Mitochondrial outer membrane imaged with SPIFFI (left) versus widefield microscopy (right). EPFL CC BY SA - 4.0
A new fluorescence microscopy technique that generates super-resolution images from a single camera exposure could help researchers study rapid movements within cells that are difficult to capture with existing methods.
Super-resolution microscopy has transformed cell biology by allowing scientists to see cellular structures and details that are too small to be imaged clearly with conventional light microscopes. Some methods achieve super-resolution by combining information from hundreds or thousands of image frames taken over time. However, this works best when biological samples like cells or tissues have been fixed, or preserved with chemicals. Otherwise, structures like mitochondria, microtubules, and DNA - which are constantly moving, changing, and interacting - appear blurry using existing super-resolution techniques.
Now, researchers led by Aleksandra Radenovic in the Laboratory of Nanoscale Biology (LBEN) in EPFL's School of Engineering have introduced SPIFFI: Spatial Polarization-Induced Fluorescence Fluctuation Imaging. This technique harnesses the polarization of fluorescent light to generate super-resolution images of dynamic structures and processes inside living cells from a single camera exposure. The research has been published inNature Methods.
"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 summarizes.
Capturing cellular dynamics in real time
To visualize nanometer-scale structures inside cells, researchers often label them with fluorescent molecules that glow when illuminated by a laser. This allows them to analyze tiny fluctuations in this 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," explains first author and LBEN PhD student Wei Guo.
SPIFFI takes a fundamentally different approach: it exploits the fact that fluorescent molecules emit polarized light. This means that the light waves emitted by a molecule oscillate preferentially in certain directions, depending on how the molecule is oriented. SPIFFI splits this fluorescent light into four polarization-sensitive channels, and compares the resulting images to recover previously hidden structural details.
In their experiments, the researchers showed that the method results an image resolution improvement of up to twofold, resolving structures around 160-170 nanometers in size with a single snapshot. The technique allowed the team to visualize the movements of mitochondria and microtubules, as well as cellular fusion and splitting events, which are difficult to image clearly using 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 says. "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 the optical hardware can be integrated with existing fluorescence microscopes, SPIFFI could become a practical tool for live-cell biology, neuroscience, biophysics, and drug discovery, allowing researchers to capture rapid nanoscale phenomena as they happen.
