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We developed a compact stimulated emission depletion (STED) two-photon excitation microscopy that utilized electrically controllable components. Transmissive liquid crystal devices inserted directly in front of the objective lens converted the STED light into an optical vortex while leaving the excitation light unaffected. Light pulses of two different colors, 1.06 and 0.64 μm, were generated by laser diode-based light sources, and the delay between the two pulses was flexibly controlled so as to maximize the fluorescence suppression ratio. In our experiments, the spatial resolution of this system was up to three times higher than that obtained without STED light irradiation, and we successfully visualize the fine microtubule network structures in fixed mammalian cells without causing significant photo-damage.

We have generated optical pulses of 1.2 MW peak power and 0.6 ps duration using a 1060 nm band gain-switched laser diode pulse oscillator. Optical pulses are amplified by three-stage ytterbium-doped fiber amplifiers, and remarkable reductions of amplified spontaneous emission noise and temporal duration have been accomplished based on self-phase modulation in the middle-stage amplifier. After the main amplifier, optical pulses were temporally compressed by a grating pair, and this enabled generation of subpicosecond optical pulses with over 1 MW peak power.

In vivo two-photon microscopy is an advantageous technique for observing the mouse brain at high resolution. In this study, we developed a two-photon microscopy method that uses a 1064-nm gain-switched laser diode-based light source with average power above 4 W, pulse width of 7.5-picosecond, repetition rate of 10-MHz, and a high-sensitivity photomultiplier tube. Using this newly developed two-photon microscope for in vivo imaging, we were able to successfully image hippocampal neurons in the dentate gyrus and obtain panoramic views of CA1 pyramidal neurons and cerebral cortex, regardless of age of the mouse. Fine dendrites in hippocampal CA1 could be imaged with a high peak-signal-to-background ratio that could not be achieved by titanium sapphire laser excitation. Finally, our system achieved multicolor imaging with neurons and blood vessels in the hippocampal region in vivo. These results indicate that our two-photon microscopy system is suitable for investigations of various neural functions, including the morphological changes undergone by neurons during physiological phenomena.

We have developed a method to generate sub-nanosecond 650-nm-band optical pulses. These pulses have a peak power of 40 W and a pulse energy of 13 nJ at a 1-MHz repetition rate. This technology is intended for application in stimulated-emission-depletion microscopy. Our method is based on the pulsed operation of a 1.3-µm-band semiconductor-laser optical amplifier and the second-harmonic generation of the optical pulses after amplification by a Pr-doped fiber amplifier. The resultant peak power and pulse energy of the 650-nm-band optical pulses are two orders of magnitude higher than those directly obtained from a laser diode.