photoactivated localization microscopy, PALM imaging, Single-molecule imaging, Single-molecule localization microscopy, SMLM, single-molecule microscopy
Photoactivated Localization Microscopy (PALM) is a super-resolution imaging technique that achieves nanometre-scale resolution by exploiting the properties of photoactivatable fluorophores to reveal spatial details of tightly packed molecules. This method enables the detection of individual molecules such as proteins in a cellular context. The method offers a high level of detail when depicting 3D structures in cell bodies and works like this:
Exposing fluorophores to low-power activating lasers of a certain wavelength leads to a change in their emission spectra. This conversion is implemented stochastically so that only a few fluorophores will turn into their active state. The stochastic excitation of the fluorophore ensures that each fluorescence point comes from a single fluorophore. A high-power laser beam briefly exposes these activated molecules, after which they are immediately returned to their inactive state (e.g. by photobleaching). This process is then repeated over thousands of images and the frames are merged into a super-resolution image.