Nanowire-based single-cell endoscopy
Ruoxue Yan1†, Ji-Ho Park1,2†, Yeonho Choi3,4, Chul-Joon Heo3,5, Seung-Man Yang5, Luke P. Lee3
and Peidong Yang1*
One-dimensional smart probes based on nanowires and nano-
tubes that can safely penetrate the plasma membrane and
enter biological cells are potentially useful in high-resolution1–6
and high-throughput7,8 gene and drug delivery, biosensing6,9
and single-cell electrophysiology6,10. However, using such
probes for optical communication across the cellular membrane
at the subwavelength level remains limited. Here, we show that
a nanowire waveguide attached to the tapered tip of an optical
fibre can guide visible light into intracellular compartments of a
living mammalian cell, and can also detect optical signals from
subcellular regions with high spatial resolution. Furthermore,
we show that through light-activated mechanisms the endo-
scope can deliver payloads into cells with spatial and temporal
specificity. Moreover, insertion of the endoscope into cells and
illumination of the guided laser did not induce any significant
toxicity in the cells.
Optical nanoscopy, a high-resolution technique that breaks the
diffraction barrier, has been introduced recently to study chemistry,
biology and physics in intracellular molecular processes11–13.
Although current optical techniques have now reached the subcellu-
lar level, high-precision optical imaging systems are complex,
expensive and bulky. Therefore, incorporating a nanophotonic com-
ponent into simple, low-cost, bench-top optical set-ups would min-
iaturize spectroscopic analyses, and would be particularly useful for
studying chemical and biological events inside single cells as well as
their substructures. Fluorescence sensing techniques based on sub-
micrometre tapered optical fibres have been developed for probing
living biological specimens14–17, but the requisite large and conical
fibres mean that their illumination volume remains large and they
can easily rupture cellular membranes. Nevertheless, fibre-optic
fluorescence imaging techniques have unique features that can be
incorporated into handheld optical systems and flexible endoscopes
for minimally invasive imaging of opaque biological specimens such
as tissues18. Integrating nanophotonic probes into the fibre-optic
imaging system allows us to manipulate light at the nanoscale
inside living cells for studying photoactive biological processes19,20
and for bioanalytical analyses21,22.
Previously, we have shown that subwavelength dielectric nano-
wire waveguides can efficiently shuttle ultraviolet and visible light
in air and fluidic media23,24. Nanowires are promising for interrogat-
ing intracellular environments, because their small dimensions
(100–250 nm) and mechanical flexibility minimize the damage
they inflict on cellular structures and functions. Furthermore,
because of their higher refractive index (n≈ 2.1–2.2) than that of
conventional optical fibre (n≈ 1.5), the nanowires can guide
visible light efficiently in high-index physiological liquids and
living cells (n≈ 1.3–1.5). In addition, the oxide surface of nanowires
can be functionalized readily, enabling payload delivery and specific
sensing inside cells. Recently, there have been an increasing number
of efforts to interface nanowire arrays with living cells to modulate
the physicochemical properties of the cells and deliver biomolecules
into subcellular environments25–27. By controlling nanowire diam-
eter, length and density, they can be safely internalized into the
cell without disturbing cell proliferation and differentiation.
Here, by combining the advantages of nanowire waveguides and
a fibre-optic fluorescence imaging technique, we have designed a
novel nanowire-based endoscope system (Fig. 1a) for optical
probing inside single cells and the spatiotemporal delivery of pay-
loads into intracellular sites with minimal perturbation to the
cellular system.
The nanowire endoscope was fabricated by bonding a SnO2
nanowire to the tapered tip of an optical fibre (Supplementary
Fig. S1,S2). Light travelling along the optical fibre can be effectively
coupled into the nanowire and travel to the nanowire tip, where it is
re-emitted to free space (Fig. 1b). Mechanical stability is a funda-
mental requirement for biological probes for use in intracellular
sensing and imaging. The nanowire endoscope is extremely flexible
due to its small size and high aspect ratio, yet it is mechanically
robust, and can endure repeated deformation, bending and buckling
cycles without being peeled off the fibre. Figure 1d shows that, under
208 deformation, the nanowire remained firmly attached to the fibre
tip. Also, the intensity of its tip emission did not show a significant
fall-off compared to that when it was free of mechanical defor-
mation (Fig. 1c), indicating that the optical coupling also
remained undisturbed.
Highly efficient optical coupling between the nanowire and the
optical fibre is crucial for nanowire endoscopes to function as loca-
lized light sources. To test the waveguiding capability of the nano-
wire endoscope in a physiological environment and to visualize
the intensity distribution of the endoscope emission, the endoscope
was immersed in cell culture media, which contains fluorescent pro-
teins that can be excited by the endoscope (Fig. 1e). The profile of
fluorescence intensity follows that of the endoscope emission
(Fig. 1f,g). For a highly efficient nanowire endoscope, the optical
output is closely confined to the nanowire tip, offering highly direc-
tional and localized illumination. Despite the small dimensions of
the nanowires, the large increase (33%) in environmental refrac-
tive index did not affect the optical coupling or light propagation,
benefiting from the high refractive index of the SnO2.
The advantage of using nanowires instead of tapered fibre-optics
for single-cell endoscopy is that nanowires are less invasive because
of their fine diameters and uniform geometry. As in previous studies
on silicon and gallium phosphide nanowire arrays25–27, where the
nanowires were found to penetrate the lipid membrane of cells
without causing any damage to cellular functions, the insertion of
our SnO2 nanowire into the cell cytoplasm did not induce cell
1Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720,
USA, 2Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea, 3Department of
Bioengineering, University of California, Berkeley, California 94720, USA, 4Department of Biomedical Engineering, Korea University, Seoul, 136-703, South
Korea, 5CRI Center for Integrated Optofluidic Systems, Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and
Technology, Daejeon, 305-701, South Korea. †These authors contributed equally to this work. *e-mail: p_yang@uclink.berkeley.edu
LETTERS
PUBLISHED ONLINE: 18 DECEMBER 2011 | DOI: 10.1038/NNANO.2011.226
NATURE NANOTECHNOLOGY | ADVANCE ONLINE PUBLICATION | www.nature.com/naturenanotechnology 1
© 2011 Macmillan Publishers Limited. All rights reserved.

death (Supplementary Table S1, Fig. S5), apoptosis (Supplementary
Fig. S6), significant cellular stress (Supplementary Fig. S7, Video S2)
or membrane rupture (Supplementary Fig. S8) under the exper-
imental conditions required for the spot cargo delivery and endo-
scopy studies described below. In contrast, insertion of a tapered
optical-fibre tip caused 40% of the plated cells to die, and also
induced considerable cellular stress and cell membrane rupture
(Supplementary Section II). Moreover, illuminating the intracellular
environment of HeLa cells with blue light using the nanoprobe did
not harm the cells, because the illumination volume was small
(down to the picolitre scale; Supplementary Table S2).
Having shown that the nanowires are biocompatible, we used the
endoscopes to deliver payloads into specific sites in the cell. Carbon
andboronnitride nanotube-based delivery systemshave been reported
previously1,28, in which the payloads (for example, quantum dots,
QDs) were attached to the nanotube by disulphide bond linkers,
which are cleavable in the reducing environment of the cytoplasm.
A major limitation of this passive delivery system is the long insertion
time (20–30 min) and the lack of temporal control over the delivery
process (Supplementary Fig. S9). To reduce insertion time and
improve temporal control during delivery, we attached QDs to the
nanowire tip by means of photo-cleavable linkers (Fig. 2a–c) that
a
Focusing
lens
Excitation laser
Microscope
objective
Spectrometer CCD
Spectrum
Optical fibre
Micromanipulator
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r grati
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x
z
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Nanoprobe
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c d
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Cell medium
Figure 1 | Design of the nanowire-based optical probe for single-cell endoscopy. a, Schematic illustration of the nanowire-based cell endoscope system. The
nanowire endoscope, consisting of a nanowire waveguide fixed on the tapered tip of an optical fibre, can be inserted into a single living cell at designated
positions using a three-axis micromanipulating system for spot delivery of payloads. The nanowire endoscope can be optically coupled to either an excitation
laser to function as a local light source for subcellular imaging or a spectrometer to collect local optical signals. CCD, charge-coupled device. b, Three-
dimensional schematic showing a blue laser waveguided through a nanowire endoscope constructed by gluing a SnO2 nanowire to the tip of a tapered
single-mode optical fibre. c,d, Dark-field images of an endoscope (coupled to a 442 nm blue laser) before (c) and during (d) deformation by a tungsten
needle, showing that the nanowire endoscope is flexible and robust. Yellow arrows in b–d point to the nanowire tip where the waveguided light was emitted
into free space. e–g, Intensity profile of the nanowire endoscope emission. Schematic drawing (e), dark-field (f) and fluorescence (g) images of a nanowire
endoscope immersed in cell culture medium, which illuminated fluorescent proteins with blue light emitted from the nanowire tip. f and g are top views of
the real nanowire endoscope device illustrated in e. A 442 nm long-pass filter was applied to remove the excitation beam from the fluorescence image (g). Scale
bars, 50mm.
LETTERS NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2011.226
NATURE NANOTECHNOLOGY | ADVANCE ONLINE PUBLICATION | www.nature.com/naturenanotechnology2
© 2011 Macmillan Publishers Limited. All rights reserved.