Converting lateral scanning into axial focusing to speed up three-dimensional microscopy

ABSTRACT: In optical microscopy, the slow axial scanning rate of the objective or the sample has traditionally limited the speed of volumetric imaging. Recently, by conjugating either a movable mirror to the image plane in a remote-focusing geometry or an electrically tuneable lens (ETL) to the back focal plane, rapid axial scanning has been achieved. However, mechanical actuation of a mirror limits the axial scanning rate (usually only 10–100?Hz for piezoelectric or voice coil-based actuators), while ETLs introduce spherical and higher-order aberrations that prevent high-resolution imaging. In an effort to overcome these limitations, we introduce a novel optical design that transforms a lateral-scan motion into a spherical aberration-free axial scan that can be used for high-resolution imaging. Using a galvanometric mirror, we scan a laser beam laterally in a remote-focusing arm, which is then back-reflected from different heights of a mirror in the image space. We characterize the optical performance of this remote-focusing technique and use it to accelerate axially swept light-sheet microscopy by an order of magnitude, allowing the quantification of rapid vesicular dynamics in three dimensions. We also demonstrate resonant remote focusing at 12?kHz with a two-photon raster-scanning microscope, which allows rapid imaging of brain tissues and zebrafish cardiac dynamics with diffraction-limited resolution. 3D microscopy: clever mirrors reflect bioimages in a heartbeat A new optical design based on the concept of remote focusing can benefit researchers looking to capture high-speed images of biological processes in action. While most optical microscopes refocus by mechanically adjusting the distance between the sample and the objective, remote focusing setups use movable mirrors to quickly scan the focal spot of a laser along the optical axis. Dr. Reto Fiolka at the University of Texas Southwestern Medical Center in Dallas, United States, and colleagues now demonstrate that a tweak to this concept—fixed mirrors that are scanned by a moving laser—can provide aberration-free images at millisecond time scales. Experiments revealed that combinations of step-shaped and sloped mirrors enabled samples such as beating zebrafish hearts to be imaged three-dimensionally with frame rates up to an order of magnitude higher than previous remote-focusing approaches.

SUBMITTER: Chakraborty T 

PROVIDER: S-EPMC7501866 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature