New Microscope Captures Full 3D Molecular Dynamics

When two instruments are thoughtfully combined, they can achieve outcomes that neither could accomplish independently.

Such is the case with a new hybrid microscope developed at the Marine Biological Laboratory (MBL). This microscope allows researchers, for the first time, to simultaneously image multiple molecules—such as labeled proteins within cells—in full three-dimensional detail. The findings were recently published in the journal Proceedings of the National Academy of Sciences.

The innovation merges a dual-view light sheet microscope (diSPIM), known for its exceptional ability to image along the depth (axial) axis, with polarized fluorescence technology, a technique essential for determining molecular orientations.

This powerful combination opens up numerous applications. For example, proteins often perform their biological functions by interacting with other molecules and altering their 3D orientation in response to environmental changes. The new microscope can record these subtle shifts in 3D protein orientation. "There's real biology that might be hidden if you look only at the position of a molecule," explains Talon Chandler, first author of the study, currently at Chan Zuckerberg Biohub San Francisco and formerly a University of Chicago graduate student who conducted part of this research at MBL.

Another notable application addresses a longstanding challenge at MBL and elsewhere: imaging molecules within the spindle structure of dividing cells.

“With traditional microscopy, including polarized light, you can easily study the spindle when it's positioned perpendicular to your viewing direction. But once the plane is tilted, readings become ambiguous,”

Rudolf Oldenbourg, co-author and senior scientist at MBL.

The new microscope overcomes this issue by correcting for tilt, clearly capturing both the 3D orientation and position of spindle microtubules.

Moving forward, the research team aims to speed up their imaging system to monitor dynamic changes in live samples more effectively. As fluorescent probe technology continues to evolve, they anticipate imaging an even broader range of biological structures.

A Collaborative Vision

The inspiration for the hybrid microscope emerged in 2016 during brainstorming sessions at MBL among leading experts in microscopy.

Hari Shroff of HHMI Janelia, an MBL Whitman Fellow and former researcher at the National Institutes of Health (NIH), had been utilizing a custom-built diSPIM microscope at MBL, constructed together with Abhishek Kumar, now based at MBL.

This dual-view microscope illuminates and images samples from two perpendicular directions, providing better control over polarization and overcoming depth-resolution limitations associated with single-view microscopes. Shroff and Oldenbourg realized this configuration could also address another limitation of polarized microscopy—poor illumination along the propagation direction of the polarized light.

“If we had two orthogonal views, we could better detect polarized fluorescence along that direction,” Shroff explains. “We thought, why not adapt the diSPIM to capture polarized fluorescence measurements?”

Patrick La Rivière, a professor at the University of Chicago specializing in computational imaging algorithms, had already been collaborating with Shroff at MBL. La Rivière introduced his graduate student, Talon Chandler, to MBL, where Chandler embraced the integration of these two technologies as his doctoral research. Chandler spent the following year tackling this challenge in Oldenbourg’s lab.

To manipulate polarization angles precisely, the team—including early contributor Shalin Mehta, formerly at MBL—equipped the diSPIM with liquid crystal components.

“I spent significant time figuring out how to reconstruct images effectively and exploring what could be extracted from the data we started collecting,” Chandler recalls.

Co-author Min Guo, then working in Shroff’s NIH lab, also played a crucial role, contributing extensively to the team's efforts to achieve comprehensive 3D reconstructions of molecular orientations and positions.

“There was a tremendous amount of collaboration between the MBL, the University of Chicago, and NIH throughout this process,” Chandler emphasizes.