Fast, Focused, and Far-Reaching: A New 3D, Two-Photon Imaging Technique
Material below is adapted from the SfN Short Course Acousto-optical Scanning–Based High-Speed 3D Two-Photon Imaging in Vivo, by Balázs Rózsa, MD, PhD, Gergely Szalay, and Gergely Katona. Short Courses are daylong scientific trainings on emerging neuroscience topics and research techniques held the day before SfN’s annual meeting.
Oftentimes when imaging the brain, a wider view is sacrificed for finer detail, or vice versa. Although the ideal imaging technique could resolve both signals propagating within single neurons and signals sent across a whole network of hundreds of neurons, the two views present different challenges. Imaging in single cells must be able to distinguish activity in adjacent compartments (the soma compared to the dendrite, for example) while imaging networks of hundreds of cells must be able to measure simultaneous activity in cells that are hundreds of micrometers apart. Because of this, most current technologies are optimized for just one of the two views. A microscope that could tackle both would need to be fast yet fine-detailed, focused yet far-reaching.
Recently, researchers have created a high-speed, three-dimensional, two-photon system that meets all these requirements. The system uses an acousto-optical deflector (AOD), which diffracts the laser beam with sound waves. The two-photon technology provides depth, enabling exploration deeper into the cortex. All together, the microscope has a millimeter scanning range and a sub-millisecond speed, both far-reaching and fast.
The system has two main modes, a random-access scanning mode (for calcium imaging across hundreds of neurons) and a trajectory scanning mode (for measuring calcium spikes along the length of a dendrite). Four AODs are used (two for the horizontal scanning lens and two for the vertical scanning lens) to compensate for possible drifting of the focal spot. Drift can also be corrected by picking a landmark (such as a particularly bright glial cell), regularly checking its position, and recalibrating the microscope as need be. The special grouping of the four AODs increases the imaging range almost three-fold.
This technology scans large volumes (around 1 mm3) of tissue, ideal for experiments that require imaging regions of interest that are spread apart, whether they are multiple areas along a single dendrite, within a single cell, or across large networks. Random-access point scanning identifies points of interest to quickly scan in three dimensions, making it a convenient way to image neuronal networks. The frame scanning mode can be used to move or rotate areas in three-dimensions and can help find regions of interest. Trajectory scanning follows multiple neuronal processes simultaneously and can even be done continuously by allowing the focal spot to drift along a neuronal process.
High-speed, three-dimensional, two-photon imaging enables researchers to scan entire neuronal networks or focus on the dynamics of single dendrites. The combination of high speed and high resolution allows activity within dendritic spines to be resolved, while the combination of high speed and extensive volume permits simultaneous recording across networks. Looking ahead, there is potential for improvement to increase the field of view, better correct for artifacts, lower cost, or gain access to deeper cortical areas. For now, though, the advanced system — and the types of data it can generate — is exciting.
