Two-photon excitation microscopy is a powerful tool for studying brain neuronal activities. The imaging speed of traditional two-photon excitation microscopy technologies based on mechanical point-by-point laser scanning is relatively slow, which prevents the real-time observation of neuronal activities. Additionally, femtosecond lasers with high repetition rate are essential for high-speed two-photon excitation microscopy to achieve high signal intensity within a short pixel dwell time. We demonstrate a parallel GHz ultrafast laser scanning technology using acousto-optic deflection to exploit new potential for high-speed two-photon microscopy. The high-speed GHz ultrafast laser scanning system is built in the 920 nm wavelength range. By adjusting the temporal and spatial arrangement, 33 distinguishable parallel GHz ultrafast laser scanning beams are simultaneously generated within a frequency range of 15-31 MHz.

We adopt high-speed single-pixel parallel signal detection. The 920-nm femtosecond laser with a high repetition rate is split into two polarized beams using a polarizing beam splitter. One beam experiencing multitone-frequency modulation via an acousto-optic modulator serves as the reference beam, and the other beam is deflected by the radio frequency (RF) encoding technology. A time-domain signal with a random initial phase for each frequency drives the acousto-optic deflector to generate a one-dimensional laser beam array. The light spot is characterized by a CCD camera, then a delay line is employed to adjust the spatio-temporal overlap of the two beams to achieve interference. The electrical signals generated by the photodetector are digitally sampled by a high-speed data acquisition card and then are applied with a fast Fourier transform (FFT). Each laser beam is tagged with a specific frequency.

Frequency encoding design is performed within an RF range of 55-71 MHz to generate multitone RF driving signals with 33 frequencies, and each with a random initial phase. The duration of an arbitrary waveform cycle is set at 32.76 μs (Fig. 2). Although initially set with uniform amplitudes, the Fourier spectra of the loaded multi-frequency driving signals show variations after passing through the waveform generator and RF amplifier (Fig. 3). The acousto-optic deflector generates a one-dimensional laser beam array with relatively uniform intensities of the spots (Fig. 5), validating the correctness of the encoding scheme. After achieving spatio-temporal overlap, the photodetector detects typical beating signals with a duration of 32.76 μs (Fig. 8). A final RF spectrum of 33 uniformly spaced beating frequencies after performing FFT is obtained by multiple averaging (Fig. 10). The proposed parallel scanning technology presents promising applications in high-speed two-photon microscopy.

We design a high-speed parallel scanning system based on a 920 nm GHz ultrafast laser. The diffraction efficiency of the acousto-optic deflector is optimized, and a double-peak pattern is found within the driving frequency range of 30-90 MHz, with a 3 dB bandwidth of approximately 40.2 MHz. By designing an RF encoding scheme, the system generates 33 frequencies simultaneously. The generated diffraction laser beam array shows a nearly uniform intensity distribution. By adjusting the spatio-temporal overlap, each laser beam is frequency-tagged with a specific frequency. The RF spectrum of the beating signals after performing FFT is obtained by averaging the data multiple times to generate 33 distinguishable beating frequencies in the frequency range of 15-31 MHz. This confirms that the system can serve as a high-speed 0.9 μm two-photon laser parallel scanning light source.