Two-photon microscopy (TPM) enables deep tissue imaging of complex biological processes at high resolution. However, the ability to visualize some biological processes, such as neural activity at the sub-millisecond scale, requires high-speed imaging in addition to high resolution. To address this issue, a team of researchers from institutions in China and Germany developed a high-speed TPM system that scientists can use to observe very fast biological processes at high temporal resolution and high spatial resolution. Until now, the performance of TPM has been limited by its line-scanning frequency, measured as frames per second (fps). Line-scanning frequency refers to the rate at which the target sample can be swept along one direction by the excitation laser. A slow scanning frequency affects the microscope’s overall fps, because it also determines the speed at which the laser is swept in the other direction. This leads to a trade-off between the microscope’s temporal resolution and the size of its observation frame. In the high-speed TPM, the researchers resolved this trade-off by combining two laser scanning modes. To increase the TPM’s line-scan frequency, the researchers designed a custom acousto-optic deflector (AOD) using a tellurium dioxide (TeO2) crystal. An AOD is a special type of crystal whose refractive index can be precisely controlled by acoustic waves. The AOD has high acoustic velocity, enabling the laser to scan a line in the frame within 2.5 µs. By combining two laser scanning modes, researchers have developed a versatile two-photon microscopy system that can be used to observe extremely fast biological processes at high frame rates and high spatial resolution. Courtesy of Li et al., doi 10.1117/1.NPh.10.2.025006. To expand the maximum frame rate of the microscope, the researchers incorporated a second AOD for slow axis scanning. For stepwise magnification, from population view to subcellular view with high spatial and temporal resolution, the researchers combined the AOD with resonant-galvo scanning (RS). The use of AODs to control the scanning of the excitation laser enables laser-steering that is faster than the speeds obtained with the galvanometers used in conventional TPMs. At the same time, the option to switch to a galvanometer-based laser scanning mechanism allows large regions of a sample to be scanned at an acceptable resolution and speed, making it easier to locate small areas of interest before switching to AOD scanning. The new microscope has a maximum line-scan frequency of 400 kHz and a maximum frame rate of 10,000 fps at 250 × 40 pixels. The user can switch among a variety of scanning settings. To demonstrate the high-speed TPM, the researchers installed cranial windows on genetically engineered mice and used the TPM to observe the morphology and activity of neurons and the movement of single red blood cells. The frame rate of up to 10,000 fps was sufficient to precisely measure the velocity at which calcium propagates in neuronal dendrites and to visualize the trajectory of individual red blood cells within blood vessels. The experimental results also showed that photobleaching rates were reduced by increasing line-scan frequency. The experiments demonstrated that the combination of AOD and RS in two-photon microscopy provides both versatility and precision to support the quantitative analysis of single neuronal activities and hemodynamics in vivo. The high-speed TPM could be used for high-resolution, temporally resolved, subcellular investigations of complex neural and vascular systems, improving scientific understanding of these areas. “The new system for AOD-based scanning microscopy represents a substantial improvement in imaging speed and performance, as demonstrated in its application for calcium signal propagation and blood flow measurements in the brain in vivo,” said UC Berkeley professor Na Ji, who is also associate editor of Neurophotonics. The research was published in Neurophotonics (www.doi.org/10.1117/1.NPh.10.2.025006).