Photoactivated localization microscopy

Photo-activated localization microscopy (PALM or FPALM)and stochastic optical reconstruction microscopy (STORM) are widefield (as opposed to point scanning techniques such as laser scanning confocal microscopy) fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. The methods were proposed in 2006 in the wake of a general emergence of optical super-resolution microscopy methods, and were featured as Methods of the Year for 2008 by the Nature Methods journal.The development of PALM as a targeted biophysical imaging method was largely prompted by the discovery of new species and the engineering of mutants of fluorescent proteins displaying a controllable photochromism, such as photo-activatable GFP. However, the concomitant development of STORM, sharing the same fundamental principle, originally made use of paired cyanine dyes.One molecule of the pair (called activator), when excited near its absorption maximum, serves to reactivate the other molecule (called reporter) to the fluorescent state.Immobilized fluorescent proteins being photoactivated, excited and bleachedSuper Resolution Dynamic Imaging of Dendritic Spines Using a Low Affinity Photoconvertible Actin.Investigating Sub Spine Actin Dynamics in Rat Hippocampal Neurons with Super Resolution Optical Microscopy Photo-activated localization microscopy (PALM or FPALM)and stochastic optical reconstruction microscopy (STORM) are widefield (as opposed to point scanning techniques such as laser scanning confocal microscopy) fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. The methods were proposed in 2006 in the wake of a general emergence of optical super-resolution microscopy methods, and were featured as Methods of the Year for 2008 by the Nature Methods journal.The development of PALM as a targeted biophysical imaging method was largely prompted by the discovery of new species and the engineering of mutants of fluorescent proteins displaying a controllable photochromism, such as photo-activatable GFP. However, the concomitant development of STORM, sharing the same fundamental principle, originally made use of paired cyanine dyes.One molecule of the pair (called activator), when excited near its absorption maximum, serves to reactivate the other molecule (called reporter) to the fluorescent state. A growing number of dyes are used for PALM, STORM and related techniques, both organic fluorophores and fluorescent proteins. Some are compatible with live cell imaging, others allow faster acquisition or denser labeling. The choice of a particular fluorophore ultimately depends on the application and on its underlying photophysical properties. Both techniques have undergone significant technical developments, in particular allowing multicolor imaging and the extension to three dimensions, with the best current axial resolution of 10 nm in the third dimension obtained using an interferometric approach with two opposing objectives collecting the fluorescence from the sample. Conventional fluorescence microscopy is performed by selectively staining the sample with fluorescent molecules, either linked to antibodies as in immunohistochemistry or using fluorescent proteins genetically fused to the genes of interest. Typically, the more concentrated the fluorophores, the better the contrast of the fluorescence image. A single fluorophore can be visualized under a microscope (or even under the naked eye) if the number of photons emitted is sufficiently high, and in contrast the background is low enough. The two dimensional image of a point source observed under a microscope is an extended spot, corresponding to the Airy disk (a section of the point spread function) of the imaging system.The ability to identify as two individual entities two closely spaced fluorophores is limited by the diffraction of light. This is quantified by Abbe’s criterion, stating that the minimal distance that allows resolving two point sources is given by d = λ 2 N A {displaystyle d={frac {lambda }{2NA}}} where λ {displaystyle lambda } is the wavelength of the fluorescent emission and NA is the numerical aperture of the microscope. The theoretical resolution limit at the shortest practical excitation wavelength is around 150 nm in the lateral dimension and approaching 400 nm in the axial dimension (if using an objective having a numerical aperture of 1.40 and the excitation wavelength is 400 nm). However, if the emission from the two neighboring fluorescent molecules is made distinguishable, i.e. the photons coming from each of the two can be identified, then it is possible to overcome the diffraction limit. Once a set of photons from a specific molecule is collected, it forms a diffraction-limited spot in the image plane of the microscope. The center of this spot can be found by fitting the observed emission profile to a known geometrical function, typically a Gaussian function in two dimensions. The error that is made in localizing the center of a point emitter scales to a first approximation as the inverse square root of the number of emitted photons, and if enough photons are collected it is easy to obtain a localization error much smaller than the original point spread function.