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.
Limitations on optical microscope resolution imposed by physical laws.
A review of STORM, and related techniques that rely on imaging single molecules.
Selected Literature References
Overviews of the literature written by experts in optical design and superresolution imaging.
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.
A point-spread function engineering technique that relies on high-power lasers.
Applying structured excitation illumination to resolve high spatial frequencies.
Structured illumination excitation with theoretically infinite resolution.
Points to consider when conducting single-molecule superresolution investigations.
An advanced single-molecule technique that avoids several potential artifacts.
Patterned excitation for optical sectioning forms the basis for superresolution.
An emerging technique with great potential for superresolution imaging.
Describing superresolution with reversible saturable or switchable optical transitions.
Using opposed objectives to narrow the axial point spread function to near 100 nanometers.
GSD relies on driving excited state molecules into a dark metastable long-lived triplet state.
Single-molecule superresolution using optical highlighter fluorescent proteins.
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.
Fluorescent proteins, synthetics, quantum dots, and hybrid systems for superresolution.
Critical aspects of stage drift, molecular density, background signal and other artifacts.
Single-molecule imaging tuned to the fluctuating emission of fluorophores.
Emerging methodology is now being examined for potential applications.
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.