Scientists at the Laboratory of Biomedical Optics (LOB) at EPFL (École Polytechnique Fédérale de Lausanne) in Switzerland have developed the first microscope platform that can perform “super-resolution” imaging in both space and time — capturing unprecedented “4D” views inside living cells. The landmark paper is published in Nature Photonics and on open-access ArXiv.

Super-resolution microscopy is a technique (covered extensively on KurzweilAI) that can “see” beyond Abbe’s diffraction-of-light limit, providing unprecedented views of cells and their interior structures and organelles. The developers won the Nobel Prize in Chemistry in 2014.

But super-resolution microscopy only offers improved spatial resolution. That might suffice for static samples, like solid materials or fixed cells, but living cells are highly dynamic and depend on a complex set of constantly changing biological processes that occur across sub-second timescales. So to visualize and understand how living cells function in health and disease, high “temporal” (time) resolution is also required.

Enter the 4D microscope. A team led by Professor Theo Lasser, head of the LOB, has developed a “4D microscope” that they dubbed PRISM (Phase Retrieval Instrument with Super-resolution Microscopy). A simple add-on to existing widefield microscopes, it combines 3D super-resolution microscopy (for high spatial resolution) with fast 3D phase (time) imaging in a single instrument. Phase imaging translates phase changes (changes over time) of light — caused by changes in cells and their organelles — into conventional spatial maps of the cells.

To achieve fast 3D phase imaging, the scientists designed an image-splitting prism that allows for simultaneous recording of a stack of eight z-displaced (at different depths) images. This allows the microscope to perform high-speed, 3D phase imaging across a volume of 2.5 x 50 x 50 micrometers. The team was able to image intracellular dynamics at up to 200 Hz (200 times per second — about six faster than conventional video cameras — allowing for imaging fast-changing events).

The prism is also suited for 3D fluorescence imaging, which the scientists tested using super-resolution optical fluctuation imaging (SOFI). This method exploits the blinking of fluorescent dyes to improve 3D resolution through correlation analysis of the signal. Using this, the researchers performed 3D super-resolution imaging of stained structures in the cells, and combined it with 3D label-free phase imaging. The two techniques complemented each other, revealing images of the inner cytoskeleton architecture and organelles in living cells at different times.

Visualizing pathological protein aggregation in neurodegenerative diseases. “The technical advances enabled high-resolution visualization of the formation of pathological Professor Hilal Lashuel’s Laboratory Of Molecular And Chemical Biology Of Neurodegeneration at EPFL teamed up with Lasser’s lab to use the new technique to study the mechanisms by which protein aggregation contributes to the development and progression of neurodegenerative diseases, such as Parkinson’s and Alzheimer’s. “The technical advances enabled high-resolution visualization of the formation of pathological alpha synuclein aggregates in hippocampal neurons,” Lasheul said.

Lasser predicts that PRISM “will be rapidly used in the life science community to expand the scope for 3D high-speed imaging for biological investigations. We hope that it will become a regular workhorse for neuroscience and biology.”

The study was funded by the European Union (Horizon 2020, Marie Skłodowska-Curie Grant Agreement and AD-gut European consortium) and the Swiss National Science Foundation (SNSF).

Abstract of Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy

Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going ‘beyond the diffraction barrier’ comes at a price, since most far-field super-resolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase imaging with fluorescence to provide high-speed imaging and spatial super-resolution. The non-iterative phase retrieval relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of eight planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument. The 4D microscope platform unifies the sensitivity and high temporal resolution of phase imaging with the specificity and high spatial resolution of fluorescence microscopy.