Bionanotechnology III: from Biomolecular Assembly to Applications 609 Advances in TERS (tip-enhanced Raman scattering) for biochemical applications Regina Treffer*, Ren ´ eB¨ ohme†, Tanja Deckert-Gaudig*, Katherine Lau* 1 , Stephan Tiede‡, Xiumei Lin* and Volker Deckert*† 2 *IPHT (Institute for Photonic Technology), Albert-Einstein-Strasse 9, 07745 Jena, Germany, †Institute of Physical Chemistry, Friedrich Schiller University Jena, Helmholtzweg 4, 07743 Jena, Germany, and ‡Experimentelle Dermatologie, Universit ¨ atsklinikum Schleswig-Holstein, Ratzeburger Allee 160, 23538 L ¨ ubeck, Germany Abstract TERS (tip-enhanced Raman scattering) provides exceptional spatial resolution without any need for labelling and has become a versatile tool for biochemical analysis. Two examples will be highlighted here. On the one hand, TERS measurements on a single mitochondrion are discussed, monitoring the oxidation state of the central iron ion of cytochrome c, leading towards a single protein characterization scheme in a natural environment. On the other hand, a novel approach of single molecule analysis is discussed, again based on TERS experiments on DNA and RNA, further highlighting the resolution capabilities of this method. Introduction TERS (tip-enhanced Raman scattering) is based on the intrinsic properties of plasmonic structures to enhance the generally weak Raman signal and simultaneously achieve high lateral resolution [1,2]. A combination of scanning probe techniques [AFM (atomic force microscopy) and/or STM (scanning tunnelling microscopy)] [3] with a conventional Raman microscope is used to establish the system as an analytical tool. SPM (scanning probe microscopy) alone potentially allows imaging with single-atom spatial resolution, but the main information is the morphology of the sample. Vibrational spectroscopy generally provides molecular information and/or serves as a unique fingerprint for identification. To enhance the inherently weak Raman signal, rough or periodically structured metallic surfaces or colloidal solutions serve as plasmonic field enhancers and result in the so-called SERS (surface-enhanced Raman scattering) effect [4]. If the roughness is reduced to a single feature, as it is the case for a metal-coated or metallic SPM tip, the plasmonic structure consequently increases the spatial resolution related to the feature size, which means down to the nanometre scale. Several approaches are used to produce TERS active probes; one way is the evaporation of silver or gold on to conventional AFM tips [1]. This method results in isolated metal nanoparticles on the tip and regularly at the tip apex. In a more elaborate approach, the attachment of individual single-crystalline silver nanowires to tungsten wires using alternating current dielectrophoresis has also been reported Key words: atomic force microscopy, biochemical analysis, cytochrome c, epithelial progenitor cell (ePC), nanoscale domain, tip-enhanced Raman scattering (TERS). Abbreviations used: AFM, atomic force microscopy; ePC, epithelial progenitor cell; K15, cytokeratin 15; SERS, surface-enhanced Raman scattering; SPM, scanning probe microscopy; SPP, surface plasmon polariton; TERS, tip-enhanced Raman scattering. 1 Present address: Renishaw plc, Old Town, Wotton-Under-Edge, Gloucestershire GL12 7DW, U.K. 2 To whom correspondence should be addressed (email volker.deckert@ipht-jena.de). [5]. Closely related is the growth of hemispherical gold droplets on top of silicon nanowires, which are subsequently attached to an AFM tip [6,7]. A promising method is the application of adiabatic plasmon focusing tips to concentrate the electromagnetic field on to the apex of an SPM probe. This can be realized by etching a tip-shaft grating into metal scanning probe tips, resulting in an optical antenna that allows the coupling of an external beam into the grating and the adiabatic propagating SPP (surface plasmon polariton) conversion into a localized SPP at the tip apex [8]. Similar probes result from a combination of standard AFM cantilevers with a photonic crystal and a plasmonic waveguide. This enables focusing of the excitation laser to the apex of the waveguide and a photon confinement corresponding to the tip dimensions [9]. For the actual TERS measurements, several excitation and collection geometries have been implemented so far. The back-reflection or transmission mode requires an inverted Raman microscope in combination with an SPM. The TERS tip is illuminated from below through substrate and sample, and the back-scattered Raman signal is collected through the same objective in epi geometry [1]. Obviously, this method is limited to transparent samples. Non-transparent samples can be studied using a side-illumination [8,10] or top-illumination [11] mode. However, this flexibility with respect to the sample comes at the expense of a reduced collection angle, resulting in a lower contrast between near-field and far-field signals. Hence a TERS geometry using a parabolic mirror provides the most efficient alternative for opaque samples [12,13]. The spatial resolution of TERS can be currently estimated to be better than 10 nm [14] and models even predict resolutions on a molecular level (<1 nm) [15]. A comparable resolution with molecular specificity at present can only be achieved by label-based methods such as PALM (photo-activated localization microscopy) [16], STORM (stochastic optical reconstruction microscopy) [17] and STED (stimulated emission depletion) microscopy [18]. Biochem. Soc. Trans. (2012) 40, 609–614; doi:10.1042/BST20120033 C  The Authors Journal compilation C  2012 Biochemical Society Biochemical Society Transactions www.biochemsoctrans.org