3-labeled mouse STO cells. (TOTO-3 is a nucleic a cid stain.) Our results demonstrate the feasibility of using sinusoidally m odulated laser diodes for pump-probe imaging, creating the exciting possibi lity of high-contrast time-resolved imaging with low-cost laser-diode syste ms. (C) 2001 Optical Society of America. OCIS codes: 180.0180, 140.2020, 17 0.2520.