Two heads are higher than one, because the saying goes, and sometimes two instruments, ingeniously recombined, can accomplish feats that neither could have done by itself.
Such is the case with a hybrid microscope, born on the Marine Biological Laboratory (MBL), that for the primary time allows scientists to concurrently image the total 3D orientation and position of an ensemble of molecules, corresponding to labeled proteins inside cells. The research is published this week in Proceedings of the National Academy of Sciences.
The microscope combines polarized fluorescence technology, a invaluable tool for measuring the orientation of molecules, with a dual-view light sheet microscope (diSPIM), which excels at imaging along the depth (axial) axis of a sample.
This scope can have powerful applications. For instance, proteins change their 3D orientation, typically in response to their environment, which allows them to interact with other molecules to perform their functions.
“Using this instrument, 3D protein orientation changes could be recorded,” said first writer Talon Chandler of CZ Biohub San Francisco, a former University of Chicago graduate student who conducted this research partly at MBL. “There’s real biology that may be hidden to you from only a position change of a molecule alone,” he said.
Imaging the molecules within the spindle of a dividing cell — a longstanding challenge at MBL and elsewhere — is one other example.
“With traditional microscopy, including polarized light, you’ll be able to study the spindle quite nicely if it’s within the plane perpendicular to the viewing direction. As soon because the plane is tilted, the readout becomes ambiguous,” said co-author Rudolf Oldenbourg, a senior scientist at MBL. This recent instrument allows one to “correct” for tilt and still capture the 3D orientation and position of the spindle molecules (microtubules).
The team hopes to make their system faster in order that they’ll observe how the position and orientation of structures in live samples change over time. In addition they hope development of future fluorescent probes will enable researchers to make use of their system to image a greater number of biological structures.
A Confluence of Vision
The concept for this microscope gelled in 2016 through brainstorming by innovators in microscopy who met up on the MBL.
Hari Shroff of HHMI Janelia, then on the National Institutes of Health (NIH) and an MBL Whitman Fellow, was working along with his custom-designed diSPIM microscope at MBL, which he in-built collaboration with Abhishek Kumar, now at MBL.
The diSPIM microscope has two imaging paths that meet at a right angle on the sample, allowing researchers to light up and image the sample from each 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 twin view microscope could also address a limitation of polarized light microscopy, which is that it’s difficult to efficiently illuminate the sample with polarized light along the direction of sunshine propagation.
“If we had two orthogonal views, we could sense polarized fluorescence along that direction a lot better,” Shroff said. “We thought, why not use the diSPIM to take some polarized fluorescence measurements?”
Shroff had been collaborating at MBL with Patrick La Rivière, a professor at University of Chicago whose lab develops algorithms for computational imaging systems. And La Rivière had a brand new graduate student in his lab, Talon Chandler, whom he delivered to MBL. The challenge of mixing these two systems became Chandler’s doctoral thesis, and he spent the following 12 months in Oldenbourg’s lab at MBL working on it.
The team, which early on included Shalin Mehta, then based at MBL, outfitted the diSPIM with liquid crystals, which allowed them to alter the direction of input polarization.
“After which I spent an extended time working through, what would a reconstruction appear to be for this? What’s probably the most we are able to get better from this data that we are actually starting to accumulate?” Chandler said. Co-author Min Guo, then situated at Shroff’s previous lab at NIH, also worked tirelessly on this aspect, until they’d reached their goal of full 3D reconstructions of molecular orientation and position.
“There was tons of cross-talk between the MBL, the University of Chicago, and the NIH, as we worked this through,” Chandler said.
