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Microscopy Reference Library

Superresolution Microscopy Reference Library

The most significant advances in superresolution imaging have been achieved in what is termed far-field microscopy and involve either spatially or temporally modulating the transition between two molecular states of a fluorophore (such as switching between a dark and bright state) or by physically reducing the size of the point spread function used in the excitation illumination. Among the methods that improve resolution by PSF modification, the most important techniques are referred to by the acronyms STED (stimulated emission depletion) and SSIM (saturated structured illumination microscopy). Techniques that rely on the detection and precise localization of single molecules include PALM (photoactivation localization microscopy) and STORM (stochastic optical reconstruction microscopy. The references included in this section should be useful to investigators seeking introductory and advanced material on superresolution microscopy.

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.

Super-Resolution 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 high-resolution (HR) 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.