Transient absorption imaging of hemes with 2-color, independently tunable visible-wavelength ultrafast source.

Pump probe microscopy is a time-resolved multiphoton imaging technique capable of generating contrast between non-fluorescent pigments based on differences in excited-state lifetimes. Here we describe a fiber-based ultrafast system designed for imaging heme proteins with an independently-tunable pulse pair in the visible-wavelength regime. Starting with a 1060 nm fiber amplifier (1.3 W at 63 MHz, 140 fs pulses), visible pulses were produced in the vicinity of 488 nm and 532 nm by doubling the output of a short photonic crystal fiber with a pair of periodically-poled lithium niobate crystals, providing 5-20 mW power in each beam. This was sufficient for acquiring transient absorption images from unstained cryosectioned tissue.