- Photoactivated Localization Microscopy (PALM) is a single-molecule super-resolution imaging technique that overcomes the diffraction limit of light microscopy, allowing researchers to resolve structures with a resolution of ~10–30 nanometers.
- Introduced in 2006 by Eric Betzig, Harald Hess, and colleagues, PALM relies on the controlled photoactivation of fluorescent molecules and the precise localization of their emission patterns. Unlike point-scanning methods such as STED, PALM builds high-resolution images by sequentially imaging and localizing sparse subsets of fluorescent molecules over many imaging cycles, then reconstructing the positions into a composite super-resolved image.
- The method begins with a dense population of fluorescent molecules—either genetically encoded photoactivatable fluorescent proteins or photoswitchable synthetic dyes—distributed within the sample. Initially, all fluorophores are kept in a dark (non-fluorescent) state. A weak activation light pulse switches on only a sparse, random subset of molecules so that the individual emission patterns (point spread functions) from each do not overlap. These activated molecules are then excited and imaged until they photobleach irreversibly. Because the emission from each molecule is isolated, it can be fit to a mathematical model (typically a Gaussian function) to determine its centroid position with nanometer precision, far beyond the ~200 nm diffraction limit.
- This process is repeated thousands of times: new subsets of molecules are activated, localized, and photobleached. Over the course of the experiment, the coordinates of millions of single molecules are accumulated. The final super-resolution image is reconstructed from this coordinate map, revealing fine structural details that would otherwise be blurred together in conventional microscopy. The resolution achieved depends on the localization precision (influenced by photon count, background noise, and optical stability) and the labeling density (how completely the structure is sampled by fluorophores).
- PALM has been transformative in biological imaging because it enables nanoscale mapping of proteins, nucleic acids, and other biomolecules in fixed or, under optimized conditions, live cells. It has been used to study the spatial organization of membrane receptors, the nanoscale arrangement of cytoskeletal networks, chromatin structure in the nucleus, and the clustering behavior of signaling molecules. Live-cell PALM, although limited by slower acquisition times compared to some other techniques, has been applied to track single molecules in real time, providing valuable insights into molecular dynamics.
- However, PALM also has limitations. It requires photoactivatable or photoswitchable fluorophores, which may need genetic fusion to the target protein or chemical labeling. The technique involves long acquisition times for high-resolution reconstructions, making it less suited for capturing rapid cellular events. Additionally, achieving optimal performance demands careful control of activation intensity, sample drift correction, and computational processing.
- Despite these challenges, PALM remains one of the foundational methods in the single-molecule localization microscopy (SMLM) family, alongside STORM and MINFLUX, and continues to be a powerful tool for visualizing the nanoscale architecture of biological systems.
