Institute for Biomedical Materials & Devices (IBMD), Faculty of Science, University of Technology Sydney, Ultimo, NSW, Australia.
Biomedical Engineering, College of Engineering, Peking University, Beijing, China.
Third Institute of Physics, University of Göttingen, Göttingen, Germany.
School of Chemistry, University of Wollongong and Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia.
*e-mail: email@example.com; firstname.lastname@example.org; email@example.com; firstname.lastname@example.org
luorescent proteins and organic dyes are commonly used as
imaging probes in microscopy because of their small size and
compatibility with biological samples. The advent of super-res
olution microscopy made it possible to resolve cellular structures
and subcellular organelles on the nanometer scale, and has revolu
tionized optical microscopy in the life sciences. However, for long-
term tracking of single molecules and real-time super-resolution
imaging of subcellular structures, brighter and more photostable
probes are still needed to reveal the inner workings of the cell, as
the current molecular dyes and fluorescent proteins are often too
dim, can be optically switched in only a limited number of ways,
and undergo rapid photobleaching.
Advances in the materials sciences have provided considerable
opportunities to address the shortcomings of fluorescent dyes.
Recent developments in the tailored design of nanoscopically sized
probes with well-defined optical properties have resulted in a large
collection of luminescent nanoparticles (Fig. 1) such as semiconduc
tor quantum dots (QDots), upconversion nanocrystals (UCNPs),
polymer dots (PDots), fluorescent nanodiamonds (FNDs), carbon-
based nanodots (CDots), and nonfluorescent surface-enhanced
Raman scattering (SERS) nanoparticles. Though they are still large
compared with dye molecules (Fig. 1a) and substantial challenges
lie ahead with respect to their complicated surface biochemistry,
the many advantages of these nanoparticles have been successfully
demonstrated and have started to pave the way for the cell biology
and materials science communities to explore their full capability
in subcellular functional imaging at the nanoscale. This Perspective
focuses on the recent progress and future potential of these small
luminescent nanoparticles for use in tracking single molecules and
super-resolution imaging of subcellular structures.
Nanoparticles in super-resolution microscopy
QDots represent the first generation of inorganic fluorescent
nanoparticles used for fluorescent labeling. Compared with conven
tional dyes, they have a much larger absorption coefficient, a higher
luminescent quantum yield, and, as a result, greater brightness.
QDots exhibit narrow-band emission (spectral width < 50 nm), and
their emission color can be tuned through adjustments to their size
(quantum confinement effect), which makes these particles ideal
for multicolor imaging applications
and a range of super-resolution
imaging modes (Fig. 1b–f).
The commercially available ZnS-coated CdSe QDot 705 (from
Thermo Fisher), CdTe QDot 700 nm, and CdTe QDot 720 nm (from
PlasmaChem GmbH) have proven suitable for stimulated emission
depletion (STED) microscopy with spatial resolutions in the range
of 50 nm for single QDots, 85 nm for QDot-705-stained microtu
bule networks in HeLa cells
, and 106 nm for vimentin filaments in
. The high stability of QDots allows for extended time-
lapse imaging without any photophysical or photochemical pro
cesses that would diminish their brightness.
QDots have widespread applications in super-resolution opti
cal fluctuation imaging (SOFI)
, and multicolor QDots (QDot 525,
QDot 625, and QDot 705) have recently been used to achieve higher
labeling densities to improve both temporal and spatial resolu
. New developments in material engineering have dramatically
sped up the kinetics of QDot blinking to facilitate real-time optical
microscopy with SOFI, thus enabling the use of spinning-disk confo
cal microscopy in association with bleaching/blinking-assisted local-
ization microscopy and SOFI to obtain 3D super-resolution images
UCNPs represent an entirely new class of multiphoton probes
that rely on high densities of multiphoton emitters in small parti
. Each particle contains thousands of codoped lanthanide ions
that form a network of photon sensitizers and activators, which
upconvert near-infrared photons into visible and ultraviolet ones.
The large anti-Stokes spectral separation between excitation and
emission renders these probes highly useful in background-free and
reported that thousands of emitters per
nanoparticle can be activated by microscopes, resulting in a bright
ness that makes UCNPs suitable as single-molecule probes. Highly
doped UCNPs were found to easily facilitate an optical population
inversion at their intermediate metastable levels
. As a result, a
low saturation intensity of ~0.19 MW cm
in upconversion STED
microscopy was recorded, with a maximum resolution of 28 nm
(λ /36) for optical imaging of single 13-nm UCNPs. High-speed
(100-µ s dwelling time) super-resolution imaging of cellular cyto
skeleton protein structures with a resolution of 80 nm has also been
Nanoparticles for super-resolution microscopy
and single-molecule tracking
*, Peng Xi
*, Baoming Wang
, Le Zhang
, Jörg Enderlein
* and Antoine M. van Oijen
We review the use of luminescent nanoparticles in super-resolution imaging and single-molecule tracking, and showcase novel
approaches to super-resolution imaging that leverage the brightness, stability, and unique optical-switching properties of these
nanoparticles. We also discuss the challenges associated with their use in biological systems, including intracellular delivery
and molecular targeting. In doing so, we hope to provide practical guidance for biologists and continue to bridge the fields of
super-resolution imaging and nanoparticle engineering to support their mutual advancement.
NATURE METHODS | VOL 15 | JUNE 2018 | 415–423 | www.nature.com/naturemethods
© 2018 Nature America Inc., part of Springer Nature. All rights reserved.