Breaking the Diffraction Barrier:
Super-Resolution Imaging of Cells
Bo Huang,1Hazen Babcock,2and Xiaowei Zhuang2,3,*
1Department of Pharmaceutical Chemistry and Department of Biochemistry and Biophysics, University of California, San Francisco, San
Francisco, CA 94158, USA
2Center for Brain Science
3Howard Hughes Medical Institute and Department of Chemistry and Chemical Biology and Department of Physics
Harvard University, Cambridge, MA 02138
Anyone who has used a light microscope has wished that its resolution could be a little better.
Now, after centuries of gradual improvements, fluorescence microscopy has made a quantum
leap in its resolving power due, in large part, to advancements over the past several years in
a new area of research called super-resolution fluorescence microscopy. In this Primer, we explain
the principles of various super-resolution approaches, such as STED, (S)SIM, and STORM/(F)
PALM. Then, we describe recent applications of super-resolution microscopy in cells, which
demonstrate how these approaches are beginning to provide new insights into cell biology, micro-
biology, and neurobiology.
For centuries, light microscopy has greatly facilitated our under-
standing of how cells function. In fact, entire fields of biology
have emerged from images acquired under light microscopes.
For instance, more than 300 hundred years ago, Antonie van
Leeuwenhoek used his self-ground optical lenses to discover
bacteria and commence the field of microbiology. Then, ?200
years later, Ramo ´n y Cajal used light microscopes to visualize
Golgi-stained brain sections and create beautiful drawings of
neurons, which led to his ingenious vision of how information
flows in the nervous systems and helped to form modern neuro-
Indeed, one major element that makes light microscopy
so powerful in biological research is the development of various
staining methods that permit the labeling of specific molecules
and cells. For example, fluorescence in situ hybridization
(FISH) detects DNA and RNA molecules with specific se-
quences, whereas immunofluorescence labels and fluorescent
proteins allow the imaging of particular proteins in cells
(Giepmans et al., 2006). Even single molecules within a living
cell can be visualized when these labeling strategies are
combined with highly sensitive optical schemes and detectors
(Lord et al., 2010; Xie et al., 2008).
The unrivaled combination of molecule-specific contrast and
live-cell imaging capability makes fluorescence microscopy the
most popular imaging modality in cell biology. Browse through
any cell biological journal, and the impact of fluorescent micros-
copy is obvious, with > 80% of the images of cells in the book
usually acquired with a fluorescent microscope. However, the
application of fluorescence microscopy to manyareas of biology
is still hindered by its moderate resolution of several hundred
nanometers. This resolution is approximately the size of an intra-
cellular organelle and thus is inadequate for dissecting the inner
architecture of many subcellular structures.
The resolution for optical microscopy is limited by the diffrac-
tion, or the ‘‘spreading out,’’ of the light wave when it passes
through a small aperture or is focused to a tiny spot. Because
this property is intrinsic to all waves, breaking the diffraction
barrier of light microscopy has been deemed impossible for
a long time. However, such limitations have not deterred a small
group of scientists from pursuing ‘‘super-resolution’’ fluores-
cence microscopy that breaks through this seemingly impene-
The risk has paid off abundantly. Recently, these research
teams have developed several optical microscopy techniques
that have shattered the diffraction barrier, improving spatial
resolution by an order of magnitude or more over the diffraction
limit. Most importantly, these techniques are beginning to
provide insights into biological processes at the cellular and
molecular scale that were hitherto unattainable. In this Primer,
we review the technological advances in the burgeoning field
of ‘‘super-resolution fluorescence microscopy.’’ Then, we
describe the application of these techniques to various areas
of biology, which have quickly demonstrated the great promise
of this exciting new area of bioimaging.
Beating the Diffraction Limit of Resolution
When light is focused by the objective of a microscope, the
notion of light ‘‘rays’’ converging to an infinitely sharp ‘‘focal
point’’ does not happen. Instead, the light wave forms a blurry
focal spot with a finite size due to diffraction (Figure 1A). The
size of the spot depends on the wavelength of the light and
the angle at which the light wave converges; the latter is, in
turn, determined by the numerical aperture of the objective.
The width of the spot is ?0.6 l/NA, wherein l is the wavelength
of the light and NA is the numerical aperture of the lens. Simi-
larly, a point emitter, such as a single fluorescent molecule,
Cell 143, December 23, 2010 ª2010 Elsevier Inc. 1047
also appears as a blurry spot with a finite size when imaged
through a microscope. The intensity profile of this spot, which
defines the point spread function (PSF) of the microscope, has
approximately the same width as that of the focal spot
described above. Consequently, two identical emitters sepa-
rated by a distance less than the width of the PSF will appear
as a single object, making them unresolvable from each other
This resolution limit was originally recognized by Ernst Abbe
?150 years ago, and thus, it is also called the Abbe limit
(Abbe, 1873). The diffraction-limited image resolution of
dicular to the direction of light propagation (i.e., in the lateral
dimensions) and ?550 nm parallel to the direction of light prop-
agation (i.e., in the axial dimension) (Figure 1A). Many subcellular
structures are smaller than these resolution limits, and therefore,
they are unresolvable by light microscopes (Figure 1B).
For many years, several imaging techniques have pushed the
boundary of the diffraction limit of light microscopy. Among
these methods, confocal microscopy and multiphoton fluores-
cence microscopy not only enhance the image resolution, but
also reduce the out-of-focus fluorescence background, allowing
optical sectioning and thus three-dimensional imaging. In addi-
tion, infrared light experiences a lower amount of scattering
from tissues, allowing deep tissue imaging with two-photon
microscopy (Zipfel et al., 2003). 4Pi microscopy and I5M use
two opposing objective lenses to increase the effective numer-
ical aperture of the microscope and thereby improve the image
resolution (Gustafsson et al., 1995; Hell and Stelzer, 1992;
Hell, 2003). Although these methods significantly improve the
resolution, they are still fundamentally limited by diffraction and
have, in practice, achieved resolutions of ?100 nm in all three
dimensions (Hell, 2003).
The diffraction-limited resolution applies only to light that has
propagated for a distance substantially larger than its wave-
length (i.e., in the far field). Therefore, one route to bypass this
constraint is to place the excitation source or detection probe
(usually an optical fiber, a metal tip, or simply a small aperture)
near the sample (i.e., in the near field) (Synge, 1928). Indeed,
near-field microscopy has achieved resolution substantially
below 100 nm (Betzig et al., 1986; Lewis et al., 1984; Novotny
and Hecht, 2006; Pohl et al., 1984). However, the requirement
that the excitation source or detection probe be physically close
to the target object (often within tens of nanometers) has made it
difficult to look ‘‘into’’ a cell or a piece of tissue with near field
microscopy, limitingthe applications of thistechnique in biology.
It was not until recently that several novel fluorescence
microscopy approaches completely shattered the diffraction
limit of image resolution in the far field. In general, all of these
approaches generate ‘‘diffraction-unlimited images’’ by using
sions from two nearby molecules within a diffraction-limited
region. These super-resolution approaches can be divided into
two primary classes. The first category is ensemble imaging
approaches that use patterned illumination to spatially modulate
the fluorescence behavior of molecules within a diffraction-
limited region, such that not all of them emit simultaneously,
thereby achieving subdiffraction limit resolution. This category
includes stimulated emission depletion (STED) microscopy
(Hell and Wichmann, 1994; Klar and Hell, 1999) and the related
RESOLFT technology (Hofmann et al., 2005), as well as satu-
rated structured illumination microscopy (SSIM) (Gustafsson,
2005; Heintzmann et al., 2002). The second category takes
advantages of single-molecule imaging, using photoswitching
or other mechanisms to stochastically activate individual mole-
cules within the diffraction-limited region at different times.
Images with subdiffraction limit resolution are then recon-
structed from the measured positions of individual fluorophores.
This second class has been termed stochastic optical recon-
struction microscopy (STORM) (Rust et al., 2006), photoacti-
vated localization microscopy (PALM) (Betzig et al., 2006), and
fluorescence photoactivation localization microscopy (FPALM)
(Hess et al., 2006).
Although these two categories of methods use different
approaches to accomplish subdiffraction resolution, these tech-
niques also share important commonalities. In both cases,
a physical or chemical property of the fluorophore is used to
maintain neighboring molecules in different states (i.e., ‘‘on’’
Figure 1. Diffraction-Limited Resolution of Conventional Light
(A) The focal spot of a typical objective with high numerical aperture, depicted
by the cyan ellipsoid, has a width of ?250 nm in the lateral directions and
?550 nm in the axial direction. The image of a point emitter imaged through
the objective, namely the point spread function, also has similar widths. These
widths define the diffraction-limited resolution. Two objects separated by
a distance larger than this resolution limit appear as two separate entities in
the image. Otherwise, they appear as a single entity (i.e., unresolvable). These
two cases are exemplified by the two cross sections of the microtubule image,
cyan curves A and B in the right panel, at the corresponding positions
indicated by the white lines in the middle panel.
(B) The size scale of various biological structures in comparison with the
diffraction-limited resolution. (Left to right) A mammalian cell, a bacterial cell,
a mitochondrion, an influenza virus, a ribosome, the green fluorescent protein,
and a small molecule (thymine).
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