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Superresolution Microscopy

Superresolution Microscopy

A wide array of new and exciting methodologies have recently been introduced that are now collectively termed superresolution microscopy and feature both lateral and axial resolution measured in the tens of nanometers and even less. The common thread in all of the these new techniques is that they are able to resolve features beneath the diffraction limit by switching fluorophores on and off sequentially in time so that the signals can be recorded consecutively. Among the methods that improve resolution by PSF modification, the most important techniques are referred to by the acronyms STED (stimulated emission depletion), GSD Ground State Depletion, and SSIM (saturated structured illumination microscopy). Techniques that rely on the detection and precise localization of single molecules include PALM (photoactivated localization microscopy) and STORM (stochastic optical reconstruction microscopy).

Review Articles

Introduction to Superresolution Microscopy - The broad range of superresolution techniques available today are moving optical imaging of biological specimens into the realm traditionally held by electron microscopy. Rapidly evolving techniques are continually altering the perspective of this emerging field with regards to imaging in cell biology.

Photoactivated Localization Microscopy (PALM) - The principle behind photoactivated localization microscopy and related techniques (STORM and FPALM) rests on a combination of imaging single fluorophores (single-molecule imaging) along with the controlled activation and sampling of sparse subsets of these labels in time.

Practical Aspects of PALM Imaging - A number of practical considerations are mission-critical in achieving the best single-molecule localization images using PALM and related methodology. The most important considerations include instrument configuration parameters, choice of fluorescent probes, molecular density, contrast ratio, and specimen preparation.

Superresolution Structured Illumination Microscopy (SR-SIM) - An advanced imaging technique where increased resolution is obtained by discarding traditional Köhler illumination and replacing it with non-uniform excitation light patterns that feature sinusoidal intensity variations in one or more dimensions coupled with powerful image reconstruction techniques.

Interactive Tutorials

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.

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.

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.

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).

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.

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).

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.

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.

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.

Reference Library

Superresolution Microscopy Review Articles - The traditional diffraction limit in microscopy has limited applications to gross approximations of molecular positioning in cellular substructures. To address this challenge, superresolution microscopy has emerged to break the diffraction barrier and yield resolutions down to 50 nanometers or less.

Probes for Superresolution Microscopy References - Among the fluorescent probes that have proven useful for superresolution microscopy are genetically encoded fluorescent protein fusions, synthetic dyes, quantum dots, and hybrid systems that combine a genetically encoded target peptide with a separate synthetic component that is membrane permeant.

Single-Molecule Superresolution Microscopy References - Superresolution imaging using single molecule localization encompasses a number of techniques including PALM, STORM, and FPALM. The field is rapidly emerging in popularity due to the dramatic improvement in spatial resolution to enable biological processes to be described at the molecular scale.

Molecular Localization Accuracy References - In single-molecule microscopy, individual molecules that are densely packed within the resolution limit can be isolated on the basis of one or more distinguishing optical characteristics. They can then be localized to a much higher precision by determining its center of fluorescence emission through a statistical fit of the point-spread function.

Practical Aspects of PALM Imaging References - A collection of review articles and research reports on the successful implementation of single-molecule superresolution imaging. Addressed are choice of probes, stage drift, background noise, data registration, image assembly, aberrations, and details of specimen preparation.

PALM with Independently Running Acquisition (PALMIRA) - By recording non-triggered, spontaneous off-on-off cycles without synchronization of the excitation illumination with the EMCCD camera system, a technique termed PALM with independently running acquisition (PALMIRA) was developed to accelerate image acquisition speed.

RESOLFT Concept References - RESOLFT is a general concept that describes breaking the diffraction barrier using reversible saturable or switchable optical transitions. The principle was first detailed with STED and GSD microscopy, where the diffraction barrier is broken by a saturated optical transition (depletion) between two states of a fluorescent probe.

Stimulated Emission Depletion Microscopy (STED) References - Stimulated emission depletion microscopy is a technique that relies on the depletion of the excited state fluorophores surrounding the objective focal spot in order to significantly narrow the dimensions and increase resolution through point-spread function engineering.

4Pi Microscopy References - The ingenious technique of 4Pi microscope employs juxtaposed dual objectives to produce excitation light at a common focal plane. The resulting constructive and destructive interference reduces the possible axial resolution to approximately 100 nanometers, significantly reduced from the typical 400 to 700 nanometers observed in confocal microscopy.

Standing-Wave and Interference Microscopy References - Standing-wave and interference (InM) techniques employ axially structured illumination to spatially modulate the excitation light in a widefield instrument configuration. Excitation patterns for interference microscopy often contain nodes and anti-nodes within the focal plane where the beams are constructively interfering.

Ground State Depletion Microscopy References - Ground state depletion (GSD) microscopy is a RESOLFT technique that exhibits a time-sequential readout from within the diffraction zone at defined coordinates using reversible saturable or photoswitchable transitions. GSD requires lower laser intensities that STED or similar techniques because it employs the metastable triplet state.

Dark States in Single-Molecule Superresolution References - The application of reversible photoswitching between bright and metastable dark states with traditional synthetic fluorophores and fluorescent proteins is promising to become a useful technique for single-molecule superresolution imaging in cell biology.

Superresolution Structured Illumination Microscopy (SR-SIM) - Lateral resolution can be increased over the classical Abbe limit by a factor of two (approximately 100 to 120 nanometers) without discarding any emission light using laser-generated spatially structured illumination coupled to a widefield fluorescence microscope in what is termed superresolution (SR) SIM.

Three-Dimensional Superresolution Imaging References - The Abbe diffraction limit in optical microscopy restricts resolution to approximately 200 nanometers in the lateral plane and 500 to 700 nanometers axially. A number of superresolution techniques have recently been defined that significantly reduce localization precision to much smaller values in both dimensions.

Live-Cell Superresolution Imaging References - One of the ultimate goals of superresolution microscopy is to investigate the dynamics of interacting proteins in living cells at spatial resolutions that far exceed those of traditional diffraction-limited techniques. Emerging methodologies for observing living cells at high resolution are just beginning to surface.

Saturated Structured Illumination Microscopy (SSIM) - In saturated structured illumination (SSIM) and saturated patterned excitation (SPEM) microscopy, the saturated excitation produces narrow line-shaped dark regions in the zero nodes that are surrounded by high levels of fluorescence signal to generate a "negative" imprint of the features being imaged.

Near-Field Scanning Optical Microscopy (NSOM) - Near-field microscopes circumvent the diffraction barrier by exploiting the unique properties of evanescent waves. Resolution is limited only by the physical size of the aperture rather than the wavelength of illuminating light, such that lateral and axial resolutions of 20 nanometers and 2 to 5 nanometers, respectively, can be achieved.

Optical Highlighter Fluorescent Protein Original References - Optical highlighter fluorescent proteins, which include the photoactivatable GFP (PA-GFP), the green-to-red photoconverter Kaede, and the photoswitchable Dronpa, allow direct and controlled activation of distinct molecular pools of the fluorescent proteins within the cell. Listed in this section are key references to many of the original articles describing the discovery and properties of optical highlighters.

Photoactivation and Photoconversion - The ability to selectively initiate or alter fluorescence emission profiles in fluorescent proteins has resulted in the creation of a new class of probes for exploring protein behavior and dynamics in living cells. As the fluorescence intensity or spectral alterations of highlighters generally occur only after photon-mediated conversion, newly synthesized non-photoactivated protein pools remain unobserved and do not complicate experimental results. This section provides sources for selected review articles and original research reports on optical highlighter fluorescent proteins.

Contributing Authors

Mats G. L. Gustafsson, Eric Betzig, and Harald F. Hess - Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, 20147.

George H. Patterson - Biophotonics Section, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, 20892.

Jennifer Lippincott-Schwartz - Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892.

Hari Shroff - Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, 20892.

Samuel T. Hess - Department of Physics and Astronomy and Institute for Molecular Biophysics, University of Maine, Orono, Maine, 04469.

Matthias F. Langhorst, Joerg Schaffer, and Bernhard Goetze - Carl Zeiss MicroImaging GmbH, GB BioSciences, Koenigsallee 9-21, 37081 Goettingen, Germany.

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.