Fluorescence microscopy, a favorite tool of biologists, magnifies and images light-emitting objects with a resolution down to one-quarter of a micrometer, enabling the study of fine structural details of cellular architecture and dynamics. Key insights into events occurring inside cells, tissues, and whole organisms have thereby been achieved, such as the shape of intracellular transport vehicles (1
), the mechanism(s) for tissue remodeling (2
), and the movement of cancer cells within a diseased organism (3
). Contributing to the dramatic rise in fluorescence microscopy has been the advent of genetically encoded fluorescent proteins (FPs) acting as endogenous labels, allowing almost any protein or peptide to become fluorescent inside cells and thereby to be visible within a biological context (4
). The wide availability of sensitive cameras, inexpensive lasers, and high-quality optical lenses and filters has further permitted acquisition of superior images with spatiotemporal characteristics appropriate for addressing a diverse array of biological questions.
Despite its revolutionary impact, fluorescence microscopy faces a limit in its resolving capability—the diffraction of light. Arising from the wavelike character of light diffracting while passing through a lens, this limitation prevents objects smaller than ~250 nm along the x
axis and ~500 nm in the z
axis from being seen as anything but a blur. As many subcellular structures have features much smaller than this size—including microtubules, actin fibers, ribosomes, transport vesicles, and the intramembrane organization of organelles—the need to break the diffraction barrier, imaging below the size limitation it defines, has been the holy grail of light microscopy for many years (5
Recently, two distinct conceptual strategies have overcome light's diffraction barrier, allowing the analysis of biological structures at the superresolution level. One strategy, referred to here as illumination-based superresolution, uses nonlinear optical approaches to reduce the focal spot size, as in stimulated emission depletion (STED) fluorescence microscopy (6
) and saturated structured illumination microscopy (SSIM) (7
). By modifying the excitation light pattern to yield a smaller spot size, STED and SSIM can resolve fine structural details of biological specimens, such as the shape of mitochondrial membranes (8
) and chromosomal and nuclear envelope organization (9
), down to ~30 nm in the x
direction in the case of STED and ~100 nm in the case of SSIM.
The second strategy for overcoming light's diffraction barrier uses photoswitchable molecules to resolve dense populations of molecules with superresolution. This approach employs stochastic activation of fluorescence to switch on individual photoactivatable molecules and then images and bleaches them, temporally separating molecules that would otherwise be spatially indistinguishable. Merging all the single-molecule positions obtained by the photoactivation and imaging/bleaching cycles yields a final superresolution image. Referred to here as probe-based superresolution, this approach was independently developed by three groups and given the names photoactivated localization microscopy (PALM) (10
), fluorescence photoactivated localization microscopy (FPALM) (11
), and stochastic optical reconstruction microscopy (STORM) (12
). Whereas PALM/FPALM use photoactivatable or photoconvertible FPs as probes, STORM uses synthetic fluorophores as probes, as in the photoswitchable fluorophore combination of Cy3 and Cy5. Probe-based superresolution allows biological structures to be defined with nanometric accuracy, similar to illumination-based superresolution. However, it additionally permits molecules comprising subcellular structures to be individually identified at high densities and their distributions and dynamics to be analyzed. This opens many new possibilities for addressing mechanistic questions regarding biological function, including the mapping of molecular machinery, its stoichiometry, and dynamics.
Both illumination-based and probe-based superresolution imaging approaches permit biologists to now visualize structures and processes of cells at or near the molecular level. The order-of-magnitude improvement in spatial resolution achieved over previous light microscopy methods means that the new approaches have enormous potential for addressing numerous biological questions requiring resolutions below 250 nm. So far, these approaches have provided details on the fine architecture of cell structures such as mitochondria, lysosomes, focal adhesions, microtubules, and coated vesicles (8
). Dynamic processes have also been studied, including the movement of focal adhesion complexes (14
) and bacteria polarity complexes (15
), as well as single molecules on the plasma membrane (16
Here, we focus on probe-based superresolution imaging achieved using the single-molecule localization techniques of PALM/FPALM/STORM. We begin by discussing the concept underlying its development and the photoswitchable probes that it utilizes. We then describe its current state of development, including its various applications and limitations. We end by discussing the future possibilities and challenges of probe-based superresolution imaging, and the scientific areas it is likely to strongly impact.