12 Dec 2022
Oblique plane microscopy improves axial resolution over existing methods.
A project at Johns Hopkins University has now developed an augmented LSM platform employing a diffractive light-sheet, intended to allow large-scale 4D cellular resolution imaging.
As reported in Optica, the new architecture is intended to tackle the inherent trade-off between axial resolution and field of view, which can present a hurdle to imaging of dynamic processes or whole animal studies.
"Unraveling underlying cellular structures and their interaction is fundamentally important to understanding life," said Ji Yi of Johns Hopkins University.
"However, the limitations of light diffraction make it difficult to image with 3D cellular resolution over large areas of several millimeters. We circumvent the trade-offs between field of view, depth resolution and imaging speed to achieve 4D cellular resolution over a much larger field of view than previously possible."
The project's technique, christened mesoscopic oblique plane microscopy (Meso-OPM), is based on existing research into OPM techniques, which employ oblique illumination planes and detect the image through the same microscope objective lens.
"Meso-OPM belongs to a family generally termed single objective light sheet microscopy (SOLSM)," noted the team in its paper. "In contrast to conventional LSM, which uses two objective lenses, SOLSM uses only a single primary objective lens but applies an off-axis oblique light sheet excitation to the specimen and a remote focusing system."
To this principle Johns Hopkins added a transmission grating to create a high-angle illumination light sheet, a modification designed to improve the axial resolution of the technique and enhance its ability to provide data suitable for 3D real-time imaging.
Imaging at previously unattainable rates
"The result is the ability to perform mesoscopic scale imaging over a field of view that is several millimeters wide, while still being able to resolve individual cells in 3D," said Yi.
In trials, Meso-OPM was applied to two large-scale model systems: living zebrafish larvae 3 to 4 millimeters long, and mouse brain slices in which the cells are kept alive, both models labelled with fluorescence proteins. The platform recorded calcium dynamics over the entire zebrafish nerve system at volume rate of 2 hertz, a rate "previously unattainable due to insufficient axial resolution or limited FOV."
The next steps will involve improving the light collection efficiency to further increase the imaging speed, along with incorporating multiphoton imaging as a route to better penetration depths.
• In a further advance for oblique LSM techniques, Leica Microsystems has entered into a partnership with Applied Scientific Instrumentation (ASI) to commercialize SOLSM techniques and exploit their advantages for biological imaging.
According to Leica, the system combines implementation of OPM principles with a Swept, Confocally Aligned Planar Excitation (SCAPE) microscope design, in which light-sheet optical sectioning is combined with confocal de-scanning, Leica is a licensee for both technologies.
SCAPE can be used for imaging of calcium signalling in freely behaving organisms such as c. elegans worms, according to Leica. Blood flow in beating zebrafish hearts as well as large cleared and uncleared tissue sections can also be imaged using this modality.
Leica Microsystems has now granted ASI a sub-license to these patents and is supporting the development of a commercial system.
"This important collaboration with Leica Microsystems enables us to offer this uniquely powerful and flexible technology to advanced users," said John Zemek of ASI. "Together, we provide an open platform ready for customization while significantly lowering the challenges that are intrinsic to the assembly of a single-objective light-sheet microscope."