Enhanced two-photon excited fluorescence from green fluorescent proteins by ultrafast fluctuations in intense light pulse

Abstract

The parametric down-conversion process in optical parametric generators causes bunching of light due to ultrafast intensity fluctuations, which enhances the efficiency of nonlinear interactions between light and matter. However, the bunching effect in a sufficiently intense light pulse light required for biological nonlinear imaging has not yet been investigated. We demonstrate enhanced two-photon excited fluorescence by ultrafast fluctuations in intense pulse using a wavelength-tunable optical parametric generator consisting of a periodically poled lithium niobate crystal pumped by nanosecond pulses at a wavelength of 532 nm and emitting pulses with a peak power of about 1 kW. The emission wavelength is tuned to about 927 nm, which is optimal for two-photon excitation of green fluorescent protein. The effect of bunching by ultrafast intensity fluctuations in the pulse is evaluated by an autocorrelator using a green fluorescent protein solution as a two-photon absorber. We found an about 1.9-fold enhancement compared with the coherent state of light. Using this calibrated optical parametric generator, we perform two-photon imaging of green fluorescent protein in brain tissue within a timescale of seconds. These experimental results using intense pulses demonstrate that the bunching effect by ultrafast fluctuations can enhance nonlinear imaging in biology and medicine.