Atomic Force Microscopy Meets Biophysics, Bioengineering, Chemistry, and Materials Science

Abstract Briefly, herein the use of atomic force microscopy (AFM) in the characterization of molecules and (bioengineered) materials related to chemistry, materials science, chemical engineering, and environmental science and biotechnology is reviewed. First, the basic operations of standard AFM, Kelvin probe force microscopy, electrochemical AFM, and tip‐enhanced Raman microscopy are described. Second, several applications of these techniques to the characterization of single molecules, polymers, biological membranes, films, cells, hydrogels, catalytic processes, and semiconductors are provided and discussed.


Introduction
According to Web of Science, 104 522 items are associated with the term "atomic force microscopy" (AFM) since 1987 (searchc arriedo ut in December 2018).T his large number shows that AFM is well establishedi nt he scientific community. After refining the search,t he number of items related to both scientifica nd technological fields, for example, materials science multidisciplinary (32 389), applied physics (28 921), chemistry physical (21175), physics condensed matter (14 762), and chemistry multidisciplinary (13 887), is still large.
AFM, [1] as part of the scanning probe microscopy family of techniques, hasb een utilized for the last three decades to characterize (bio)moleculesa nd (bio)materials. [2][3][4][5][6] In comparison with other characterization techniques, such as electron microscopy (transmission and scanning), AFM seemst ob e more versatile. For example, it permits dynamic processes to be followed att he micro-and nanoscale in aqueous environments at different temperatures. In addition, AFM can be combined with other characterization techniques, such as fluorescence microscopy or Raman/IR spectroscopy,t oo vercome its own limitations (e.g.,a bsolute distance or chemical fingerprint determination). [7,8] For furtherr eading, the following reviewss ummarize the versatility and measuring properties of AFM in different fields. Francis et al. discussed the contribution and versatility of the technique appliedt ob iological and biomedical systems. [9] Handschuh-Wang et al. described, in ac ompactw ay,n ew advancesi nt he combination of opticalt echniques with AFM, explaining their basic principles and pointingo ut different applications. [10] An extensive article presenting the history,d evelopment,a nd prospects of high-speed AFM was writtenb y Ando. [11] Researchers interested in high-vacuum AFM should focus on the detailed article written by Giessibl. [12] Patel and Kranz, [13] in av ery complete work, discussed how tip modification deliveredc hemical information.T hey also explained the use of electrochemical imaging and their applications. Finally, Gross et al. recently reportedp rogress and challenges of highresolution scanning probe microscopy with functionalized tips in the elucidation of molecular structures. [14] 2. AFM-Based Characterization Techniques

Topography imaging and mechanical machines
If spectroscopy and opticalt echniques are based on the interaction between light and matter,A FM is mainly defined by the interaction of matter with matter.B riefly,t he main elements of an AFM are as canner,p robe (a bare cantilever or ac antilever with as harp tip or ac olloidal particle), photodetector,a nd computer ( Figure 1). The interaction forces acting between the tip and sample bend the cantilever according to the Hooke law.B ending is monitored and detected by ap hotodiode that collectst he reflection of al aser beam at the backside of the cantilever,w hile scanning or recording force curves. Ap iezo scannerm oves either the sample or the tip in three dimensions. The surface properties of the sample are then obtained from the tip/samplei nteraction and the final image is delivered by ac omputer.
Generally,t he most used imaging modes are contact and tappingm ode. Other researchers refer to static and dynamic modes with all of their possibilities:c ontact, jumping, tapping, amplitude, and frequency modulation. An importanta dvance in scanning is high-speed AFM, which was developedb yA ndo et al., [15] and deservesareview on its own.
In contact mode,w hile the sample is scanned, the value of the repulsive force between tip and sample is kept constant. Ag entlers canning option, especially for soft samples, is tapping mode because it reduces lateral forces that could damage the samples. In this mode, the cantilever is set to oscillate vertically at (or close to) its resonant frequency.O nce the tip is far away from the sample, the cantilever oscillates with constant amplitude, whereas for smaller tip-sampled istances the amplitude of the oscillations is reduced. High-resolution images can be obtained by using af eedback loop that keeps the amplitude of the cantilever oscillation at ac onstant level during scanning. Differences between the set drive phase and phase of the cantilever responsec an be used to gain an insight into viscoelastic and adhesive properties of the sample under study.I nteresting and didactic literature concerning different AFM measuringm odes can be found in the work of several authors. [16,17] Representative( biological) samples measured with these imaging modes include nanotubes, lipids,p roteins, molecular self-assemblies, or cells. [18][19][20][21][22] At this point it is worth mentioning other methodsf or beam-deflection measurements implemented by research groups with al ong tradition and expertise in AFM development.A mongt hem,t he most relevant ones are capacitive detectionb yu sing cantilevers as capacitors( their deflection produces ac hange in capacitance) [23] and piezoelectric detection (for which the deflection of piezoelectric cantilevers is detected as an electrical signal;this permits atomic resolution to be achieved). [24] Apart from scanning, AFM opens up aw ider ange of experimental possibilities upon use as a" mechanical" machine;t his is technically known as force spectroscopy.I nt his case, forcedistance (or force-time) experiments are carried out. In such experiments, the AFM tip (or ac olloidal probe) approaches and is retracted from the sample at different speeds (which might range from 30 to 10 000 nm s À1 ). Bending of the cantilever is determined as af unction of the displacement of the piezo scanner,w hereas the force sensed by the cantilever is calculated based on Hooke's law (the bending or deflection of the cantilever is multipliedb yt he spring constant). [25] The force-distance curvesc an be divided into three parts (see Figure 1, right).F irst, the approachingc urve delivers information about repulsive or attractive forces (e.g.,e lectrostatic,v an der Waals, hydration, or entropic forces). [26][27][28] Second, contact of the cantilever with the sample (the dwell time can be defined by the researcher) permits mechanical properties (e.g., the Young modulus, relaxation time, and viscosity) to be investigated. [29][30][31] Finally, the retracting curve provides information about nonspecific adhesion forces, ligand-receptor forces,  Af lexible cantilever (in this case, with as harp tip) interactsw ith the sample (through attractive/repulsive forces),a nd therefore, is the AFMsensing element. The deflectionoft he cantilever due to interactions is measured after detecting the reflected laser beam with ap osition detector (a four quadrant photodiode). The piezoelectric scanner can be moved with nanoscale resolution along either the x-y or x-y-z axes, depending on the commercial device. Right: Representative force-distance curver ecorded for al iving cell. The measurement starts with the cantilever at rest (sensing zero force). Then, the cantilever approaches the cell to deliver information about molecular and colloidal forces.Finally,t he cantilever is retracted from the samplet oc onvey informationaboutthe existence of adhesive forces, molecular unfolding events, and tethers. (Imagea dapted from Ref. [33].) tethers, and molecular unfolding. [3,32,34] To quantify the interaction forces, the spring constant of the cantileverh as to be evaluated before startingt he experiments, whereas the contact point between tip and sample has to be determined for every recorded force curve. [35,36] Modification of the tip (or colloidal particle) throughw ell-defined chemistry leads to ap articulart ype of force spectroscopy (chemical force microscopy)a nd an ew form of imaging based on molecular recognition. [37,38] In both cases, "two molecules" interacts pecifically.T hese experiments permit the study and determination of particular interactions (e.g.,h ydrophobic-hydrophobic, ligand-receptor).
Ab reakthrough in high-resolutioni maging is the work of Schulera nd co-workers. [39] They were able to resolve the structure at an atomic level of asphaltene molecules by combining AFM with scanning tunneling microscopy.S uch results opened up the possibility of investigating, in detail, the structureo f compounds used in molecular electronicso rp hotovoltaic devices.

Electrical modes (Kelvin-probe force microscopya nd electrochemistry)
Another interesting possibility is to use conducting probes (tips or particles) to combine classical scanning with electrical surfacemapping. This is the case for Kelvin-probe force microscopy (KPFM). With this technique, the conducting cantileveri s scannedo ver as ample at constanth eight to obtain the contact potential difference, V cpd (which is determinedb yt he work functions of both tip and sample;F igure 2). Measurement of the contact potentiald ifference is performed by applying an oscillation (of frequency w)i nt he bias voltage to the probe and sample. In this way,acapacitor is formed. Simultaneously, the cantilever is oscillated at its resonance frequency, w 0 ,a nd a typical noncontact contact mode feedback acts on the phase, so that the frequencys hift can be monitored and registered. If the dependence of the bias voltage with time is V bias = V DC + V AC sin (wt), with steady DC and oscillating AC components,t he frequency shift exhibits two harmonic components (at w and 2 w)o fe xpressions: DF w % (V DC ÀV cpd )V AC sin (wt)a nd DF 2w % (V AC ) 2 cos (wt). Now,i fasecond feedback mechanismi sa ble to change V DC such that DF w = 0, V DC will be equal to the contact potentiald ifference, V cpd ,w hich is the magnitude of interest. In general,m apping of the work function provides information about corrosionp henomena, catalytic activity,o re lectrical properties of junction devices. [40][41][42] The last measuring mode discussed in this section is related to electrochemical experiments. By adding as pecial sample holderw ith three electrodes to the standard AFM configuration, it is possible to follow redox reactions that are taking place in electrolyte solutionso na ne lectrode surface, as well as to characterize the morphology of the surfaceo ft he electrode. [43] Basically,t he sample acts as aw orkinge lectrode, while the nonconducting tip monitors variations in the sample as af unctiono ft ime. Experimentally,i ti sr ecommended to use dilute electrolyte solutions and to avoid corrosion effects on the AFM scanner and AFM tip. Ac ontemporary extension of this measuring mode is the use of as pecial metal-coatedt ip that is insulated, except at its apex, which is utilized as an electrode to detect species in an electrolyte solution.

Spectroscopy mode (tip-enhanced Raman spectroscopy)
Raman spectroscopy permits vibrational and rotationalm odes to be monitored, providing as tructural fingerprint by which molecules can be identified. [44] Therefore, the combination of AFM (which can be operated in liquid conditions) with Raman spectroscopy can be usefult oi nvestigate both topographical and chemicalc hanges at the nanoscale for aw ide range of (biological) samples, such as fibril proteins, [45] carbon nanotubes, [46] DNA, [47] and cells. [48] More specifically,t ip-enhanced Ramans pectroscopy (TERS) [49] uses an apertureless probe to enhancet he Raman signal emitted by the sample molecules, which are separated from the probe by af ew nanometers, and am etallict ip that is irradiated along its apical axis by al aser with aw avelength in the visible region (l = 500-650 nm). Because the tip is sufficiently close to the sample, field enhancement is possible, leadingt om olecular excitation and registration of local Ramans pectra. Field enhancement is relatedt o excitation of metal plasmons of the tip, which acts as an antenna.T he maximum excitation range is about 20 nm in the vertical direction and2 0-50 nm in the lateral one. Once the laser is aligned to the tip (its wavelength should match the resonanceo ft he surface plasmons), the sample stage scans the sample underneath the tip, without disturbing the initial laser alignment on the tip. TERS can be used in different setups, and therefore, is able to operate in either reflection (convenient for nontransparent samples)o rt ransmission (this mode

Selected Examples of Applications
Although the previousm ethodological section contains basic references to introduce the reader to different measurement possibilities of AFM, in this section, ac ollection of articles (many of them published in the last four years), considered to be scientific and technically interesting for the readero fChem-SusChem,h ave been selected. The sectioni sd ivided by (model)s ystem and contains different examples that were investigated with the four methodologies described in the previous section (high-resolution imaging, force spectroscopy,e lectrical modes, and tip-enhanced Raman spectroscopy).

Molecules and polymers
Ar ecent example of the strength of AFM, in terms of high resolution, has been reported by Buchholz and co-workers. [50] The authors, by combining AFM with circulard ichroism (CD) spectroscopya nd dynamic light scattering, investigated the blood protein beta 2-glycoprotein, which presents two main conformations (opena nd closed). AFM images revealed that lysine acetylation promoted al arger population of proteins in an open conformation,w hich playedarole in the autoimmune disease antiphospholipids yndrome. Another example of the resolution capability of the atomicf orce microscope can be found in the work of Milhiet et al. [51] In this study,anew methodt ol ocate transmembrane proteins in lipid bilayers was tested with AFM. The authors could demonstrate that proteins were incorporated within the lipid bilayer through their hydrophobic domains. Other dynamic processes can be also moni-tored with standard AFM imaging. Moreno-Cencerrado et al. followed the formation of 2D bacterial proteins crystals as a functiono ft ime for different protein concentrations (Figure 4). [52] Such measurementse nabledt he testing of theoreticalc rystal-growth models.
Av ery instructive work concerning protein immobilization strategies for performing single-molecule force spectroscopy experiments hasb een presented by Becke and co-workers. [53] They provided ap rotocolt hat described the covalent binding of proteins to silicon surfaces by straightforward chemistry (e.g.,t he use of silanes, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide). Furthermore, they determined the interaction force (about 50 pN) between adhesin RrgA (from Streptococcus pneumoniae)a nd fibronectin. This study shows the relevance of molecular orientation in force spectroscopy measurements.
The combination of force spectroscopy measurements with tips that are chemically modified permits recognition experiments to be performed on model systems. The work of Parreira andc o-workersi llustrated the powero ft his approach for studying bacterial adhesion. [54] They reported on the binding force between adhesin BabA (from Helicobacter pylori)a nd a glycanr eceptor.T he strategy used involved the immobilization of BabA on the AFM tip, whilet he glycanr eceptor was linked to biotin self-assembled monolayers.T his study is also interesting because of analysis of the theoretical bond kinetics. Hence, the main findings showedt hat two bond populations described the binding process.
Among recent work on the mechanical unfolding of proteins, the study performed by Yu et al. is remarkable. [55] They reported on instrumental limitations that might not allow existing intermediate states to be detected while performing force spectroscopy measurements. Through optimization of the experimental setup, it wasp ossible to obtain microsecond resolution. In this way,t he authors could mechanically unfold bacteriorhodopsin moleculesi nn ative lipid bilayers to find new intermediate states.T heir setup permitted the unfolding of about two amino acids to be observed over microseconds (usual experiments record the unfolding of 6t o6 0a mino acids over milliseconds). Furthermore, they were ablet or econstruct the folding free-energy landscape.
Recently,T ERS was utilized to characterize protein glycosylation and glycans. [56] The authors pointedo ut the complexity of data processing (e.g.,m ultivariate analysis)b ecause of the potential dependence on the orientation of the protein. Furthermore, the resultsi ndicatedt hat the technique could discriminate between native and glycosylated forms of RNase.
TERS has also been successful in the detection of nucleobases. Treffer and co-workersp erformed experimentso ns inglestranded adenine and uracil polymers. [57] Their resultsc onstitute an advance because spectral contributions of the nucleobases could be distinguished on as trand.I na ddition, if the tip was movedl aterally in steps of ab ase-to-based istance, the collected spectra gave sequential information.

Membranes, fibers, and cells
Supported lipid bilayersa re one of the most studied model membranes. For this particularc ase, AFM imagingh as offered many possibilities to researchers. One of the latest published works deals with the monitoring of phase transitions of lipid mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-racglycerol) and1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPG:POPE). Borrell et al. reported on differences in the shift of the meltingt ransitionf or liposomes (measured in bulk through differentials canning calorimetry) and lipid bilayers (observed throughA FM). [58] Recently,U nsay et al. published an interesting academic work on the use of AFM imaging and force-distance curves on lipid bilayers. [59] The authors described ap rotocol to build supported lipid bilayerso ns olid supports (e.g.,mica), and reported on the topography and mechanical properties of (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin (SM)/cholesterol (Chol)) lipid bilayers. AFM, in combination with total internal reflection fluorescence microscopy,w as used to image, in real-time,c ellulose hydrolysis on nanometer-scalef ibers. [60]  In this regard, AFM has delivered important information about cellulose fibers. Lahiji and co-workers investigated the topography and elastic and adhesivep roperties of wood-derived cellulose nanocrystals under different humidity conditions. [61] They implemented finite elementc alculations to estimate at ransverse elastic modulus in the order of GPa,a nd found variations in thickness from 3t o8nm. Another interesting and more focuseds tudy on the elasticity of cellulose was performed by Hellwig et al, [62] who determinedt he mechanical properties of wet cellulose beads of different charge densities by recording force-distance curves with ac olloidal probe (i.e., gold particles of about 20 mm). The resulting data were computed by meanso ft he Derjaguin-Müller-To porov (DMT) model,w hich included the adhesion force between the sample surfaceand AFM probe in the analysis.
Investigatingamore complex system,M uraille et al. indented one type of lignified cell wall and compared the results with those obtainedf rom lignocellulosic films;t hus showing the role of lignin on the mechanical properties of cell walls. [63] Another work concerning the mechanical characterization of wood cell walls was carriedo ut by Cassdorf et al., [64] who could distinguish between the cell wall layers of the compound middle lamella of spruce wood by meanso fm echanical measurements.
For about 20 years, AFM (imaging and force spectroscopy) has also been applied to bacteria and humanc ells. Recently, El-Kirat-Chatele tal. investigated the adhesion of bacteria (which presented different phenotypicalt raits) on antifouling coatingst ob eu sed on ship hulls. [65] The main findings indicated that adhesion forces at the population level did not always correlate with individual adhesion forces because some bacteria were susceptible to phenotypich eterogeneity amid their population. If one considers bacteria as colloidalo bjects, the surfacepotentialcould play an important role, through electrostatic interactions, on adhesion phenomena. Such surfacep otential (or surface charge density) can be determinedb y means of KPFM.B irkenhauer and Neethirajan illustrated how to characterizet he surface potential of Staphylococcus aureus on steel and gold surfaces (functionalized with poly-l-lysine). [66] Imaginga nd force spectroscopy also provided interesting results in the investigation of human cell lines. Nowadays, it can be said that AFM is ad iagnostic tool, which can differentiate healthyf rom unhealthy cells. [67] Ar ecent and very complete work on cell mechanics was performed by Efremova nd coworkers. [68] The authors proposed am ethodt oa cquire the viscoelastic properties of cells (and hydrogels) for arbitrary loading. They tested the method on different cancerous cell lines and inducedt ransitions from epithelial to mesenchymal states, and performed finite element simulations to modela nd validate the experimental results. Force spectroscopy at cellular level can be used to elucidate the main molecules responsible for the adhesiono fb ladder cancerc ells to endothelial cells. Duperray et al. quantified adhesion forces and identified the key ligands involved in the process. [69] Complementary experiments to target membrane receptors in healthy and cancers cells can be carried out with TERS. In particular,X iao and Schultz showed that TERS could differentiate between different membrane receptors (integrins)t hat bound to the same arginine-glycine-aspartate-phenylalanine-cysteine (RGD) sequences. [70] In ap reviouss tudy,W ang and Schultz demonstrated that the Ramans pectrumo ft he a v b 3 integrin receptor could be detectedi nt he membrane of colon cancer cells. [71] The measurements trategy consisted of exposing the cells to nanoparticles (NPs) functionalized with cyclic RGD (c-RGD) ligands before collecting the Ramans ignal. Figure 5s hows the variation of the TERS intensity and spectra for particle/celli nteractions.

Hydrogels, catalysis, corrosion, and semiconductors
Interest in hydrogels has increased in recent decades due to their "smart" response to changes in environment( e.g.,t emperature, ionic strength, humidity). Researchers working on tissue engineering, [72] plasmonics, [73] or biosensing and drug delivery [74] have taken advantage of the physicochemical properties of hydrogels. Vamsi and Yadavalli characterized the mechanicalp roperties of poly(ethyleneg lycol) diacrylate based hydrogels, which are important for biomedical applications, at the micro-and nanoscale. [75] The authors quantified the effect of monomer molecular weight, initiator concentration, and hydration rates on the mechanical properties of the hydrogels through force-distance curves.
Catalysis is important fors cientific and technological fields such as biology,m edicine, environmental chemistry,o rc hemical engineering. The study of catalytic phenomenaa tt he micro-and nanoscale requires ac ombination of AFM scanning and spectroscopy.H arvey and co-workers integrated Raman spectroscopy with AFM to study the photo-oxidation of rhodamine 6G over Al 2 O 3 -supported AgNP. [76] The experimentsw ere carried in ac ell in which gas and temperature could be varied. Ar ecent article on electrochemical AFM features demonstrated how ac onducting tip sensed the electronic properties of cobalt (oxy)hydroxide phosphate. [77] Another use of electro-chemicalA FM wasi ts application in an investigation of corrosion on aluminuma lloys in solution. Davoodi et al. proposed a probe that could work either as cantilever or as microelectrode. In their experiments, the electrochemical current was registered by using the redox mediator I À /I À 3 . [78] Recently,s tudies that extend an understanding of heterogeneity on graphite/graphenes urfaces for electrochemical applications have been conducted by Nellist et al. [79] They used a nanoelectrode (with ac onical Pt tip) in combination with tapping AFM mode to characterize surface topography,n anomechanics, and nanoelectronic properties.
KPFM has also been au seful toolt oi nvestigate photovoltaics and solar conversion technologies. In both cases, high energy conversion efficiency and low-cost processing are key goals. In this context, Jiang et al. investigated charget ransport and separation in perovskite solar cells. [80] Their resultsi ndicated the existenceo fap-n junction structure at the TiO 2 /perovskite interface. The results also showedt hat improved carrier mobility was essential to increaset he efficiency of perovskite solar cells. Finally,n onvolatile random access memory devices are able to retain information if the poweri st urned off. Ferroelectric thin films are suitable candidates to build such memory devices.S ua nd Zhang used KPFM to investigate the surfacep otentiald ependence on voltage and duration upon application to copper-doped ZnO films (see Figure 6). [81] They concludedt hat the copper-doped ZnO films exhibited enhanced bipolar charge-trapping properties, which could be an advantage for nonvolatile memory applications. The difference can be understood in terms of the doping-induced shift of the Fermi level, whichp rovides ag ood representation of the characteristics of the semiconductor material.

Summary and Outlook
In the last three decades, atomic force microscopy (AFM) has expandedi ts use to many scientific and technological fields. This short Review has presented four measuring modes based on AFM. Although the described methods have been successfully applied to study differentp roblems, there is room fori mprovement,f or example, high-speed imaging has openedu pn ew possibilities for exploration (as pointedo ut before, this alone should be the topic of as eparate review). The appropriate model to interpret mechanical experiments performed on softmatter systems is stillr equired. In addition, ac riticali ssue for tip-enhanced Raman spectroscopy (TERS) experiments is opti-  Kelvin-probef orce microscopy (KPFM) has not yet been widely utilized in biological and colloidal systems, mainly because it is used in air.Ad ifficult challenge would be to elucidate the surface potential in biosystems because it is affected by interactions with the medium. This task is certainly relevant because surfacep otentials are important for the interaction of particles, biointerfaces, cells and tissues.
Finally,asummary of the main properties and challenges associated with AFM, KFPM,and TERS is provided in Ta ble 1.  [a] functionsi nair,vacuum, and liquid (between % 5a nd 60 8C); atomic resolutioninhighvacuum( sub-nanometer in liquid);ing eneral, lateral molecular resolution ( % 1-5 nm) and sub-nanometer verticalr esolution ( % 1nm) 3D surface profile to deliver directi nformationa boutthe interaction forces between tip and sample; useful for studying mechanical propertieso fbiomaterials at the nanoand microscale; does not require either vacuumo rpossibly damaging sample treatment (e.g.,m etal/carbonc oatings) smaller scan images ize (mmrange)c ompared with scanninge lectron microscopy (mm range); slow scan time could lead to thermal drift (partially solved with highspeed AFM); AFM images can be affected by piezoelectric hysteresis(closed-loop scannersmight eliminate this problem); unsuitable tips can produce imagea rtifacts KFPM [b] simultaneous mappingo ft opographya nd potential( or work function); mainly used for materials science for metallic and semiconducting structures; allows measuremento ft he contactp otential difference with laterala nd potential resolutions below 50 nm and 10 mV,r espectively; surface potentialc ontrol is important for materials and biological systems (e.g.,c rucial for the interaction of living cells with exogenous devices); surface potentialw hile studying biological samples is affected by the environmental medium (and surface contaminants); measurement in liquid requires AFM tips insulated at their apex to prevent current leakage; proper comparison and interpretation of KFPMd ata withr esults obtained from electrophoreticmobility and streamingpotential; cheap and robust electrodef abrication TERS [c] nanometer spatial resolution ( % 30 nm); chemical fingerprint recognition (identification and classification of materials); there are still unresolved questions about mode selectivity (this might limit certain applications); could benefit from the ability to use visible light for excitation ability to investigate samples in solutions; tip reproducibility and references amples; deeperunderstanding of the TERS process; statisticald ata analysis [a] Introducedb yB innig and co-workers in 1986. [1] [b] Introducedb yN onnenmacher and co-workers in 1991. [82] [c] Introduced by Stçckle and co-workers [83] and Anderson [84] in 2000.