Laser-Scanning Microscopy Method Achieves Rapid 3D Imaging

A technique developed by researchers from Tohoku University and Osaka University rapidly takes 3D images without moving the observation plane, which is typically necessary in conventional laser-scanning microscopes.

A major issue when using laser-scanning microscopy for 3D imaging is the time-consuming nature of the process. It involves repeated 2D image acquisition, which requires the observation plane to be constantly changed.

The axially resolved detection realized by wavefront engineering for light needle microscopy. A laser-scanning microscopy technique to rapidly acquire 3D images without moving the observation plane could speed image acquisition in various research and industrial fields. Courtesy of Yuichi Kozawa et al.

The method developed by the researchers uses a laser, which they refer to as a “light needle,” that is spot-elongated along the axial direction and used as the illumination source in laser scanning microscopy. In general, the use of such a light needle is a common approach that produces deep-focus images that capture the extended depth range of specimens without blurring. However, the approach only provides a 2D image that lacks depth information about a specimen.

Using a technique based on computer-generated holography, the researchers proposed to manipulate fluorescence signals emitted from specimens. The team manipulated the wavefront of the fluorescence signals to laterally shift the image position, depending on its axial position. With this method, 3D volumetric images could be captured at a speed equal to the frame rate of 2D raster scanning, which greatly improves the acquisition speed for 3D volumetric imaging in the framework of point-scanning-based imaging.

The system constructed a 3D image from a single 2D scan of a light needle for the depth range of 20 µm. It also recorded 3D videos of dynamic motions of micron-size beads suspended in water, which is rarely achieved by existing laser-scanning microscopes, according to the researchers.

Tests also realized quick 3D image acquisition for thick biological samples at a speed more than 10× that of the traditional modality. The researchers said the technique could speed image acquisition in various research and industrial fields in which the 3D image observation and evaluation are essential. The team plans to further extend the applicability of the proposed method to downsized systems, targeting its use in practical applications.

The research was published in Biomedical Optics Express (www.doi.org/10.1364/BOE.449329).