Photoactivated localization microscopy (PALM) is a superresolution technique that dramatically improves the spatial resolution of the optical microscope by at least an order of magnitude (featuring 10 to 20 nanometer resolution), which enables the investigation of biological processes at close to the molecular scale. The technique relies on the controlled activation and sampling of sparse subsets of photoconvertable fluorescent molecules, either synthetic or genetically-encoded. This interactive tutorial explores the sequential steps involved in creating a PALM image.
The tutorial initializes with an pre-activated fluorescent protein specimen appearing in the Specimen window and a dark ribbon structure of the protein Eos positioned on the right-hand side of the tutorial window. In order to operate the tutorial, use the PALM Process slider to transition through the various steps in gathering an image. As the slider is moved from one position to another, the action occurring in that particular stage is explained by text boxes in the tutorial and below. In order to choose an alternative specimen, use the Choose a Specimen pull-down menu.
Using photoactivatable fluorescent proteins, it is possible to selectively switch on thousands of sparse subsets of molecules in a sequential manner. The basic principle behind PALM is to start with the vast majority of the molecules in the inactive state (in effect, not contributing fluorescence emission). A small fraction (less than 1 percent) is photoactivated or photoconverted using a brief pulse of ultraviolet or violet light to render that subset fluorescent. The activated molecules are then imaged and localized to produce nanometer-level precision coordinates, followed by removal from the larger set of unactivated molecules by photobleaching. In the next step, a second fraction of molecules is photoactivated, localized, and eliminated by photobleaching. The process is repeated many thousands of times until the molecular coordinates of all labeled molecules are obtained. The PALM image is a composite of all the single molecule coordinates. As new fluorescent probes for PALM are developed, the photoconversion and readout wavelengths are likely to ultimately span the entire ultraviolet, visible, and near-infrared spectral regions.
Contributing Authors
Adam M. Rainey, Tony B. Gines, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.