To overcome the limitations of high-speed volumetric imaging due to slow axial scanning rates or aberrations introduced by the z-scanning mechanism, scientists at UT Southwestern have introduced a novel optical design that transforms a lateral scan motion into a scan in the third dimension. The technique allowed the microscope to achieve a laser focusing rate of 12 kHz and allowed the observation of fast dynamics inside cells and the beating hearts of zebra fish embryos.

The speed of imaging in high-speed volumetric imaging is tightly tied to how fast the position of the imaging system’s focus can be changed, particularly in the third dimension.

Traditionally, refocusing is done either by mechanically moving the microscope objective or the sample, both of which lead to low third-dimensional scanning speeds as the speed of moving physical objects is limited by inertia.

A potential method of alleviating this issue is through remote focusing, which refocuses by changing the wavefront of the optical system. However, most existing technologies to that end necessitate a trade-off between resolution and speed.

To overcome that, a team led by Reto Fiolka, a professor in the Department of Cell Biology, and Lyda Hill, of the Department of Bioinformatics, developed an optical design that employs well-established lateral scan technologies, and transformed that lateral scanning motion into refocusing in the third dimension.

Taking the concept of aberration-free remote focusing, and rather than moving a corresponding remote mirror in the third dimension, the researchers scanned a laser spot laterally with a high-speed galvanometer over a stationary mirror. If the distance between the stationary mirror and the objective lens is not constant along the scan direction, a defocus is introduced as is necessary for remote refocusing. On the return path, the lateral scan component is perfectly compensated to be able to achieve a pure scan motion in the third dimension.

Two implementations were adopted to realize this concept: one using a step mirror, and the other using a tilted planar mirror. The former allows arbitrarily large axial step sizes over a finite number of steps, and the latter allows for an arbitrary number and size of axial steps and is capable of continuous scanning in the third dimension, albeit over a more limited scan range.

The scanning technology allows one order of magnitude acceleration while keeping the high spatial resolving of the technology. In a second application, the researchers implemented their scanning technology in a 2-photon raster scanning microscope and performed high-resolution volumetric imaging with a scan rate in the third dimension of 12 kHz. The technology is fully compatible with acousto-optical deflectors, and therefore theoretically capable of scanning on the submicrosecond timescale in the third dimension. Using Lissajous scanning patterns, the researchers forecast the ability for volumetric imaging at kHz rates.

The team believes the technology will open major applications for intravital imaging, particularly in neuroscience.

The research was published in Light: Sciences & Applications (www.doi.org/10.1038/s41377-020-00401-9).