‘One-scan’ microscopy method rapidly creates 3D images

10 Mar 2022

Rapid 3D image acquisition technique, without moving observation plane, developed by Tohoku and Osaka universities.

Conceptual illustration of the “One-scan” microscopy method.

A newly-proposed 3D microscopy technique enables rapid 3D image acquisition; branded “One-scan”, the method involves an elongated light spot that resembles a “needle” which captures three-dimensional images of a specimen.

The new approach, developed by researchers from Tohoku University and Osaka University, both Japan, can rapidly take 3D images without moving the observation plane – something that is necessary in conventional laser scanning microscopes.

The work is reported in Biomedical Optics Express.

Light microscopy is ubiquitous and vital for various fields including life science and medical diagnosis. As many biological cells or tissues are structurally complex, 3D observation is crucial. Laser scanning microscopy is a representative and well-established approach that enables 3D observation by scanning a focal spot on the sample.

One major challenge is that it is a time-consuming procedure because it involves repeated 2D image acquisition that requires changing the observation plane.

The researchers used a laser spot elongated along the axial direction, referred to as “light needle”, as illumination in laser scanning microscopy. In general, the use of such a light needle is a common approach that produces deep-focus images capturing the extended depth range of specimens without blurring. However, this approach only provides a 2D image, which does not include any depth information of a specimen.

Computer-generated holography

The solution proposed by the researchers was manipulating fluorescence signals emitted from specimens through a technique based on computer-generated holography (CGH). They devised a hologram to be applied to fluorescence emitted from different depth positions inside the sample.

Video-rate acquisition of 1 µm fluorescence beads in water.

This hologram was designed to produce laterally shifted and spatially separated images at the detector plane depending on the depth position of objects. With this technique, the depth information can be recorded as the lateral information simultaneously, allowing for the construction of 3D images without changing the observation plane.

Using this principle, the researchers developed a microscope system equipped with a spatial light modulator, a computer-controlled apparatus to project the CGH. The developed microscope system constructed a 3D image from a single 2D scanning of a light needle for the depth range of 20µm.

The system has recorded 3D movies of the dynamic motion of micron-sized beads suspended in water, something rarely achieved by existing laser scanning microscopes, says the team. The researchers also demonstrated the prompt 3D image acquisition for thick biological samples with a speed more than 10 times as fast as the conventional technique.

The team concludes that their proposed technique will notably speed up image acquisition in various research and industrial fields, where the 3D image observation and evaluation are essential. The researchers are now planning to further extend the applicability of the proposed method to downsized systems, targeting its use in practical applications.