nanophotonic approaches for nanoscale imaging and single-molecule detection at ultrahigh...

Nanophotonic Approaches for Nanoscale Imaging andSingle-Molecule Detection at Ultrahigh ConcentrationsMATHIEU MIVELLE,1 THOMAS. S. VAN ZANTEN,1 CARLO MANZO,1 AND MARIA F. GARCIA-PARAJO1,2*1ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, Castelldefels, 08860 Barcelona, Spain2ICREA-Instituci�o Catalana de Recerca i Estudis Avancats, 08010 Barcelona, Spain

KEY WORDS optical antennas; fluorescence correlation spectroscopy; membrane organization

ABSTRACT Over the last decade, we have witnessed an outburst of many different opticaltechniques aimed at breaking the diffraction limit of light, providing super-resolution imagingon intact fixed cells. In parallel, single-molecule detection by means of fluorescence has become acommon tool to investigate biological interactions at the molecular level both in vitro and in liv-ing cells. Despite these advances, visualization of dynamic events at relevant physiological con-centrations at the nanometer scale remains challenging. In this review, we focus on recentadvancements in the field of nanophotonics toward nanoimaging and single-molecule detectionat ultrahigh sample concentrations. These approaches rely on the use of metal nanostructuresknown as optical antennas to localize and manipulate optical fields at the nanometer scale. Wehighlight examples on how different optical antenna geometries are being implemented for nano-scale imaging of cell membrane components. We also discuss different implementations of self-standing and two-dimensional antenna arrays for studying nanoscale dynamics in living cellmembranes as well as detection of individual biomolecular interactions in the mM range for sens-ing applications. Microsc. Res. Tech. 00:000–000, 2014. VC 2014 Wiley Periodicals, Inc.

INTRODUCTION

One of the ultimate goals in biology is to understandthe relationship between structure, function, anddynamics of biomolecules in their natural environ-ment: the living cell. Although modern molecular biol-ogy has made enormous progress in identifying a fullrepertoire of proteins, lipids, and other molecular com-ponents both inside and at the cell membrane, directvisualization of molecular interactions in living cellsremains as a major challenge.

Take as example the cell membrane, which is highlyheterogeneous in terms of structure, composition anddynamics. In recent years it has become evident thatmembrane components do not operate separately butare part of well-organized multimolecular aggregates.Indeed, many membrane receptors exhibit clear butdistinct spatial patterns, which might arise from pro-tein–lipid interactions (Lingwood and Simons, 2010;Simons and Gerl, 2010), interactions with the corticalcytoskeleton (Goswami et al., 2008; Kusumi et al.,2005), and/or with other local organizers of the cellmembrane, such as tetraspanins (Hemler, 2005;Yanez-Mo et al., 2009) or galectins (Lajoie et al., 2009).This has led to the concept that in addition to receptorexpression, spatiotemporal organization on the cellsurface is tightly controlled and crucial for function.Moreover, recent research is providing evidence thatreceptors might also exist in preassembled nanoclus-ters prior to ligand activation, in the absence of inter-actions with other molecular components (Cambiet al., 2004; Manzo et al., 2012; Sieber et al., 2007; vanZanten et al., 2009). As such, the overall result is thatmost plasma membrane receptors distribute heteroge-

neously in small domains that are diverse in terms ofsize, composition and stability. This complex, heteroge-neous arrangement has been shown to be critical tovarious physiological processes (Cambi et al., 2004,2006; Mayor and Pagano, 2007; Manes and Viola,2006). Yet, very little is known about the molecularmechanisms leading to the non-random organizationof the cell membrane, in particular, prior to cell activa-tion, since this compartmentalization occurs at thenanometer scale (Goswami et al., 2008; Kusumi et al.,2005; Lingwood and Simons, 2010; van Zanten et al.,2009) a size regime not accessible by standard micros-copy techniques as they suffer from diffraction.

In recent years, the emergence of far-field opticaltechniques able to surpass the diffraction limit of lightis advancing our understanding of the cell surfaceorganization at the nanometer scale. These techniquesmake use of specific photophysical properties of fluo-rescence probes in conjunction with tailored ways ofillumination. For instance, stimulated emission deple-tion (STED) based on reversible saturable transitionson a fluorescent dye can achieve �30 nm resolution onfixed cells (Donnert et al., 2006; Sieber et al., 2007).Alternatively, the “apparent” resolution (more

*Correspondence to: M.F. Garcia-Parajo, ICFO-Institut de Ciencies Fotoni-ques, Mediterranean Technology Park, Castelldefels, 08860 Barcelona, Spain.E-mail: [email protected]

Received 27 November 2013; accepted in revised form 27 March 2014

REVIEW EDITOR: Dr. Francesca Cella Zanacchi

Contract grant sponsor: European Commission (FP-ICT-2011-7); Contractgrant sponsor: 288263; Contract grant sponsor: Spanish Ministry of Science andAgencia de Gestion d’Ajuts Universitaris i de Recerca (AGAUR).

DOI 10.1002/jemt.22369Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com).

VVC 2014 WILEY PERIODICALS, INC.

MICROSCOPY RESEARCH AND TECHNIQUE 00:00–00 (2014)

correctly localization accuracy) can in principle reachthe molecular scale by allowing only a subset of fluo-rescent molecules (autofluorescent proteins or organicdyes) to be photoactive at a given time and ensuringthat their distance is larger than the diffraction limit.These techniques known in general as single-moleculelocalization methods (Betzig et al., 2006; Hess et al.,2006; Rust et al., 2006) allow for the reconstruction ofan image on a molecule-by-molecule basis using com-putational algorithms. Although these emerging tech-niques are already providing highly detailedinformation at the nanometer scale, they are still slow,so that applications in living cells are scarce. Never-theless, remarkable progress has been achieved inrecent years by improving the performance of photo-switchable fluorescent probes as used in localizationmethods (Jones et al., 2011; Shim et al., 2012), reduc-ing the observation area, both in fluorescence photoac-tivation localization microscopy (Hess et al., 2007) andSTED (Westphal et al., 2008), or by implementingSTED in combination with fluorescence correlationspectroscopy (FCS; Eggeling et al., 2009) (see subse-quently for more detailed description on this particularapproach).

Although in principle, single-molecule dynamicevents on in vitro conditions can be studied in a mucheasier way using several optical approaches, mosttransient interactions between proteins and/or withnucleic acids, and between enzymes and their ligandsoccur at micromolecular ligand concentrations (Holz-meister et al., 2013). Currently, single-molecule detec-tion by fluorescence for in vitro studies is commonlyperformed by confocal microscopy and far-field optics,in combination with techniques such as FCS. Unfortu-nately, due to the diffraction limit of light, the focalillumination volume in confocal corresponds to about 1fL, which implies that for detection of individual mole-cules extremely low sample concentrations, in theorder of hundreds of pM to a few nM, should be used(Holzmeister et al., 2013). As a consequence, at the rel-evant micromolar concentrations, many molecules willreside in the observation volume preventing the detec-tion of interactions at individual molecular level,which is a major limitation in the field.

Different experimental strategies are being imple-mented in recent years to allow for in vitro single-molecule detection at relevant sample concentrations,including reduction on the number of visible moleculesby reversible photoswitching of photochromic mole-cules (Eggeling et al., 2007), molecular confinementand optical observation volume reduction amongstothers (for a recent review in the field see Holzmeisteret al., 2013). One of the most obvious ways to guaran-tee that only one molecule is present in the excitationvolume is by reducing the size of the illumination vol-ume. This has been accomplished by making use ofsubwavelength apertures surrounded by an opaquemetal film, also known as zero-mode waveguides(ZMW) (Levene et al., 2003). By combining the evanes-cent character of the axial field emanating from thesestructures (less than 100 nm) together with thereduced lateral dimensions of the nanoapertures(between 50 and 200 nm in size), effective illuminationvolumes orders of magnitude smaller than in confocalcan be achieved (a few tens of zeptoliters, i.e., 10221 L)

allowing the detection of individual molecules at highsample concentrations (Levene et al., 2003). Unfortu-nately, as the dimensions of these nanostructures arereduced, very small-transmitted light is obtained. Thislow light throughput together with the finite skindepth of the metal used (normally aluminum) restrictsthe practical size of the nanoapertures to about 50–70nm.

In this review, we focus on recent advancements inthe field of nanophotonics toward nanometric opticalresolution as well as single-molecule detection bymeans of fluorescence at ultrahigh sample concentra-tions. These novel approaches, denoted as nanoanten-nas or optical antennas, might become key players inmodern biology by providing tools to study processesboth in vitro and in vivo at relevant spatial scales andphysiological concentrations. It is important to men-tion that other characterization techniques aside fromfluorescence have been also widely developed in thelast decade. In particular, the combination of Ramanspectroscopy with antennas and plasmonics has wit-nessed an enormous interest in the field since in prin-ciple they allow for single-molecule analysis in label-free conditions and native environments, being there-fore considered as complementary to fluorescence (Daset al., 2008; de Angelis et al., 2009, Wood et al., 2012).Since the main focus of this review concerns fluores-cence detection, the reader is referred to Mauser andHartschuh (2014) for a excellent recent review on theprogress of Raman spectroscopy in combination withfield enhancement as afforded by antennas.

OPTICAL ANTENNA PROBES FORSUPER-RESOLUTION IMAGING

Optical antennas convert freely propagating opticalradiation into localized energy, and vice versa. Assuch, they enable the control and manipulation of opti-cal fields at the nanometer scale, having enormouspotential for sensing and imaging applications (Gar-cia-Parajo, 2008; Novotny and van Hulst, 2011). One ofthe earliest examples of an optical antenna is the sub-wavelength aperture probe, as used in near-field scan-ning optical microscopy (NSOM) (Betzig et al., 1991;Hwang et al., 1995). The probe generally consists of asmall aperture (50–100 nm in diameter) at the end of ametal-coated tapered optical fiber (Dufrene andGarcia-Parajo, 2012; Hinterdorfer et al., 2012), whichis scanned in close proximity to the specimen understudy. The lateral resolution, down to tens of nano-meters, is essentially determined by the size of theaperture and the sample-to-probe distance. The probeilluminates the sample with an evanescent field that isstrongly localized at the vicinity of the aperture anddecreases exponentially away from the probe’s endface. Interaction of the near field with the sample sur-face induces changes in the far-field radiation, whichis collected by conventional optics and directed tohighly sensitive detectors to provide an optical image(Betzig et al., 1991; de Lange et al., 2001; van Zantenet al., 2010a). Since light of different wavelengths canbe simultaneously funneled through the same subwa-velength aperture, NSOM allows simultaneous multi-color excitation free from chromatic aberrations (deBakker et al., 2008; Enderle et al., 1997). In addition,the small excitation volume (105 vs. 108 nm3 as

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Microscopy Research and Technique

obtained in confocal microscopy) reduces dramaticallythe cytoplasm background fluorescence, enabling mul-ticolor single-molecule detection with high signal-to-background ratios (de Lange et al., 2001; Dufrene andGarcia-Parajo, 2012; Garcia-Parajo et al., 1998; Hin-terdorfer et al., 2012; van Zanten et al., 2010a) andlocalization accuracies (i.e., determination of thecenter-of-mass position of the nanoscopy fluorescentspot) better than 3 nm on intact cell membranes underphysiological conditions (van Zanten et al., 2009,2010b).

Unfortunately, conventional subwavelength aper-tures as used in NSOM have very low light through-put, so that in practice optical resolutions better than50 nm are hard to achieve. In recent years, more crea-tive optical antenna designs using sharp metallic tips,metal nanoparticles and complex geometrical nano-structures are being currently explored to localize andto boost the optical field to dimensions smaller than 30

nm in size (Fig. 1) (Garcia-Parajo, 2008; Novotny andvan Hulst, 2011).

Although modest yet, first applications on the use ofthese antenna configurations for biological imagingare starting to appear. For instance, using a goldnanoparticle-based optical antenna excited in the farfield, H€oppener and Novotny (2008) imaged singleCa21 channels on erythrocyte plasma membranes inaqueous conditions (Figs. 1a and 1b). The spatial reso-lution obtained was 50 nm, although improvements inthe antenna geometry and illumination schemes thatreduce the far field surrounding background can inprinciple improve the resolution to 10 nm. Other geo-metries include the fabrication of monopole antennascarved around a subwavelength aperture as used inNSOM. This concept takes advantage of the local illu-mination properties of the aperture to drive theantenna to resonance, enhancing and confining theoptical field at the end of the tip, without background

Fig. 1. Super-resolution imaging using optical antennas. a: Goldnanoparticle attached to a tapered optical fiber. The antenna isexcited in the far field. b: Near-field image of individual plasma-membrane-bound Ca21 pumps (PMCA4) on erythrocyte membranesas imaged with the gold-nanoparticle antenna. c: Monopole antennacarved at the apex of an aluminum-coated tapered optical fiber. Exci-

tation of the antenna is performed through the NSOM probe. d:Image of individual fluorescently labeled antibodies imaged by theantenna probe shown in (c). Cross sections of two encircled featuresshow responses of 26 nm and 53 nm. a and b: Adapted from H€oppenerand Novotny 2008 and (c and d) adapted from van Zanten et al.(2010c).

NANOPHOTONIC APPROACHES FOR BIOLOGY 3


contribution from the far field (Taminiau et al., 2007;van Zanten et al., 2010c). Comparable concepts havebeen also reported by developing geometries that arecapable to coupling the far-field light into near field,without background illumination over a wide range ofwavelengths. Of particular interest are the pioneeringworks of de Angelis et al. (2009) and Bao et al. (2012)to reach adiabatic compression by efficiently creatingphoton-to-plasmon coupling structures, suppressingfar-field background contributions and achievingexcellent nanometric resolutions and extremely highsensitivity. Although demonstration of functionalityhas been shown for Raman spectroscopy (de Angeliset al., 2009) and photoluminescence spectroscopy onsemiconductor nanowires (Bao et al., 2012), there is noreason as to why these optimal designs could not beused in biological applications based on fluorescencecontrast. Indeed, in the field of fluorescence, monopoleantennas have been used to detect individual mole-cules on polymer thin films (Taminiau et al., 2007) andto image individual proteins in intact cell membraneswith 30 nm spatial resolutions and virtually no back-ground, allowing localization accuracies in the order of1 nm (van Zanten et al., 2010c) (Figs. 1c and 1d).Although true resolution down to the nanometer scaleis achieved by means of monopole antenna probes,enhancement of the electromagnetic field is only mod-est and tightly coupled to the small dimensions to theantenna, which imposes high demands on the fabrica-tion of these nanostructures.

Bowtie nanoaperture antennas (BNA) carved at theend facet of tapered aluminum-coated optical fibersprovide a more robust and alternative design tomonopole antennas. These nanostructures consist oftwo triangle openings faced tip-to-tip and separated bya small opening gap providing a superconfined spotwith an intense local field and broadband response inthe visible regime (Guo et al., 2010). Moreover, theeffective confinement region of BNAs can be readilytuned by controlling the excitation polarization (Guo

et al., 2010; Mivelle et al., 2012). BNAs have been suc-cessfully used as nanometer-sized light sources fornanolithography (Wang et al., 2006) and as highthroughput near-field probes (Mivelle et al., 2010;Onuta et al., 2007). Recently, we showed single-molecule nanoimaging using a BNA scanning probeand revealed the full three-dimensional (3D) vectorialcomponents of the optical near field of BNAs usingindividual molecules as nanoscale optical sensors (Fig.2) (Mivelle et al., 2012). Furthermore, direct compari-son of the response upon confocal and BNA probe exci-tation for each individual molecule alloweddetermination of the field enhancement provided byBNA probes. Our results showed an approximately six-fold enhancement on the fluorescence emission of indi-vidual molecules when the BNA is properly excitedand aligned to the dipole emitter. In addition, fabrica-tion of BNA probes on tapered optical fibers near thecut-off region provides about three orders of magni-tude higher throughput than circular aperture probesof similar dimensions, making these bright nanostruc-tures ideal candidates for a large number of highlysensitive applications, including biosensing, spectros-copy, and nanoimaging of biological samples.

NEAR-FIELD APPROACHES AND OPTICALANTENNAS FOR NANOSCALE DYNAMICS IN

LIVING CELL MEMBRANES

One of the great advantages of light microscopy isthe possibility to image living cells. However, the diffu-sion of most proteins and lipids on the cell membraneoccurs in the millisecond time scale, posing a challengeto most optical imaging techniques. As a result single-molecule fluorescence approaches that provide suffi-cient time resolution do not rely on imaging but ratheron following a subset of labeled molecules as they lat-erally diffuse on the cell surface, as used in single-particle tracking (SPT) (Kusumi et al., 2005; Saxtonand Jacobson, 1997). Unfortunately, because mem-brane lipids and some protein components such as

Fig. 2. Single-molecule nanoimaging using a bowtie antenna probe.a: Bowtie nanoaperture carved at the apex of a metal-coated taperedoptical fiber. The gap region is 50 nm. The antenna is excited throughthe NSOM probe and the fluorescence emission collected in the farfield using a polarization-detection scheme. b: Single-molecule pat-terns as obtained from imaging individual molecules using the bowtieantenna excited with an field polarized along the antenna arms (seeinset). The color-coding on the figure indicates the detected polariza-tion emission of individual molecules (from green to red according to

the in-plane orientation, from x to y, respectively). Notice that theindividual fluorescence patterns have different shapes, resultingfrom the 3D near-field components of the field emanating from thebowtie (Mivelle et al., 2012). c: Intensity patterns and cross-sectionsof two fluorescence features as encircled in (b). The red pattern corre-sponds to an in-plane molecule while the two-lobe pattern corre-sponds to a z-oriented molecule. The optical resolution is �80 nm.Adapted from Mivelle et al. (2012).

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glycosylphosphatidylinositol-anchored proteins canundergo very rapid diffusion, it is challenging to followtheir motion using camera-based SPT approaches(Fujiwara et al., 2002). An alternative approach thatprovides higher temporal resolution is FCS. Themethod relies on parking the illumination spot on aspecific region of the sample. Intensity fluctuationsresulting from the diffusion of individual componentstransiting the illumination volume are recorded andautocorrelated in time to provide information on theirdiffusion behavior (Kim et al., 2007). Although confocalFCS provides the temporal resolution needed toresolve fast dynamic events, the large illumination vol-ume of the diffraction-limited confocal spot hides diffu-sion heterogeneities taking place at the nanoscopiclength scale (He and Marguet, 2011). Recently, STEDnanoscopy has been combined with FCS to detectnanoscopic anomalous diffusion, such as of single lipidor protein molecules in the plasma membrane of livingcells. Combining a (tunable) resolution down to �20nm with FCS, Eggeling et al. (2009) showed thatsphingolipids or “raft”-associated proteins were transi-ently (�10 ms) trapped at the nanoscale in cholesterol-mediated molecular complexes. Indeed, one of themajor advantages of STED is that it allows to vary theeffective illumination area by changing the power ofthe STED depletion beam, providing easy access to dif-fusion coefficients for different observation areas (Egg-eling et al., 2009; Ringemann et al., 2009). However,STED-FCS suffers from several drawbacks thatrequire further technological developments before fullimplementation in living cells, including the highpower density of the STED depletion beam (10–100MW/cm2), the difficulty to extend the technique to mul-tiple colors, and the still diffraction-limited axial reso-lution that contributes to residual background inparticular for 3D applications. The latter can be some-what corrected by the use of pinholes, albeit at the costof lower signal-to-noise ratios (Ringemann et al.,2009).

These limitations can in principle be all overcome bythe use of metallic nanostructures, either fabricated onglass substrates as 2D arrays, or at the apex of NSOMprobes. 2D arrays rely on the fabrication of subwave-length apertures in a metal film deposited on a glasssubstrate (called ZMW). The utility of 2D subwave-length apertures in live cell membrane research hasbeen demonstrated using different aperture sizesuncovering a relationship between the transient timesof the molecules traversing the illumination volumeand the illumination area (Wenger et al., 2007; Wawre-zinieck et al., 2005). Using a similar approach, it wasalso shown that gangliosides partition in compart-ments of 60 nm in size consistent with their associa-tion in nanoscale lipid raft platforms (Wenger et al.,2007). More recently, ZMW were used to determine thesubunit stoichiometry of the pentameric neuronal nico-tinic acetylcholine receptors on living cell membranes(Richards et al., 2012). Unfortunately, despite its greatpotential for live cell membrane investigations, 2Dsubwavelength apertures do not in general, exhibitoptical enhancement, imposing a size limit of about50–70 nm to the useful structures that can be used forFCS-type of applications. Moreover, an important limi-tation of this method is directly related to the need for

cell membranes to adhere to the substrate, causingmembrane invaginations, curvature effects and/orunspecific adhesion of the membrane close to the aper-ture edges (Samiee et al., 2006). To overcome theselimitations, planarized apertures of 50 nm in diameterhave been recently developed by means of filling themetal apertures with silicon oxide and used to recordFCS data on supported lipid and plasma membraneswithout penetration of the sample into the aperture(Kelly et al., 2011).

An alternative way to fully eliminate membraneinteractions with the nanostructures is by using self-standing apertures in combination with a NSOMapproach. In this configuration, the nanoaperture isheld stationary above the sample surface (with a dis-tance separation of about 10 nm so that no interactionwith the membrane occurs) and intensity fluctuationsarising from the diffusing molecules are recorded inthe far field using conventional optics. The first dem-onstration of NSOM-FCS was performed in 2008 byVobornik et al. by measuring the mobility of fluores-cent lipids in supported lipid bilayers (Vobornik et al.,2008) and then followed by Naber’s group determiningthe diffusion of ligands transported axially throughsingle nuclear pore complexes (Herrmann et al., 2009).Recently, we demonstrated for the first time the feasi-bility of performing NSOM-FCS on living cells by sta-bly maintaining the NSOM probe in close proximity tothe sample, obtaining vertical distance fluctuationsbelow 1 nm (Manzo et al., 2011). We used this configu-ration to measure the diffusion of different lipids onliving CHO cells (Figs. 3a and 3b). While the lipid ana-log phosphatidyl-ethanolamine (PE) showed free,Brownian diffusion on the cell membrane, with transittimes that linearly scaled with the illumination area(Fig. 3c), the sphingolipid SM showed a clear deviationfrom Brownian diffusion when the illumination areawas reduced from confocal to NSOM (Fig. 3d), consist-ent with cholesterol-induced lateral confinement (Egg-eling et al., 2009).

There is a large potential for the use of NSOM-FCSsince it combines membrane (proximal) specificity andhigh signal-to-noise ratios. The limitations of conven-tional NSOM probes in terms of light throughput canbe easily overcome by the use of antenna probes, usinggeometries such as bowties. Indeed, as we alreadyshowed, BNA probes provide 3–4 orders of magnitudelarger signal comparable to conventional NSOM aper-tures of the same size (Mivelle et al., 2012). Moreover,BNAs have broad-band emission allowing for multi-color excitation and dual-color cross-correlation spec-troscopy in ultrasmall volumes. These devices in thefuture could be used to provide exquisite informationon the coupling between the inner-outer leaflets of themembrane, signaling complex formation and the inti-mate relation between membrane nanodomains andthe actin cytoskeleton.

It should be also mentioned that 2D antenna config-urations bear similar potential for studies on livingcell membranes. So far, most efforts are being devotedto the fabrication of large arrays of antennas (neces-sary for inspection of multiple cells on a single sub-strate) and how to overcome unwanted interactionsbetween the immediate cell membrane and the metalnanostructures. Recently, a combination of colloidal



chemistry together with plasma processing has beendeveloped to fabricate millions of bowtie antennas on asingle substrate (Lohmu€uller et al., 2012). First proof-of-principle experiments were performed using modellipid bilayers and showing unhindered diffusion ofindividual proteins embedded in the bilayer. Althoughmany technological challenges still lie ahead, it is clearthat the field is moving forward driven by the excitingand unique possibilities offered by optical antennas.

OPTICAL ANTENNAS FOR IN VITRO SINGLE-BIOMOLECULE DETECTION

As already mentioned, subwavelength apertures onmetallic films have been successfully used for single-molecule detection in solution at 10 mM sample concen-trations (Levene et al., 2003). Impressive in vitro stud-ies include high throughput fluorescence-based DNAsequencing (Eid et al., 2009), protein–protein interac-tions at 5 mM concentrations (Miyake et al., 2008) andstudies on the bacterial translation machinery(Uemura et al., 2010).

Because of the strong spatial confinement and fieldenhancement afforded by optical antennas, these devi-ces are starting to emerge as superior alternatives forsingle-biomolecule detection at high concentrations. In2009, Moerner and colleagues demonstrated fluores-cence intensity enhancement of more that three ordersof magnitude using gold-bowtie antennas (Kinkhab-wala et al., 2009). This large enhancement resultedfrom an effective increase in the excitation field on thegap region of the bowtie (10 nm gap) by two orders ofmagnitude, in combination with an enhancement ofthe quantum yield of the emitter by a factor of 10(Kinkhabwala et al., 2009). Yet, in order to achievesuch signal enhancements, molecules have to be pre-

cisely positioned in the gap region of the antenna,imposing several practical constrains and restrictingthe utility of antennas for broad applications in life sci-ences. To circumvent this limitation, an extremelyattractive strategy has been recently reported by Tin-nefeld and coworkers (Acuna et al., 2012). Theapproach used DNA-based self-assemble structures(also known as DNA origamis) as scaffold on whichtwo gold nanoparticles were placed to form dimerantennas. The main advantage of this method is thatthe structure offers handles to place the biomolecule ofinterest at the right position within the hotspot of theantenna (Acuna et al., 2012). Using this concept, fluo-rescence enhancement of more than two orders of mag-nitude was demonstrated on gap antennas of 23 nm insize, allowing at the same time single-molecule meas-urements at 500 nM.

Recently, we introduce an “nanoantenna-in-box”platform especially designed for enhanced single-molecule analysis in solutions at high concentrations.The design consists of a nanoantenna dimer formed bytwo gold-hemispheres placed in a rectangular nanoa-perture (Fig. 4) (Punj et al., 2013). The rationalebehind this design is that in any nanoantenna experi-ment on molecules in solution, the observed fluores-cence signal is a sum of two contributions: theenhanced fluorescence from the few molecules in thenanoantenna gap region (hotspot) and a fluorescencebackground from several thousands of moleculeswithin the diffraction-limited confocal volume. Assuch, the different components of the “antenna-in-box”have complementary roles: a central gap-antenna cre-ates the hotspot for enhancement, while the surround-ing nanoaperture screens the background bypreventing direct excitation of molecules diffusing

Fig. 3. NSOM-based fluorescence correlation spectroscopy in livingcells. a: In FCS mode, the NSOM probe is kept stationary over themembrane of the living cell so that only molecules that laterally dif-fuse through the small illumination area of NSOM are excited. b:Schematics showing the comparison between confocal (left) andNSOM (right) illumination areas. Green denotes the illuminationarea in both cases, while the black curves illustrate the diffusion ofindividual molecules experiencing transient arrest and heterogeneity

in their mobility. c: Correlation curves as function of lag time asobtained from the lipid PE for different illumination conditions: con-focal, and NSOM probes of 180 nm and 120 nm in diameter. d: Corre-lation curves as function of lag time for SM, obtained with confocaland with an NSOM probe of 120 nm in diameter. Symbols in (c) and(d) correspond to experimental data, while the lines are fittings to thecurves using anomalous behavior (Manzo et al., 2011). Reproducedfrom Dufrene and Garcia-Parajo (2012).

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away from the central gap region. This configurationmaximizes the signal-to-background discrimination bysingling out the fluorescence signal from the hotspotwhile several thousands of non-excited molecules arepresent in the confocal volume. Using this approach,we reached fluorescence enhancement values up to1,100-fold (Punj et al., 2013). Furthermore, we moni-tored the diffusion of different individual biomolecules(DNA and proteins) in detection volumes in the zeptoli-ter range, corresponding to single-molecule detectionat concentrations higher than 15 mM (Fig. 4). The com-bined huge fluorescence enhancement and ultrasmalldetection volume renders these type of optical antennadevices ideal for the design of massively parallel sens-ing platforms for single-biomolecule analysis at micro-molar concentrations.

CONCLUSIONS AND PERSPECTIVES

In this review, we have highlighted first excitingresults where optical antennas have been alreadyextended to biological applications both at the level ofnanoimaging and detection of dynamic events in vitroand in living cells. Concepts from FCS implemented inultraconfined volumes as afforded by optical antennasallow nowadays measurements of ultrafast dynamicsat the nanoscale in living cell membranes as well as

detection of individual molecules at the mM range.While these exciting results convincingly demonstratethe potential of these nanostructures, they also showimportant technological challenges that need improve-ment before their routine application in life sciences.While current antenna designs (either self-standing or2D) are ideally suited for cell membrane investigation,inner-cellular structures are more difficult to assessusing these geometries. Recent exciting developmentsaiming at the fabrication of 3D nanostructures will inprinciple have the potential of detecting single mole-cules events inside living cells and/or in real 3D envi-ronments (de Angelis et al., 2011, 2013).

Self-standing optical antennas require the combina-tion of a scanning probe configuration (either NSOMor AFM) for manipulating and positioning the antennaclose to the sample. On the positive side, the methodprovides the flexibility of placing the antenna at thedesire location for maximum coupling to the fluores-cent emitter, while avoiding direct unwanted interac-tions with the sample. However, these antennaconfigurations are commonly placed at the apex oftapered optical fibers, which are fragile and requirehigh technical skills for probe manipulation. Besides,accurate control of the tip-to-sample separation isimperative in order to prevent unwanted interactions

Fig. 4. Antenna-in-box for single-molecule detection at mM concen-trations. a: Scheme of the antenna-in-box configuration. The dimerantenna is fabricated inside of a nanoaperture square carved in agold-film deposited on a glass substrate. b: Scanning electron micros-copy (SEM) image of the fabricated antenna-in-box. The gap region ofthe dimer antenna is �19 nm. c: Detection volume and concentrationfor which there is, on average, an individual molecule in the nanoan-

tenna detection volume. d: Normalized fluorescence correlation func-tions measured on an antenna-in-box of 15 nm gap size, withexcitation polarization parallel to the antenna axis. The samples areAlexa Fluor 647-free dye (red), Annexin 5b (orange), 51 bp doublestranded DNA (green), and protein A (blue). The points correspond toexperimental data and the solid lines to a numerical fit. Adaptedfrom Punj et al. (2013).



between antennas and the membrane. Finally, illumi-nation spot size variation as conveniently afforded bySTED (Eggeling et al., 2009; Ringemann et al., 2009)is significantly more tedious to implement withantenna probes since it requires the exchange of multi-ple antennas with different sizes. 2D optical antennasare on the other hand much easier to handle, and theycan be made of different sizes on the same substrate.However, they are less flexible in terms of coupling theantenna to the fluorescent dye. Moreover, unwantedeffects due to physicochemical interactions of the fluo-rescent molecules (for in vitro studies) or the cell mem-brane (for in vivo applications) with the antennascomplicate the analysis of the data and the throughputof successful experiments. While for in vitro studiessmall arrays of antennas might be sufficient, live cellresearch imperatively requires the use of large anten-nas arrays. Current efforts are therefore focused onimplementing techniques that allow the fabrication oflow-cost, large-throughput and highly reproducible(down to the nm scale) antenna arrays. Driven by theirenormous potential, we expect that current technologi-cal obstacles will be soon overcome and that biology-compatible antenna geometries will be readily avail-able to the biological community in the coming years.

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