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Interactive Tutorials

Superresolution Microscopy

Photoactivation 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.

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Stimulated Emission Depletion (STED) Microscopy

Superresolution microscopy using stimulated emission depletion (STED) creates sub-diffraction limit features by altering the effective point spread function of the excitation beam using a second laser that suppresses fluorescence emission from fluorophores located away from the center of excitation.

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Superresolution Structured Illumination Microscopy (SR-SIM)

SR-SIM is capable of achieving a lateral resolution of 50 to 60 nanometers and an axial resolution ranging from 150 to 300 nanometers. The technique relies on superimposing different grid orientations on the specimen to generate raw images, which are reconstructed into high resolution derivatives.

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The RESOLFT Concept

The theoretical foundation necessary for achieving resolution beneath the diffraction barrier, which is actually composed of a family of physical concepts, was first advanced by Stefan Hell and associates with their introduction of the idea of reversible saturable (or switchable) optical fluorescence transitions (RESOLFT).

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The PALM Concept

Photoactivated localization microscopy (PALM) 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.

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The Stimulated Emission Depletion (STED) Concept

Point-spread engineering techniques designed to circumvent the diffraction barrier all rely on a time-sequential readout of fluorescent probe photoswitching. The first technique successfully applied to superresolution biological imaging of fixed cells was the RESOLFT method named stimulated emission depletion (STED).

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Depletion Lasers in STED Microscopy

In STED microscopy, the specimen is illuminated by two synchronized ultrafast co-linear sources consisting of an excitation laser pulse followed by a red-shifted depletion laser pulse that is referred to as the STED beam. Pulsed lasers are used to produce radially symmetric depletion zones.

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Saturated Structured Illumination Microscopy

Saturated structured illumination microscopy is a superresolution technique where non-linearity arises from saturation of the excited state. SSIM and related methodology can readily be implemented on a widefield microscope with a single laser system and standard fluorophores.

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Superresolution Microscopy with STED

STED takes advantage of the RESOLFT concept by significantly modifying the shape of the excitation point-spread function by manipulating the phase, pulse width, and intensity of the excitation and depletion lasers. This interactive tutorial explores how images are constructed using STED microscopy.

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