Fig. 1. The schematic diagram of the pixel-excitation-gating enhanced GHz femtosecond pulse TPE microscope. The pixel-excitation-gating excitation is implemented by modulating the excitation GHz pulse train with an acousto-optic modulator (AOM), which is driven by a modulation waveform signal synchronized to the galvo-based raster scanning. The right bottom inset shows the temporal characteristics of the driven signals for the X-galvo, arbitrary waveform generator (AWG), and AOM. DAQ, data acquisition; PMT, photomultiplier tube; L, lens; Obj, microscope objective; DM, dichroic mirror.

Fig. 2. (a) The lateral resolution of the TPE microscope, measured using 200 nm fluorescent microspheres, under GHz femtosecond pulse TPE. The inset shows the image of a single 200 nm microsphere, averaged from 50 Z-stack frames. TPE images of 3 µm red fluorescent microspheres, excited by (b) GHz femtosecond pulses without pixel-excitation-gating, (c) with pixel-excitation-gating of 50% duty cycle and (d) 25% duty cycle. In the three cases, the same average power of 40 mW is applied. “R” indicates the raw image, while “G” indicates the image processed with a 2-pixel-radius Gaussian filter.

Fig. 3. TPE imaging of the mouse kidney section with a region size of 245 µm × 245 µm, captured (a) without and (b) with pixel-excitation-gating of 25% duty cycle. All the images were captured at a 30 mW average power. (c) and (d) Line plots of the image along the convoluted tubule, as indicated by the yellow lines in (a) and (b), respectively.

Fig. 4. TPE imaging of neurons in a mouse brain slice with a region size of 327 µm × 327 µm, captured (a) without and (b) with pixel-excitation-gating of 25% duty cycle. All the images were captured at a 40 mW average power. “R” denotes the raw images, while “S” denotes the image averaged from 50 raw images. (c) Line plots of the images along the soma (indicated by L1) and dendrites (indicated by L2), as shown in (a) and (b). The solid and dashed lines indicate the cases with and without pixel-excitation-gating, respectively.