Photoactivated Localization Microscopy (PALM)

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.

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.

source: zeiss

A unique strategy for overcoming the diffraction barrier employs photoswitchable fluorescent probes to resolve spatial differences in dense populations of molecules with superresolution. This approach relies on the stochastic activation of fluorescence to intermittently photoswitch individual photoactivatable molecules to a bright state, which are then imaged and photobleached. Thus, very closely spaced molecules that reside in the same diffraction-limited volume are temporally separated. Merging all of the single-molecule positions obtained by repeated cycles of photoactivation followed by imaging and bleaching produces the final superresolution image. Techniques based on this strategy are often referred to as probe-based superresolution, and were independently developed by three groups in 2006 and given the names photoactivated localization microscopy (PALM), fluorescence photoactivation localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM). All three methods are based on the same principles, but were originally published using different photoswitchable probes.

source: zeiss