This movie shows how the new method can resolve the 3D orientation and position of cellulose in plant cells. The cells and biomolecules are reconstructed in three different ways (left to right): a coarse-grained representation developed by the authors called orientation distribution functions (ODFs), a peak-cylinder visualization, and a density reconstruction. As the movie progresses, the camera’s viewing axis rotates around the object. Credit: Chandler and Guo et al.

Researchers have developed a new method to simultaneously image the 3D orientation and position of biomolecules, giving scientists a new view into how structures are arranged inside cells.

Scientists study where biomolecules are located and how they are oriented to understand their function. It is difficult to clearly see the position and orientation of these molecules in cellular samples with conventional optical microscopes, so scientists use a technique called polarized fluorescence microscopy. This method uses polarized light to detect the excitation and emission patterns of fluorescent dyes attached to the biomolecules, which can be used to infer the position and orientation of the underlying structures.

This technique works well for taking 2D measurements, but researchers had a hard time making it work in three dimensions, particularly along the z, or optic, axis. Currently, researchers take many 2D measurements and stitch them together to get a 3D picture of a group of biomolecules, but this still results in a picture with less information along the optic axis.

Now, a team of Janelia researchers and collaborators report that they have developed a new method that can simultaneously measure the molecules’ position and orientation in three dimensions, providing a complete 3D view of the arrangement of biomolecules in a sample.

The new system uses a polarized dual-view light sheet microscope, which has two detection paths that allow researchers to simultaneously image a sample from different perspectives. They can also change the direction of the illumination light polarization in each of these paths.

In addition, the team developed a new way to represent groups of oriented molecules, giving researchers more information about the actual measurements that are being made and how the microscope could be affecting them. This allowed the team to determine the angular resolution limit of their imaging system, enabling them to iterate on their design and figure out that they needed to tilt the light sheet to be able to clearly image the molecules’ orientation in 3D.

The team used their new system to show the 3D orientation and position of membranes in vesicles, cellulose in plant cells, and actin in cancer cells. They also observed actin in mouse cells grown on nanowire grids, showing a correlation between the actin alignment and the orientation of the cell.

The team hopes to make their system faster so that they can observe how the position and orientation of structures in live samples change over time. They also hope development of future fluorescent probes will enable researchers to use their system to image a greater variety of biological structures.

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Citation

Talon Chandler, Min Guo, Yijun Su, Jiji Chen, Yicong Wu, Junyu Liu, Atharva Agashe, Robert S. Fischer, Shalin B. Mehta, Abhishek Kumar, Tobias I. Baskin, Valentin Jaumouillé, Huafeng Liu, Vinay Swaminathan, Amrinder S. Nain, Rudolf Oldenbourg, Patrick J. La Riviere, and Hari Shroff. “Volumetric imaging of the 3D orientation of cellular structures with a polarized fluorescence light-sheet microscope.” PMID: 39982748