Despite the advantages of traditional fluorescence microscopy, the technique is hampered in ultrastructural investigations due to the resolution limit set by the diffraction of light, which restricts the amount of information that can be captured with standard objectives. In the past few years, a number of novel approaches have been employed to circumvent the diffraction limit, including near-field scanning optical microscopy (NSOM), stimulated emission depletion microscopy (STED), stochastic optical reconstruction microscopy (STORM) and structured illumination microscopy (SIM). These techniques have all achieved improved lateral (x-y) resolution down to tens of nanometers, more than an order of magnitude beneath that imposed by the diffraction limit, but each method has a unique set of limitations.
The Diffraction Barrier in Optical Microscopy
Limitations on optical microscope resolution imposed by physical laws.
Single-Molecule Super-Resolution Imaging
A review of STORM, and related techniques that rely on imaging single molecules.
Selected Literature References
Superresolution Review Articles
Overviews of the literature written by experts in optical design and superresolution imaging.
3D Superresolution Techniques
Probing both the lateral and axial dimensions at resolutions beneath the diffraction limit.
A single-molecule superresolution technique that utilizes conventional fluorophores.
Single-molecule superresolution using photoswitchable carbocyanine dyes.
Stimulated Emission Depletion (STED)
A point-spread function engineering technique that relies on high-power lasers.
Superresolution Structured Illumination
Applying structured excitation illumination to resolve high spatial frequencies.
Interference Superresolution MicroscopyInterference techniques that rely on standing wave interference for superresolution.
Saturated Structured Illumination
Structured illumination excitation with theoretically infinite resolution.
Superresolution Practical Aspects
Points to consider when conducting single-molecule superresolution investigations.
PALMIRA Superresolution Imaging
An advanced single-molecule technique that avoids several potential artifacts.
Structured Illumination Microscopy
Patterned excitation for optical sectioning forms the basis for superresolution.
Selective Plane Illumination
An emerging technique with great potential for superresolution imaging.
The RESOLFT Concept
Describing superresolution with reversible saturable or switchable optical transitions.
4Pi Superresolution Microscopy
Using opposed objectives to narrow the axial point spread function to near 100 nanometers.
Ground State Depletion (GSD) Microscopy
GSD relies on driving excited state molecules into a dark metastable long-lived triplet state.
Single-molecule superresolution using optical highlighter fluorescent proteins.
Standing Wave Microscopy
Axial resolution enhancement using interference between two standing waves.
Fundamental principles underpinning the techniques of PALM, STORM, and GSDIM.
Investigations are beginning to address dynamics using superresolution microscopy.
Superresolution Microscopy Probes
Fluorescent proteins, synthetics, quantum dots, and hybrid systems for superresolution.
Superresolution Localization Accuracy
Critical aspects of stage drift, molecular density, background signal and other artifacts.
Blinking Superresolution Microscopy
Single-molecule imaging tuned to the fluctuating emission of fluorophores.
Specialized Superresolution Methods
Emerging methodology is now being examined for potential applications.
Near-Field Scanning (NSOM)
Probing specimens with an evanescent wavefield for superresolution.
Joel S. Silfies and Stanley A. Schwartz - Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York, 11747.
Sunita Martini, Stephen P. Price, Alex B. Coker, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.