Multifarious control of two-photon excitation of multiple fluorophores achieved by phase modulation of ultra-broadband laser pulses.

We propose two-photon excited fluorescence (TPEF) microscopy employing a novel phase modulation technique of ultra-broadband laser pulses, which allows the relative excitation of an individual fluorophore with respect to other fluorophores. This technique is based on the generation of multi-wavelength pulse train, which independently interacts with each fluorophore. Our technique is applied to dual-color imaging of cells expressing two types of fluorescent proteins. We achieve the selective excitation of one over the other and vice versa. The product of the maximum contrast ratios exceeds 100. We also demonstrate yielded equal excitation rates in the simultaneous excitation. By the selective excitation of a donor fluorescent protein, fluorescence resonance energy transfer imaging is also achieved.