Existing superresolution fluorescence microscopy methods such as STED and PALM have trouble imaging molecules with spatial resolution better than 20 to 40 nm. A new superresolution method called MINFLUX combines aspects of those earlier methods to resolve molecules just 6 nm apart with nanometer precision. (Science 2016, DOI: 10.1126/science.aak9913).
STED and PALM (and the related technique STORM) distinguish densely packed features by allowing isolated molecules to fluoresce while those around them remain dark. The methods differ in how they determine the position of that fluorescence. In STED, a laser beam focused into a doughnut shape turns off fluorescence beneath the beam but not at the doughnut’s center. In PALM, fluorescent molecules are randomly switched on and off repeatedly so that only a few well-separated ones are activated at a time. The position of the activated molecules is determined with a camera.
“The strength of STED is that the doughnut always determines where the molecules are on and off and hence where the signal comes from,” says team leader Stefan W. Hell of the Max Planck Institute for Biophysical Chemistry, who won the 2014 Nobel Prize in Chemistry for STED. On the other hand, “the strength of PALM is that you’re working with individual molecules.” But those molecules must emit many thousands of photons in order for scientists to get enough fluorescence intensity to determine their positions precisely.
In the new method, Hell, Francisco Balzarotti, also at the Max Planck Institute for Biophysical Chemistry, and coworkers use a doughnut-shaped laser beam for exciting fluorescence instead of turning it off. Because there’s no light intensity in the doughnut hole, a molecule located entirely within the hole won’t fluoresce. But a molecule that’s even slightly offset from the hole does fluoresce. How much it fluoresces depends on how far it is from the beam’s zero-intensity center. By scanning the doughnut in a defined pattern over the sample, the researchers can determine the precise position of a molecule from how its fluorescence emission changes.
“Because we can relate the position of the molecule to the ‘zero’ of the doughnut, many fewer photons are required to find out the position of the molecule than in PALM,” Hell says.
The team constructed two arrays of fluorescent molecules at defined distances from one another: 11 nm in one array and 6 nm in the other. MINFLUX determined the molecules’ positions with a precision of 2.1 nm and 1.2 nm, respectively.
In addition to improved spatial resolution, the MINFLUX measurements are fast enough and require such low laser intensity that the researchers used the method for tracking individual molecules in live cells at multiple time scales.
“It’s a big deal that this method can be used for imaging but also for tracking at short and long time scales,” says Christy F. Landes, a superresolution expert at Rice University. “Not all methods are useful for these three very different imaging applications.”