Yi-Cheng Fang

In vivo two-photon imaging of mouse hippocampal neurons in dentate gyrus using a light source based on a high-peak power gain-switched laser diode

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.

Generation of synchronized picosecond pulses by a 1.06-µm gain-switched laser diode for stimulated Raman scattering microscopy.

We propose that a gain-switched laser diode (GS-LD) can be used as a picosecond laser source for stimulated Raman scattering (SRS) microscopy. We employed a 1.06-µm GS-LD to generate ~13-ps pulses at a repetition rate of 38 MHz and amplified them to >100 mW with Yb-doped fiber amplifiers. The GS-LD was driven by 200-ps electrical pulses, which were triggered through a toggle flip-flop (T-FF) so that the GS-LD pulses were synchronized to Ti:sapphire laser (TSL) pulses at a repetition rate of 76 MHz. We found the timing jitter of GS-LD pulses to be approximately 2.7 ps in a jitter bandwidth of 7 MHz. We also show that the delay of electrical pulses can be less sensitive to the optical power of TSL pulses by controlling the threshold voltage of the T-FF. We demonstrate the SRS imaging of polymer beads and of HeLa cells with GS-LD pulses and TSL pulses, proving that GS-LD is readily applicable to SRS microscopy as a compact and stable pulse source.

Synchronized and timing-stabilized pulse generation from a gain-switched laser diode for stimulated Raman scattering microscopy

We present a picosecond laser source based on a gain-switched laser diode (GS-LD) that can be applied to stimulated Raman scattering (SRS) microscopy. A 1.06-μm GS-LD was used to generate 14-ps pulses at a repetition rate of 38 MHz. The GS-LD was driven by 200-ps electrical pulses, which were triggered through a toggle flip-flop (T-FF). As a result, the GS-LD pulses were subharmonically synchronized to Ti:sapphire laser (TSL) pulses at a repetition rate of 76 MHz. We investigated the timing jitter of GS-LD pulses and found it to be less than 2.5 ps. We also show that the trigger delay can be less sensitive to the optical power of TSL pulses by controlling the threshold voltage of the T-FF. As a result, GS-LD pulses sufficiently overlapped with TSL pulses even when we scanned the wavelength of the TSL pulses. We demonstrate the SRS imaging of HeLa cells with GS-LD pulses and TSL pulses, proving that GS-LD is readily applicable to SRS microscopy as a compact and stable pulse source.

Improvement of two-photon microscopic imaging in deep regions of living mouse brains by utilizing a light source based on an electrically controllable gain-switched laser diode

In vivo two-photon microscopy is an advantageous technique for observing living mouse brains at high spatial resolutions. We previously used a 1064 nm high-power light source based on an electrically controllable gain-switched laser diode (maximum power: 4 W, repetition rate: 10 MHz, pulse width: 7.5 picoseconds) and successfully visualized EYFP expressing neurons at deeper regions in H-line mouse brains under living conditions. However, severe damages were frequently observed when the laser power after the objective lens was over 600 mW, suggesting that a higher average power might not be suitable for visualizing neural structures and functions at deep regions. To increase fluorescent signals as a strategy to avoid such invasions, here, we evaluated the effects of the excitation laser parameters such as the repetition rate (5 - 10 MHz), or the peak power, at the moderate average powers (10 - 500 mW), by taking the advantage that this electrically controllable light source could be used to change the repetition rate independently from the average power or the pulse width. The fluorescent signals of EYFP at layer V of the cerebral cortex were increased by approximately twofold when the repetition rate was decreased from 10 MHz to 5 MHz at the same average power. We also confirmed similar effects in the EYFP solution (335 μM) and fixed brain slices. These results suggest that in vivo two-photon microscopic imaging might be improved by increasing the peak power at the same average power while avoiding the severe damages in living brains.

Advanced easySTED microscopy based on two-photon excitation by electrical modulations of light pulse wavefronts

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.

Simultaneous multi-optical parametric oscillations and broad up-conversion on χ (2) nonlinear photonic crystals

Parallel excitation of optical parametric oscillations was observed on PPLT dispersed with tri-QPM structures in the transverse direction to a nano-second pump beam. It led to simultaneous IR generation from 1000 to 1150nm with slope efficiency > 30%. When a nonlinearly-chirped PPLT was subject to such IR pump, it exhibits simultaneous broad green generation from 500 to 560nm with single pass efficiency ∼10%. This was due to broad spectral coverage of QPM-SHG and SFG in the chirp structures.

Experimental investigation of timing jitter of a 1.06-μm gain-switched laser diode for stimulated Raman scattering microscopy

The timing jitter of 13-ps gain-switched laser diode pulses at 1.06 μm is measured to be 3.9 ps, which is further reduced by external injection. We successfully demonstrate stimulated Raman imaging using the pulses.

Advanced semiconductor-laser light pulse sources for multiphoton microscopy

We have developed novel light pulse sources based on the picosecond-pulse semiconductor laser technology. The light pulses are amplified to 100-kW peak power, and this enables the deep-site in vivo imaging of mouse brain tissues.