New Imaging Method Captures Live Cells in Super-Resolution

Super-resolution microscopy has changed cell biology by letting scientists see structures too small for conventional light microscopes to capture clearly. Some super-resolution methods work by combining information from hundreds or thousands of image frames collected over time, an approach that works best on cells or tissues that have been fixed, or preserved with chemicals. In living samples, structures such as mitochondria, microtubules, and DNA are constantly moving and interacting, and they appear blurry under existing super-resolution techniques. 

Now EPFL researchers led by Aleksandra Radenovic have introduced a new technique, called SPIFFI, short for Spatial Polarization-Induced Fluorescence Fluctuation Imaging. Published in Nature Methods, the method uses the polarization of fluorescent light to generate super-resolution images of moving structures and processes inside living cells from a single camera exposure. “Our experiments show that SPIFFI can capture fast-moving processes within cells, while enabling high-throughput, multi-dimensional imaging beyond the limits of conventional microscopes,” Radenovic said.

To visualize nanometer-scale structures inside cells, researchers typically label them with fluorescent molecules that glow under laser light, then analyze small fluctuations in that fluorescence across hundreds or thousands of images taken over time. “Essentially, previous approaches used temporal information to resolve spatial resolution, but this doesn’t work very well on living cells,” said first author Wei Guo.

SPIFFI instead takes advantage of the fact that fluorescent molecules emit polarized light, meaning the light waves they give off oscillate in particular directions depending on how the molecule is oriented. The technique splits this fluorescent light into four polarization-sensitive channels, then compares the resulting images to recover structural details that would otherwise stay hidden. 

In their experiments, the researchers found that the method improves image resolution by up to twofold, resolving structures around 160 to 170 nanometers in size in a single snapshot. The technique let the team visualize the movement of mitochondria and microtubules, as well as cellular fusion and splitting events, processes that are difficult to image clearly with multi-frame approaches. “With previous techniques, taking many images would only result in one super-resolved frame. With SPIFFI, every frame is super-resolved, meaning we can now produce super-resolution videos of live cells,” Guo said. “We also seamlessly integrated SPIFFI images with existing fluctuation-based methods for post-processing, achieving resolutions of about 80 nanometers.”

The researchers are now working to make the SPIFFI imaging setup more compact and to combine the technique with three-dimensional imaging technologies. Because its optical hardware can be integrated with existing fluorescence microscopes, the team says SPIFFI could become a practical tool for live-cell biology, neuroscience, biophysics, and drug discovery, letting researchers capture rapid nanoscale phenomena as they happen.