Nonfluorescent Optical Probing of Single Molecules and

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Nonfluorescent Optical Probing of Single Molecules and Nanoparticles Thomas Jollans, Martin Dieter Baaske, and Michel Orrit J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.9b00843 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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The Journal of Physical Chemistry

Nonfluorescent Optical Probing of Single Molecules and Nanoparticles Thomas Jollans, Martin D. Baaske, and Michel Orrit∗ Huygens–Kamerlingh Onnes Laboratory, Leiden University, Postbus 9504, 2300 RA Leiden, The Netherlands E-mail: [email protected]

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Abstract Notwithstanding its unique power for imaging and investigation of transparent condensed and biological matter, fluorescence presents severe limitations: it requires special fluorescent labels, which are prone to photobleaching, and the photon streams it provides are relatively weak. In the past 10 to 20 years nonfluorescent optical methods have appeared, which can also provide information on matter at the nanoscale, while presenting different limitations. In the present paper, we review some of these methods, with special emphasis on work from our group. We consider mostly the optical detection and study of single immobilized or transiently bound molecules and nanoparticles through their scattering, the heat they dissipate in the environment upon light absorption, or their coupling to auxiliary optical resonators such as whispering-gallery modes.

Introduction Optical micro-spectroscopy remains one of the most direct and powerful methods to investigate matter at nanometer scales, to explore and clarify the molecular structure, dynamics and processes at work in physical chemistry, materials science, or bioscience. Much of the current knowledge of molecular materials at the nanoscale has been garnered through the fluorescence of suitable labels, dispersed in, or attached to, the structures of interest. Fluorescence is the workhorse technique in cellular biology. Its exquisite sensitivity reaches down to single-molecule detection 1 and enables super-resolution microscopy. 2 Single-molecule methods enable exploration of molecular dynamics, free from time- and ensemble-averaging. 3 For all their success, however, fluorescence techniques face limitations: they require labeling of the targeted molecules, which may alter their interactions and functions; fluorescence signals are restricted in intensity and detection rate, as well as in spectral selectivity, which directly restricts the number of different labels observable in the same sample. The urge to overcome the restrictions of fluorescence and to directly observe unaltered molecules in action has led 2


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several groups to explore fluorescence-free optical methods, which we briefly review in the present article. Although they face limitations of their own, these fluorescence-free methods cannot or do not have to rely on photon-counting detectors. Therefore, they potentially provide higher light intensities and larger detection bandwidths than fluorescence. Optical interaction of light with matter can give rise to non-optical signals (e.g., a photovoltaic current) that we don’t consider here, or to optical signals. Because it changes the color of photons, fluorescence is easy to isolate from narrow-band (laser) excitation with suitable spectral filters. However, other properties of light may also be modified by interaction with matter, such as the polarization or the propagation wavefront. Processes affecting the latter are generally referred to as scattering. One usually distinguishes scattering processes according to the time-dependence of the scattering potential. Truly static defects lead to truly elastic scattering, i.e., scattering without any change of frequency. Elastic scattering should thus be distinguished from Rayleigh scattering in fluids 4 , where density fluctuations are never truly static, but relax on viscosity-dependent time scales. Acoustic waves in solid or fluid matter lead to Brillouin scattering lines, whereas higher-frequency vibrations lead to Raman scattering. As Brillouin and Raman scattering (the surface-enhanced variant 5 excepted) are usually too weak to detect single small objects, we will focus on elastic and Rayleigh scattering here, i.e. scattering without a significant frequency (or color) change. The optical theorem 6 states that interference of the scattered wave with the incident wave is responsible for the extinction signal, i.e., for a loss of intensity in the incident wave after it has interacted with the object of interest. For single nanoscale objects, the changes they induce, seen either in scattering or extinction, are extremely weak in relative values. Their observation thus requires very large numbers of detected photons to overcome shot noise. But, because the intensity that a nanoparticle or a molecule can withstand is limited, naïve measurements of scattering or extinction are rendered impractical by the extremely long integration times they require; more subtle techniques are needed. Essentially, two strategies have been developed to solve this problem, depending on

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whether the objects to be detected absorb or are purely dielectric. In the first case, one uses a probe beam to optically detect a consequence of absorption that vanishes in the absence of absorption, for example heat dissipated by the particle upon light absorption of a heating beam with another color. This method is called photothermal microscopy. 7–15 The change of intensity of the scattered probe beam is thus used as proxy for absorption of the heating beam. The other strategy, applicable to non-absorbing as well as absorbing objects, is to improve noise rejection in direct scattering (i.e., with a single probe beam), which is only possible with bright-field scattering on realistic samples in condensed matter. This technique has recently been popularized under the name iScat but has been implemented in different guises with various optical designs 16–19 . Initial experiments at low temperatures 16 benefited from the resonance enhancement of the scattering cross section and have been improved significantly in recent years 20 . However, the difficulty of measuring scattering in cryogenic environments has limited their applications so far. Alternatively, optical resonators can be used as efficient detectors for small objects: whispering gallery modes 21,22 or plasmonic structures 23 can be used alone or in combination 24–26 to detect and study small objects. In the present article, we start with a short discussion of the signal-to-noise ratio in bright-field scattering, and discuss some of the most usual methods: photothermal microscopy, interferometric methods in microscopy, coupling to plasmonic or all-dielectric cavities. Although we focus on immobilized particles, these methods can also be applied to diffusing particles and to on-the-fly measurements 27 .

Signal, Background and Noise in Scattering Experiments We consider the optical detection of a small object, which we call the scatterer and which we will often approximate as a point electric dipole. In the most general scheme, we illuminate this object and look at the field Esc it scatters. Together with the scattered field, we will also collect at least a part of the incident field, which we call Eref . We then measure an intensity

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Im that results from a superposition of these two fields: 2

2

Im ∝ |Eref + Esc |2 = E ref + E sc + 2E ref E sc cos φ,

(1)

where the bars indicate the (positive) amplitudes of the fields. We have assumed both fields to be spatially matched (see below), and φ is a phase angle between the reference and scattered field, which depends on the geometry of the experiment, on the position of the scatterer, and on the frequency of the excitation laser. The simple expression in eq. (1) allows us to discuss fundamental issues of signal, background, and noise. We first assume that detection noise is limited to photon or shot noise, i.e. the number fluctuation of detection events in an ideal photon-counting detection chain. In this case, we immediately see from eq. (1) that, without scatterer, the background intensity 2

Bm = E ref gives rise to noise that scales as the square root of the detected intensity, i.e., as E ref . The signal, which we identify as the change of detected intensity when we illuminate 2

the scatterer, Sm = E sc + 2E ref E sc cos φ, changes in a more complex way depending on the interference between reference and scattered fields: 2

(i) For very weak scatterers, the scattered intensity E sc can often be neglected. Therefore, for an ideal detection, both the signal and the background noise scale as E ref : the signalto-noise ratio is independent of the reference field. In other words, the signal-to-noise ratio cannot be improved by increasing (or decreasing) the amplitude of the reference field. Of course, in a practical experiment, other noise sources must be considered, and the amplitude of the reference field is a crucial parameter to optimize, as briefly outlined below. (ii) If the reference field can be eliminated altogether, the signal-to-noise ratio can in principle become arbitrarily large. This is the case for fluorescence, where a spectral detection filter completely suppresses the illumination light, while transmitting a fluorescence signal proportional to the absorbed intensity. In that case, the background and the noise associated with it disappear completely, and the fluorescence signal is detected on (ideally) a

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dark background. The same argument would seem to apply to dark-field scattering. Unfortunately, as the intensity scattered by a single molecule or nanoparticle is exceedingly small, even weak background will easily overcome it. Such background is caused by Rayleigh and Brillouin scattering by the matrix surrounding the scatterer, by defects of the sample and of the optical components, or by stray reflections at the surfaces of the setup’s lenses and mirrors. We now consider deviations from the ideal scheme above: (i) Noise sources: in a simple optical detection experiment, noise arises not only from quantum fluctuations of the optical signal itself, characterized by its noise equivalent power 28 √ σph = 2BP hν, B being the detection bandwidth, P the detected power and hν the photon energy. Important additional noise sources are power fluctuations of the excitation laser (laser noise σlaser ), detection noise arising either from the detection electronics (dark noise σdark ) or excess noise from the electron avalanche often involved in the amplification process √ for photon-counting devices. 29 This latter is given by σexc = F − 1·σph , F being an effective factor describing the total noise created by photon noise and excess avalanche noise of the 2 2 = F · 2BP hν). Excess noise is usually negligible for analog + σexc detector 30 , so that σph

detectors which do not rely on avalanches to amplify the signal. (ii) Mode matching: eq. (1) supposes that the reference and scattered fields interfere at a given point in space. However, in real experiments, the fields are integrated over a significant part of the wave front in order to optimize the signal collected from a small object. Interference then takes place between extended wave fronts which in general do not coincide, e.g. the spherical wave scattered by a point dipole and an incident Gaussian reference beam. 31 The overlap of the two interfering fields introduces a mode-matching factor which reduces the amplitude of the interference term. Noting the field distributions of each wave in the detector plane as Eref (x, y) = E ref Fref (x, y) and Esc (x, y) = E sc Fsc (x, y), where Fref and Fsc are square-normalized functions bearing the spatial dependence of the fields, we

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find a complex mode overlap factor ZZ α=

∗ Fref (x, y)Fsc (x, y)dxdy,

(2)

so that the intensity becomes ZZ Im =

2

2

|Eref + Esc |2 dxdy = E ref + E sc + 2E ref E sc |α| cos φ.

(3)

The amplitude |α| ≤ 1 reduces the interference term, whereas the argument of the complex overlap factor may modify the angle φ. In conclusion, to optimize the signal-to-noise ratio in scattering experiments, different strategies must be pursued simultaneously to reduce fundamental and experimental noise sources. To reduce statistical fluctuations of the number of detected photons, one should apply the highest intensities and integration times permitted by the photostability of the objects under study. Laser power fluctuations can be removed to a large extent by optical subtraction, either in an interference setup 18,32 or by clever design of the optical path 19 . The detected intensity resulting from the interference of reference and scattered fields must be adapted to the best regime of the detector. This can be done in different ways, for example in reflection by careful index-matching at the glass-sample interface 12 or in transmission by interferometry 18 . Finally, the interference between reference and scattered fields should be improved by matching the two modes as much as possible, for example by introducing a weakly transmitting mirror (fig. 3.1) or mask to reduce the reference field. 33,34

Photothermal Techniques One of the main challenges in any scattering-based detection technique is the background created by stray scatterers such as dust, impurities in microscope oil, or the environment the measurement is being performed in. This problem of selectivity can be particularly daunting

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in an intrinsically scattering environment such as a cell. sample

AOM heating laser

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Figure 1: (a) simplified scheme of a photothermal microscope. (b) cartoon representation of thermal lens creation in a photothermal microscope’s focus. Photothermal microscopy, a highly selective technique for detecting objects which absorb light of a chosen wavelength or set of wavelengths, relies on an indirect measurement. Here, we focus on the all-optical scattering-based variant. 7 Closely related to a large, wellestablished family of photothermal and photoacoustic techniques, 15,35–39 it uses a two-color approach to detect and localize absorbers: A resonant heating laser beam is absorbed by an object in the sample, which transforms a significant part of the absorbed energy into heat. The heat diffuses out of the particle and into the surrounding medium, where it establishes a localized, time-dependent temperature gradient and a corresponding refractive index gradient. This local refractive index gradient acts as a (thermal) lens and can be detected through the scattering of a second (probe) laser beam 7,11,13,40 (see fig. 1). Therefore, the method is closely related to the scattering signal discussed above. The position of the probe focus with respect to the thermal lens influences the angle φ, and should be varied to optimize the photothermal difference signal. Whereas the heating intensity is limited by photodamage and/or saturation of the absorber, the scattered probe signal depends only on a broadband refractive index change. Therefore, the probe wavelength can be judiciously chosen out of the spectral absorption range of the absorber to minimize damage and saturation by the probe, thereby allowing for high probe intensities and accordingly low photon noise. 11 8


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To discriminate the signal from background scattering, photothermal and related techniques modulate the heating beam at a certain frequency, generally in excess of 100 kHz. The corresponding oscillating component of the scattered probe beam is then extracted to recover the actual signal—generally with a lock-in amplifier. 7 The technique is sensitive to absorbing objects only, and extremely robust in the face of even substantial non-absorbing scatterers. This makes noble-metal nanoparticles, which are only weakly luminescent but strongly absorbent, ideal contrast agents even in noisy biological environments , 10,41 though other contrast agents have been proposed 42 and label-free live-cell photothermal imaging has been demonstrated . 43 In very carefully optimized experiments using judiciously chosen media and samples, absorption measurements down to single molecules 12,44 have been

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Figure 2: (a) Photothermal signal-to-noise ratio as a function of the incident probe power measured for 20 nm gold NPs in water on glass. (SNR ∝ P 1/2 ) (b) Photothermal SNR measured for 20 nm NPs in different fluids as a function of calculated photothermal strength for these fluids, ΣPT = n|∂n/∂T |Cp−1 , scaled with respect to glycerol. (c) Normalized histograms of SNR for 20 nm gold NPs in glycerol: (dark grey) deposited on glass; (light grey) deposited on a 100 nm thick thermal isolation layer of PMMA—figure from Gaiduk et al. 11

The signal–to–noise ratio achievable in a photothermal measurement depends not only on optical considerations, but also on the choice of modulation frequency and environment. Tuning the frequency allows one, ideally, to limit ubiquitous 1/f noise while matching the thermal diffusion length to the optical probe volume. Adapting the environment, both medium and substrate, allows one to affect the strength of the refractive index gradient through ∂n/∂T and limit invisible heat loss into the substrate (see fig. 2). Moving away 9



from “conventional” media, ∂n/∂T and the SNR can be increased dramatically by working near the critical point of e.g. xenon or carbon dioxide, where most material properties (including the refractive index) vary violently with the thermodynamic variables of state. There, detection of dissipated powers ∼ 64 pW has been demonstrated, nearly two orders of magnitude better than the ∼ 3 nW seen in glycerol. 45

Interferometric Methods Interference-based scattering microscopy (iScat, see fig. 3) utilizes the interference between the field scattered by an analyte and the field reflected by the glass slide (eq. (1), final term). The interference term scales linearly with the volume of the analyte V and the magnitude of the reflected field. In comparison to dark-field methods, which only detect the scattered intensity Es2 ∝ V 2 , this technique yields improved contrast for small analytes. This method has enabled the spectroscopy of single gold nanoparticles with radii down to 10 nm 17 and the extinction-based imaging of a single quantum dot. 19 It was further successfully used in conjunction with single-molecule fluorescence microscopy to track translational and rotational motion of single virus particles (Simian Virus 40). 46 Moreover, iScat has enabled the detection of single proteins as small as Bovine Serum Albumin (BSA, molecular mass: 65 kDa) binding to a cover slide surface and their localization with 5 nm accuracy. 47 The phase factor cos φ of the interference term also depends on the distance between the scatterer and the interface on which the reference reflection occurs. This can be used for three-dimensional tracking of nanoparticles. The motion patterns obtained that way not only allow for determining the potential of electrostatic traps 48 they also provide a new means to access and investigate dynamic biological processes like the movement of single proteins during diffusion in the plasma membrane, transport along filopodia and endocytosis. 49 Instead of backward scattering, coherent forward scattering can also be used for tracking single virus particles in three dimensions. 50

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The imaging contrast that can be obtained via iScat is technically limited by the dynamic range of the camera used to record the images. This, however, can be mitigated by artificially reducing the collection efficiency for the reflected field, while enabling high throughput of the scattered field by placing transmission/reflection masks in the infinity conjugated path of the microscope to spatially filter the respective fields (fig. 3.1). 33,34 Moreover, the method has enabled the observation of microtubule motion, 51 the imaging of single proteins secreted from a single cell 52 and the monitoring of nanoparticle-labelled lipids diffusing inside lipid membranes spanned over pores. 53 Furthermore, it has been used to track nanoparticles on the membrane of live cells 54 and for quantitative determination of single-molecule mass (see fig 3.2). 55 While using the reflected field as reference provides a common path for the interfering fields and thus makes for a more stable device—it is not intrinsically necessary. Using a Michelson type configuration to provide for the interference allows one to control the intensity and polarization state of the reference field, enabling the detection of nanoparticles moving through nanoscale fluidic channels. 18,56,57

Cavity-Based Techniques Another kind of optical tool that enables the label-free observation of nanoscopic entities is represented by optical microcavities. A specific family of microresonators, which relies on the confinement of light via total internal reflection (TIR) on the outer interface of an axially symmetric cavity are the so-called Whispering Gallery Mode (WGM) resonators. 21,22 Named after a similar acoustic phenomenon observed in the whispering gallery at St Paul’s Cathedral in London, these devices are able to confine light in micrometer-sized mode volumes for extensive periods of time, ranging from nano- to microseconds (i.e. 106 -109 optical oscillations). An electromagnetic wave undergoing TIR exhibits an evanescent tail that extends into the lower refractive index medium beyond the interface, consequently making

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1

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Figure 3: Interferometric scattering microscopy. 1, adapted from Cole et al. 33 : (1b) setup of a typical iScat microscope; incident beam, focused into the back focal plane of the objective, and the reflection on the sample slide in darker blue, optical path of light scattered by the analytes in light blue. In this case the setup contains a partial reflector (PR) which couples the light into the microscope and attenuates the reflected beam while transmitting most of the scattered field in order to optimize the field overlap factor α and with it the imaging contrast. (1a, 1c) Working principle: spatial intensity distribution of the scattered light in the back focal plane. The position of the PR is represented by the circle/gray area. 2, adapted from Young et al. 55 : iScat images that allow determination of the molecular mass of scattering analytes. (2A) Schematic of the experiment indicating the adsorption of analyte molecules with different mass. (2B) iScat image of BSA (Bovine Serum Albumin) molecules adsorbing to a glass slide. (2C) Images showing the contrast increasing for BSA oligomers consisting of one to four molecules. The corresponding occurrence statistics are represented in the top panel of (2D) alongside the respective scatter plots (bottom).

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the resonators susceptible to refractive index perturbations in the surrounding medium. As the light field probes the surrounding medium repeatedly during successive cavity round trips even minute perturbations by nanoscopic entities can be detected as changes in the cavity’s resonance spectrum. There, the excess polarizability of the entity with respect to the surrounding medium gives rise to a shift in the WGM’s resonance frequency, 58 allowing for the detection of nanoparticles and viruses. 59,60 In addition, losses caused via scattering and absorption of light by the entity can be observed as broadening of the WGM’s resonance. 61–64 Precise quantification of single analyte properties, however, requires the accurate determination of its position as linewidth broadening and frequency shift depend on the analyte’s location within the WGM’s field distribution. The necessary information can be obtained by scanning the cavity with a probe beam and observation of the cavity’s photothermal response 24,65 or via simultaneous frequency tracking of multiple non-degenerate WGM’s with different polar mode numbers 66,67 . A WGM-based method that does not require this information relies on analyte-induced mode splitting: Light scattered by nanoparticles is efficiently coupled into the counter-propagating cavity mode. This leads to the formation of two standing wave modes with either their intensity node or anti-node at the scatterer’s location and thus to distinct frequencies and linewidths. 61,68–70 The ratio of the differences in frequency and the differences in linewidth between the split modes is independent of the local field strength and thus allows for the direct determination the analyte’s polarizability. 71,72 A recent study shows that further sensitivity enhancements, especially for perturbations by small scatterers, are possible using exceptional points. 74 Mode-splitting-based sensing, however, goes hand in hand with the highest demands regarding cavity Q factors, and suggests the choice of active/lasing microcavities as sensors, to compensate for nanoparticleinduced losses via optical gain. 74–77 The frequency shift a WGM undergoes when perturbed by an analyte scales with the electric field intensity at the location of the analyte and with the analyte’s polarizability. Consequently, the near-field enhancement provided by plasmonic nanoparticles resonantly

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Figure 4: Single-molecule detection with plasmonically-enhanced optical microcavities. (a) Typical layout of a prism-coupled WGM single-molecule sensor (adapted from Baaske et al. 63 ). Light from a tunable laser is evanescently coupled into a microsphere cavity via total internal reflection on the prism’s surface. The microsphere cavity is modified by adsorption of gold nanorods on its surface. (b) Typical WGM transmission spectra exhibiting Lorentzian dips where the wavelength of the tunable excitation laser matches a WGM resonance (red: unperturbed state, blue: the resonance shifted upon perturbation). (c) Sketch illustrating transient (left) and the permanent interaction (right) of an analyte molecule with receptors molecules on the surface of gold nanorods inside a WGM’s evanescent field. The wavelength traces displayed in (d) and (e) show typical wavelength shift patterns (spikes and steps) associated with transient and permanent analyte–receptor interactions, respectively (adapted from Kim et al. 73 ). excited by WGMs has allowed boosting WGM sensitivity to a level at which single molecules (see fig. 4) can be resolved. 63,73,78,79 This increase, however, comes at the cost of a strongly decreased sensing volume, now limited to the volume where the field strength around the plasmonic NP is highest (≈ 104 nm3 ). This imposes severe restrictions on the total number of molecules detectable by assays of the one-way-binding type. Modification of the NPs with chemical agents that only allow the analyte of interest to bind is necessary in order to ensure detection specificity. Consequently, significant statistics of single-molecule events can only be obtained if transient, highly specific interactions between the target and receptor molecules (tethered to the plasmonic NP) are monitored, 63 which also enables strong positive and negative controls. Nonetheless plasmonically-boosted WGM sensors can be used to monitor single-molecule surface modification processes and enzyme conformational changes associated with single-molecule interactions in real time and over a wide range of environmental conditions. 73,79 WGM-based recognition of single Zinc and Mercury ions interacting 14


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with gold nanorods has been reported, 80 however, the precise physical mechanism underlying this observation has yet to be determined. Recently, the capacity of WGM-based resonators to act as single-particle photothermal spectrometers has been demonstrated 24,65,81 : Heat dissipated by single nanoparticles upon absorption of a probe beam locally heats a microcavity, altering its refractive index profile and thus the WGM’s frequency. At wavelengths where the probe wavelength coincides with a WGM resonance, the photothermal absorption spectrum exhibits distinct Fano patterns on top of the particle’s absorption line. The shape of each individual Fano pattern reflects the frequency detuning of the nanoparticle’s resonance with respect to the resonance of the corresponding WGM.

Plasmonics In a perfect conductor, charge carriers would respond to any electric field instantly. In real metals, this clearly can’t be the case, not least because a frequency-independent response would be inconsistent with, among others, energy thresholds in the photoelectric effect. In the simple Drude–Sommerfeld family of models, the frequency dependence of the electric and optical properties of a metal can be reduced to two parameters, the plasma frequency and the Drude time. 82,83 In the bulk, this primarily represents the frequency of a charge oscillation with a large wavelength; 84 at the interface with a dielectric, however, the electron plasma supports captive resonances in its neighborhood 85 known as surface plasmon resonances (SPR). While SPRs are significant in thin films, localized SPRs (LSPRs) in metal nanoparticles and, more generally, three-dimensional nanostructures, have a much stronger and more versatile optical response. 86 The strong plasmonic response is what gives solutions of gold nanoparticles their distinctive red appearance—described by Faraday 87 and first explained fully by Gustav Mie 88 —and what gives many medieval stained glass windows their color. 89

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The strong and stable optical response of metal nanoparticles makes them suitable for use as optical labels with which to track biomolecules. In contrast to fluorophores, they do not suffer from photoblinking or -bleaching, at the cost of significantly larger sizes. As such, they have been used e.g. to track phospholipids and proteins diffusing in cell membranes 49,90 and to detect sub-steps of the rotation of F1 -ATPase. 91 Since the SPR is a surface effect, it is highly sensitive to the refractive index of the dielectric medium near the surface. LSPRs couple primarily to a near field with a size scale of some nanometers, which is especially enhanced in regions of high curvature by the lightning-rod effect. 92 This coupling of a property measurable in the far field, the surface plasmon resonance, to the optical properties in a zeptoliter volume, is what gives single metal nanoparticles their power for sensing. 93 While nanoscopic sensors are impractical for many applications, 94 they allow for the detection of rare events and potentially tiny concentrations of analytes. In particular, a single-gold-nanorod-based sensor detecting the arrival in, and departure from, the near field of individual molecules has been demonstrated . 23,95 When two plasmonic nanoparticles approach each other to distances comparable with the size of the near field, the coupling between the two, which can be thought of as plasmon hybridization, 86 is highly dependent on the distance (see fig. 5). This opens the use of a pair of nearby nanoparticles as a plasmonic nano-ruler. 96 Suitably attached to a biomolecule, such a ruler can be used to monitor conformational dynamics in real time ; 97 recently, Ye et al. demonstrated continuous monitoring of the conformational dynamics of a single protein for 24 h. 98 If two nanoparticles are linked with well-characterized elastic molecules, a nanoruler becomes a plasmonic nanospring which can be used as a force sensor with optical readout. 99

Conclusion and Outlook In this paper we have reviewed current optical methods providing single-molecule or singlenanoparticle sensitivity, while not relying on fluorescence detection. In practice, all these

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Figure 5: Spectral signature of two nearby plasmonic nanoparticles coupling (plasmonic nanoruler). (a) Nanoparticles attached to a glass surface/to each other with BSA– biotin/biotin–spreptavidin. Silver (b) and gold (c) have different colors depending on whether they are individual particles (b/c left) or pairs (b/c right). (inset: TEM.) (d) Representative scattering spectra of single particles and pairs of silver (top) and gold (bottom). Taken from Sönnichsen et al. 96 .

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methods rely on light scattering in one or the other guise. Scattering has three main advantages over fluorescence: (i) Scattering signals are stable, they do not blink nor bleach. Indeed, as scattering relies on the molecule’s optical polarizability, it essentially reflects the molecule’s integrity and chemical stability, rather than the complex electron-vibration coupling processes affecting fluorescence. (ii) Bright-field scattering signals are often intense. Scattering is not limited by nanosecond fluorescence lifetimes, and can often be detected with higher efficiency than the fluorescence of single molecules. Non-absorbing objects can be illuminated by strong laser beams without photodamage, provided the laser wavelength is chosen properly. Absorbing molecules or particles, in particular metal nanoparticles, usually sustain much higher intensities and illumination doses than fluorescent dyes. (iii) Scattering makes it possible to avoid labels altogether. Non-labelled species placed in a suitable medium, e.g. a protein in solution, will scatter light. Of course, the absence of labels comes at a cost, the lack of specificity of scattering. Two molecules with the same polarizability will give indistinguishable scattering signals. One could restore specificity through chemical functionalization or through specific chemical recognition, which is the solution favored by life throughout evolution. However, just as with fluorescent labels, these additional chemical interactions may alter the properties and functions of the objects to be detected. We have described three main non-fluorescent optical methods. (i) Photothermal microscopy is based on the absorption of specific objects, which can be considered as possible labels for molecules or nanoparticles. Just as fluorescent dyes, absorbing metal nanoparticles can be fabricated in different colors, thanks to variations in metal composition, size and shape of the nanoparticles. Absorbing labels can be imaged on a virtually dark background thanks to modulation techniques, because the optical absorption of most media of interest, including living cells, is very low when the heating wavelength is chosen in a proper transparency window. Compared to the fluorescent labels so ubiquitous in cell biology, these absorbing labels are often much more stable, but also much bulkier. (ii) Bright-field scattering exploits the tiny changes of a focused wave front by a small scatterer. Interferometric scattering (iScat)

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is the most advanced modality of this method. It provides unique sensitivity and accuracy for in vitro conditions, in the absence of other scatterers, or at least of any time-dependent scattering processes. In that case, background is removed through highly sensitive image subtraction techniques. The iScat method has been compared to mass spectrometry in liquid phase, but it is also particularly useful to study interactions and assembly of single molecules in vitro. Such studies would be much more difficult, if possible at all, in a complex system such as a live cell, because of the lack of molecular specificity. (iii) It is possible to engineer light waves to enhance light-matter interactions with respect to those at the focus of a microscope. Those methods use optical cavities in a broad sense. A plasmonic hot spot can act as a cavity with low quality factor (Q) and high confinement, whereas optical cavities such as whispering gallery modes present a high Q and a (relatively) large mode volume. Whereas those two types of cavities have their own specific advantages, they can also be combined to benefit from both a high Q and a small mode volume, mitigating the original compromises. 25,26 The main advantage of cavity-enhanced detection is its extreme sensitivity, but its disadvantages are its complexity of operation compared to the previous methods, and the difficulty involved in interfacing the optical cavity or cavities with soft matter or biological systems of interest, such as live cells. Specificity also remains an issue for these methods, as they still require either specific binding or labelling with specific molecular groups, which may alter the biomolecule’s properties and functions. A further issue is the quantification of scattering signals, which is possible only if the precise structure of the electric field is controlled or known. Unlike in photothermal or iScat microscopy, the magnitude of the optical signal in cavity-enhanced scattering cannot be obviously correlated to the detected object, unless its position is controlled very precisely. The same type of difficulty arises in surface-enhanced Raman scattering and excitation and/or emission enhancement schemes, two methods we did not discuss here. A possible solution to the problem of label-induced alteration of a molecule’s properties is to do away altogether with the immobilization requirement. This solution is exploited in

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fluorescence correlation spectroscopy (FCS) versus single-molecule immobilization. We believe this scheme would open interesting avenues for the transient detection of diffusing single molecules and nanoparticles by scattering or temperature-induced (photothermal) signals. A similar scheme would exploit transient scattering or photothermal signals of plasmonic structures or cavities caused by diffusing absorbing or purely dielectric objects. Such methods would require very high time resolution. They would exclude the immobilization of single molecules, but just like FCS, they would rely on the excellent statistical significance provided by large numbers of single-molecule signals. The high time resolution would be made possible by the much higher scattering intensities than those achieved in fluorescence. Of course, a scattering correlation scheme would still leave two problems open: (i) the inability to correlate single-molecule dynamics on timescales longer than the diffusion time, and (ii) that of the specificity of scattering signals. The latter point could be addressed by measuring additional dimensions of the scattering bursts. This could be done in real time during each burst, as in the multiparameter analysis of fluorescence bursts proposed by Seidel’s group 100 . Alternatively, different quantities could be measured at different times on the same samples. First steps in this directions have been proposed in scattering media by Hiroi and Shibayama 101 and with plasmonic gold nanorods by Sönnichsen’s group. 27 The search is on for convenient optical signals to provide additional dimensions during the scattering bursts.

Acknowledgement The authors acknowledge funding by the Netherlands Organisation for Scientific Research (NWO) and the Zwaartekracht program NanoFront (TJ, MO). The project of transient scattering by dielectric particles has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement no. 792595 (MDB).

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Thomas Jollans received his M.Phys. from the University of Oxford in 2015, where he completed a project on single-virus particle tracking using near-TIRF microscopy. He is currently pursuing a PhD in the single-molecule optics group at the Leiden Institute of Physics, using photothermal techniques to study heat transfer and boiling at the nanoscale, focusing in particular the dynamics of plasmonic vapor nanobubbles.

Martin D. Baaske is currently a post-doctoral researcher in the single-molecule optics group at the Leiden University’s Institute of physics, where he holds a Marie SkłodowskaCurie fellowship. Previously he was Ph.D. student at the Max Planck Institute for the Science of Light in Erlangen and received his Ph.D. in physics from the Friedrich-AlexanderUniversität Erlangen-Nürnberg in 2017 for his studies on microcavity based single-molecule detection.

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Michel Orrit studied at E. N. S. in Paris and obtained his Ph.D. in Bordeaux. He observed the first fluorescence signal from a single molecule in 1990. Since then, single-molecule fluorescence revolutionizes cell biology and material science. In Leiden since 2001, Orrit uses single molecules to remove ensemble averaging and study dynamics free from synchronization. He received the Edison-Volta Prize (2016), and the Spinoza Prize (2017).

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Nonfluorescent Optical Probing of Single Molecules and

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