New Microscope Can Image, At Once, Full 3D Orientation & Position of Molecules
WOODS HOLE, Mass. --- Two heads are better than one, as the saying goes, and sometimes two instruments, ingeniously recombined, can accomplish feats that neither could have done on its own.
Such is the case with a hybrid microscope, born at the Marine Biological Laboratory (MBL), that for the first time allows scientists to simultaneously image the full 3D orientation and position of an ensemble of molecules, such as labeled proteins inside cells. The research is published this week .
The microscope combines , a valuable tool for measuring the orientation of molecules, with a dual-view light sheet microscope (), which excels at imaging along the depth (axial) axis of a sample.
This scope can have powerful applications. For example, proteins change their 3D orientation, typically in response to their environment, which allows them to interact with other molecules to carry out their functions.
91לUsing this instrument, 3D protein orientation changes can be recorded,91ם said first author of CZ Biohub San Francisco, a former University of Chicago graduate student who conducted this research partly at MBL. 91לThere91יs real biology that might be hidden to you from just a position change of a molecule alone,91ם Chandler said.
Imaging the molecules in the spindle of a dividing cell 91ד a longstanding challenge at MBL and elsewhere -- is another example.
91לWith traditional microscopy, including polarized light, you can study the spindle quite nicely if it91יs in the plane perpendicular to the viewing direction. As soon as the plane is tilted, the readout becomes ambiguous,91ם said co-author Rudolf Oldenbourg, a senior scientist at MBL. This new instrument allows one to 91לcorrect91ם for tilt and still capture the 3D orientation and position of the spindle molecules (microtubules).
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.
A Confluence of Vision
The concept for this microscope gelled in 2016 through brainstorming by innovators in microscopy who met up at the MBL.
of HHMI Janelia, then at the National Institutes of Health (NIH) and an MBL Whitman Fellow, was working with his custom-designed diSPIM microscope at MBL, which he built in collaboration with Abhishek Kumar, now an adjunct scientist at MBL.
The diSPIM microscope has 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, and illuminate with more control over polarization than other microscopes.
In conversation, Shroff and Oldenbourg realized the dual view microscope could also address a limitation of polarized light microscopy, which is that it91יs difficult to efficiently illuminate the sample with polarized light along the direction of light propagation.
91לIf we had two orthogonal views, we could sense polarized fluorescence along that direction much better,91ם Shroff said. 91לWe thought, why not use the diSPIM to take some polarized fluorescence measurements?91ם
Shroff had been collaborating at MBL with , a professor at University of Chicago whose lab develops algorithms for computational imaging systems. And La Rivière had a new graduate student in his lab, Talon Chandler, whom he brought to MBL. The challenge of combining these two systems became Chandler91יs doctoral thesis, and he spent the next year in Oldenbourg91יs lab at MBL working on it.
The team, which early on included , then based at MBL, outfitted the diSPIM with liquid crystals, which allowed them to change the direction of input polarization.
91לAnd then I spent a long time working through, what would a reconstruction look like for this? What is the most we can recover from this data that we are now starting to acquire?91ם Chandler said. Co-author , then located at Shroff91יs previous lab at NIH, also worked tirelessly on this aspect, until they had reached their goal of full 3D reconstructions of molecular orientation and position.
91לThere was tons of cross-talk between the MBL, the University of Chicago, and the NIH, as we worked this through,91ם Chandler said.
Citation
Talon Chandler et al. (2025) Volumetric imaging of the 3D orientation of cellular structures with a polarized fluorescence light-sheet microscope. Proc. Natl. Acad. Sci.,
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The Marine Biological Laboratory (MBL) is dedicated to scientific discovery 91ד exploring fundamental biology, understanding marine biodiversity and the environment, and informing the human condition through research and education. Founded in Woods Hole, Massachusetts in 1888, the MBL is a private, nonprofit institution and an affiliate of the .
Selected media coverage:
Marine Biological Laboratory Images Cell Components in Full 3D | Optics.org
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