Marine Biological Laboratory images cell components in full 3D

25 Feb 2025

Combination of polarized and light-sheet methods reveals unexpected cellular coordination.

Fluorescence-based imaging methods have recorded many details of cell dynamics and behavior, but the full 3D orientation and position of fluorescently marked ensembles has remained difficult to determine.

Imaging the molecules in a dividing cell's "spindle," a protein structure that extends from one side of the cell to the other, has been a particular long-standing challenge. Traditional microscopy can image the spindle if it lies perpendicular to the viewing direction, but struggles if the plane is tilted.

A project at the Marine Biological Laboratory (MBL) affiliated with the University of Chicago has now developed a hybrid microscope that could reveal new information about the structures of proteins within cells.

As reported in PNAS the technique combines polarized fluorescence microscopy, good at measuring the orientation of molecules, with a dual-view light-sheet microscope (diSPIM, from selective-plane illumination microscope) which can image along the depth or the axial axis of a sample.

"We identified an unmet need for measurements that can be used to recover the three-dimensional orientation and position of fluorescent ensembles," noted the team in its paper. "A dual-view light-sheet system should provide an excellent platform for measuring their orientation."

The University of Chicago has previously shown how a multiple light-sheet approach can reveal a cell's response to stress, and the new platform builds on prior work at MBL into a diSPIM platform capable of time-lapse volumetric imaging of living samples at sub-cellular resolution.

A diSPIM approach involves two imaging paths that meet at a right angle on the sample, allowing researchers to illuminate and image the sample from both perspectives. This dual view can compensate for the poor depth resolution of any single view, with more control over polarization than other microscopes.

Hidden cell biology revealed

Researchers investigated if this dual view microscope could also address the difficulty of efficiently illuminating a sample with polarized light along the direction of light propagation, a long-standing limitation of polarized light microscopy.

"If we had two orthogonal views, we could sense polarized fluorescence along that direction much better," said Hari Shroff from MBL. "Why not use the diSPIM to take some polarized fluorescence measurements?"

Addition of liquid crystal modules to the platform gave the researchers control over the light's transverse polarization, changing the direction of input polarization as desired. Plots of polarization-dependent efficiency of excitation could then be made, alongside the detection of emitted fluorescence.

In trials using first labeled cellulose and actin targets and then cells grown on nanowire grids, the project observed unexpected coordination across scales, with actin orientations at the smallest measurable scale proving to be correlated with the large-scale orientation of the entire cell.

"Using this instrument, 3D protein orientation changes can be recorded," said Talon Chandler of MBL and CZ Biohub San Francisco, a research non-profit funded by Mark Zuckerberg and Priscilla Chan. "There’s real biology that might be hidden to you from just a position change of a molecule alone."