The advent of two-photon microscopy (TPM) has brought about a significant change in the field of biology by facilitating the observation of intricate biological processes in living tissues at a superior resolution. Unlike conventional fluorescence microscopy techniques, TPM employs low-energy photons to stimulate fluorescent molecules for observation. This permits a much deeper penetration into the tissue and ensures that the fluorescent molecules, or fluorophores, remain undamaged by the excitation laser.
Despite its advantages, TPM has limitations when it comes to recording extremely rapid biological processes, even with cutting-edge technology. One such limitation is the line scanning frequency, which is measured in frames per second (FPS) and determines how quickly the excitation laser can sweep across the sample in one direction (for example, horizontally). A low scanning frequency also impacts the overall FPS of the microscope, as it influences the speed at which the laser can be swept across the other direction (vertically). As a result, there is a tradeoff between the microscope’s temporal resolution and the size of the observation frame.
To overcome this issue, a team of researchers from China and Germany has recently developed an exceptionally high line scanning frequency in their TPM setup. Their report, published in Neurophotonics, suggests that this microscope system has been specifically designed to capture fast biological processes at a high spatial as well as temporal resolution.
The new TPM proposed by the research team is distinct from conventional TPMs in several ways, one of which is its use of acousto-optic deflectors (AODs) to control the scanning of the excitation laser. AODs are crystals whose refractive index can be accurately manipulated by acoustic waves, allowing for precise redirection of a laser beam. Crucially, AODs permit faster laser-steering compared to the galvanometers used in conventional TPMs.
To achieve a high line scanning frequency, the research team constructed a custom AOD with an incredibly high acoustic velocity using a tellurium dioxide (TeO2) crystal. This design allowed the laser to scan a line in the frame in a mere 2.5 microseconds, resulting in a maximum line scanning frequency of 400 kHz. Similarly, an AOD was used to achieve an acceptable slow scanning frequency in the other direction.
To enhance the versatility of their microscope, the team incorporated the option to switch to a galvanometer-based laser scanning mechanism when required. This enabled rapid scanning of larger regions of the sample at a reasonable resolution and speed, making it simpler to locate small areas of interest before switching to AOD scanning.
The research team carried out multiple proof-of-concept experiments using the newly developed TPM. They implanted cranial windows on genetically modified mice and employed the TPM to observe the structure and activity of neurons as well as the movement of individual red blood cells (RBCs). By using the appropriate AOD configuration and frame size, the system achieved a frame rate of up to 10,000 FPS, which was adequate to accurately measure the speed at which calcium spreads in neuronal dendrites and visualize the path of individual RBCs in blood vessels.
Dr. Na Ji, Associate Editor of Neurophotonics and Luis Alvarez Memorial Chair in Experimental Physics at UC Berkeley, was impressed by the findings and stated, “The new AOD-based scanning microscopy system represents a significant improvement in imaging speed and performance, as evidenced by its success in capturing calcium signal propagation and blood flow measurements in the brain in vivo.”
In the future, the new TPM design will enable researchers to observe fast biological processes, providing an improved understanding of these phenomena.