Single-molecule surface-enhanced Raman spectroscopy with nanowatt excitation.

We demonstrate the possibility of single molecule (SM) detection via surface-enhanced Raman spectroscopy (SERS) in two seemingly challenging and unexpected cases: first with ultra-low excitation powers of the order of nanowatts and second in as-synthesized and not deliberately-aggregated silver colloid solution. The experiments are carried out using the bi-analyte method on a methylated form of Rhodamine 6G and one of its isotopologues excited at 514 nm close to the electronic resonance. This study spectacularly highlights the fact that SM-SERS detection is much more common and easier to achieve than typically thought, in particular in the case of resonance Raman excitation.

Polypeptide multilayer self-assembly studied by ellipsometry.

A polypeptide nanofilm made by layer-by-layer (LbL) self-assembly was built on a surface that mimics nonwoven, a material commonly used in wound dressings. Poly-L-lysine (PLL) and poly-L-glutamic acid (PLGA) are the building blocks of the nanofilm, which is intended as an enzymatically degradable lid for release of bactericides to chronic wounds. Chronic wounds often carry infection originating from bacteria such as Staphylococcus aureus and a release system triggered by the degree of infection is of interest.

Tunable SERS using gold nanoaggregates on an elastomeric substrate.

We report on the self-assembly of colloidal gold nanoparticles on a stretchable, elastomeric membrane, and the use of this membrane as a base substrate for far-field confocal Raman measurements. Surface-enhanced Raman scattering (SERS) enhancement for such a substrate was estimated as 10(6) to 10(7). Atomic force microscopy has been used to study the changes in nanoparticle topography when the membrane is stretched.

Strong correlation between molecular configurations and charge-transfer processes probed at the single-molecule level by surface-enhanced Raman scattering.

Single-molecule (SM) electrochemistry studied by surface-enhanced Raman scattering (SERS) with high spectral resolution reveals a picture in which the frequency of Raman modes is correlated with the electrochemical process through the interaction with the surface. Previously unexplored phenomena can be revealed by the synergy of electrochemistry and SM-SERS, which explores in this case subtler spectroscopic aspects (like the frequency of a vibration within the inhomogeneous broadening of a many-molecules Raman peak) to gain the information. We demonstrate, among other things, that the interaction with the surface is correlated both with the molecule vibrational frequencies and with the ability of single molecules to be reduced/oxidized at different potentials along the electrochemical cycle.

Tiny peaks vs mega backgrounds: a general spectroscopic method with applications in resonant Raman scattering and atmospheric absorptions.

A simple method using standard spectrometers with charge-coupled device (CCD) detectors is described to routinely measure background-corrected spectra in situations where the signal is composed of weak spectral features (such as Raman peaks or absorption lines) engulfed in a much stronger (by as much as ∼10(5)) broad background. The principle of the method is to subtract the dominant fixed-structure noise and obtain a shot-noise limited spectrum. The final noise level can therefore be reduced as desired by sufficient integration time.

Direct measurement of resonance Raman spectra and cross sections by a polarization difference technique.

Resonant Raman (RR) spectroscopy, despite its many promising applications in analytical chemistry and biology, remains an experimental challenge (compared to standard Raman) primarily because of the presence of large fluorescence backgrounds overwhelming the RR signals. The observation of RR spectra of fluorophores therefore requires the use of specialized, picosecond-time-resolved setups. Here, we present and demonstrate a method, based on polarization-difference, by which RR spectra and cross sections can be measured using the most standard Raman setup with continuous wave excitation and CCD-based detection.

Single-molecule SERS detection of C60.

Single-molecule Surface-Enhanced Raman Scattering (SERS) detection of buckminsterfullerene (C(60)) is achieved by using different isotopologues of the molecule with a distribution around an average isotopic substitution ((12)C → (13)C) of ~30%. The distribution of different isotopologues creates a broad (~20 cm(-1)) average SERS signal within which single-molecule SERS spectra of individual isotopic realizations of the molecule can be distinguished. The SERS enhancement factors for SM-SERS C(60) events are typically in the range of ~10(8), suggesting a limitation imposed by either photobleaching or surface interactions with the (Ag) metallic colloids to reach the highest SERS hot-spots (which can typically have larger maximum enhancements).

Single-molecule surface-enhanced Raman spectroscopy.

A general overview of the field of single-molecule (SM) surface-enhanced Raman spectroscopy (SERS) as it stands today is provided. After years of debates on the basic aspects of SM-SERS, the technique is emerging as a well-established subfield of spectroscopy and SERS. SM-SERS is allowing the observation of subtle spectroscopic phenomena that were not hitherto accessible.

Combined SPR and SERS microscopy in the Kretschmann configuration.

A novel hybrid spectroscopic technique is proposed, combining surface plasmon resonance (SPR) with surface-enhanced Raman scattering (SERS) microscopy. A standard Raman microscope is modified to accommodate the excitation of surface plasmon-polaritons (SPPs) on flat metallic surfaces in the Kretschmann configuration, while retaining the capabilities of Raman microscopy. The excitation of SPPs is performed as in standard SPR-microscopy; namely, a beam with TM-polarization traverses off-axis a high numerical aperture oil immersion objective, illuminating at an angle the metallic film from the (glass) substrate side.

A scheme for detecting every single target molecule with surface-enhanced Raman spectroscopy.

Surface-enhanced Raman spectroscopy (SERS) is now a well-established technique for the detection, under appropriate conditions, of single molecules (SM) adsorbed on metallic nanostructures. However, because of the large variations of the SERS enhancement factor on the surface, only molecules located at the positions of highest enhancement, so-called hot-spots, can be detected at the single-molecule level. As a result, in all SM-SERS studies so far only a small fraction, typically less than 1%, of molecules are actually observed.

Experimental demonstration of surface selection rules for SERS on flat metallic surfaces.

We have measured the polarization and incident angle dependence of the Surface-Enhanced Raman Scattering (SERS) signal of a nile blue monolayer adsorbed on a flat gold surface. Comparisons with predictions of electromagnetic (EM) theory indicate that the molecules are predominantly adsorbed flat on the surface. These results provide the most direct demonstration of the concept of surface selection rules in SERS, and further confirm the validity of the SERS-EM model beyond the |E|(4)-approximation.

Combining surface plasmon resonance (SPR) spectroscopy with surface-enhanced Raman scattering (SERS).

The simultaneous measurement of surface plasmon resonance (SPR) spectroscopy and surface-enhanced Raman scattering (SERS) on flat metallic surfaces is demonstrated on a relatively simple experimental setup based on the Kretschmann configuration. This setup requires only minor modifications to standard Raman microscopes, and we show that it can be applied successfully to the most common conditions of SPR spectroscopy, i.e.

Bi-analyte single molecule SERS technique with simultaneous spatial resolution.

The simultaneous combination on CCD detectors of both spectral and spatial information is used in the framework of the single molecule (SM) bi-analyte Surface-Enhanced Raman Scattering (SERS) technique, to provide a new level of understanding on the origins of SM-spectra, as well as reveal the advantages and limitations of the statistical identification of SM-events. A new and deeper interpretation of the roots of the inhomogeneous broadening of single molecule Raman peaks can be uncovered, as well as the origin of Surface-Enhanced Fluorescence (SEF) emission by single molecules. In this manner, subtler aspects of SM-SERS spectroscopy can be revealed by the additional presence of spatial information on the localization of single molecules producing the signal.

Monitoring the electrochemistry of single molecules by surface-enhanced Raman spectroscopy.

Coherent control of chemical species in complex systems is always subject to intrinsic inhomogeneities from the environment. For example, slight chemical modifications can decisively affect transport properties of molecules on surfaces. Hence, single-molecule (SM) studies are the best solution to avoid these problems and to study diverse phenomena in biology, physics, and chemistry.

Electrochemical modulation for signal discrimination in surface enhanced Raman scattering (SERS).

Electrochemical modulation to induce controlled fluctuations in SERS signals is introduced as a method to discriminate and isolate different contributions to the spectra. The modulation--which can be changed in potential range, amplitude, and frequency--acts as a controllable "switch" to turn on, off, or change specific Raman signals which can then be correlated within the spectra by different fluctuation analysis techniques. Principal component analysis (PCA), either by itself or assisted by fast fourier transform (FFT) prefiltering, are shown to provide viable tools to isolate the different components of the spectra.

Estimating the Raman cross sections of single carbon nanotubes.

The order of magnitude of Raman differential cross sections of radial breathing modes (RBMs) of individual carbon nanotubes is measured for 633 and 785 nm laser excitations. This is shown by both a calibration applied to previously published data from other authors at 785 nm and our own measurements of individual nanotubes at 633 nm excitation. We find typical values of differential cross sections of RBMs to be on the order of approximately 10(-22) cm(2)/sr for resonant nanotubes on a silicon substrate.

Quantifying resonant Raman cross sections with SERS.

We propose a method based on surface-enhanced Raman scattering (SERS) to estimate the resonance Raman cross sections of dyes. The latter are notoriously difficult (or impossible) to obtain by normal (spontaneous) constant wave Raman spectroscopy when the fluorescence quantum yield of the molecules is good and the overwhelming effect of fluorescence masks the Raman spectrum. We propose here to use the fluorescence quenching occurring in SERS conditions to overcome simply this problem.

Resolving single molecules in surface-enhanced Raman scattering within the inhomogeneous broadening of Raman peaks.

We demonstrate both the observation of either a single or a few molecules resolved within the inhomogeneous broadening of a peak in surface-enhanced raman scattering (SERS). Our results demonstrate a fundamental aspect of spectroscopy and also a possible technique to learn more about the varying interactions that single molecules can have with a given SERS substrate. Resolving more than one molecule within the inhomogeneous broadening is only possible thanks to the combination of (i) high-resolution measurements, and (ii) low temperatures (to narrow down the intrinsic homogeneous broadening as much as possible).

An iterative algorithm for background removal in spectroscopy by wavelet transforms.

Wavelet transforms are an extremely powerful tool when it comes to processing signals that have very "low frequency" components or non-periodic events. Our particular interest here is in the ability of wavelet transforms to remove backgrounds of spectroscopic signals. We will discuss the case of surface-enhanced Raman spectroscopy (SERS) for illustration, but the situation it depicts is widespread throughout a myriad of different types of spectroscopies (IR, NMR, etc.

Single-molecule surface-enhanced Raman spectroscopy of nonresonant molecules.

Single-molecule surface-enhanced Raman scattering (SERS) detection of nonresonant molecules is demonstrated experimentally using the bianalyte SERS method. To this end, bianalyte SERS is performed at 633 nm excitation using the nonresonant molecule 1,2-di-(4-pyridyl)-ethylene (BPE) in combination with a benzotriazole derivative as a partner. The results are then extended to the even more challenging case of a small nonresonant molecule, adenine, using an isotopically substituted adenine as bianalyte SERS partners.

Ultrafast nonradiative decay rates on metallic surfaces by comparing surface-enhanced Raman and fluorescence signals of single molecules.

By the simultaneous observation of surface-enhanced Raman scattering and surface-enhanced fluorescence signals from a single molecule, we can measure and quantify the modification of the total decay rate of emitters in very close proximity to metals, even down to adsorbed molecules. This modified decay rate is shown to be largely dominated by its nonradiative component, which would be extremely difficult to estimate with conventional approaches. The method provides an indirect measurement of ultrafast (approximately 25 fs) mechanisms, which would be impossible to gain with time-resolved spectroscopy of a single molecule.

Plasmon-dispersion corrections and constraints for surface selection rules of single molecule SERS spectra.

The problem of extracting information from relative intensities of Raman peaks in surface-enhanced-Raman-scattering (SERS) is intimately related to several important topics in the technique. Among them: (i) the possibility (or sometimes impossibility) of observing surface selection rules in different situations, (ii) the role of analyte resonance conditions, (iii) the crucial inclusion of plasmon-resonance dispersion corrections in the analysis of relative Raman intensities among peaks, and (iv) the connection of these phenomena with (broader) issues like surface-enhanced fluorescence (SEF). This paper deals with the underlying connections among these (apparently disconnected at first sight) topics.

Single-molecule vibrational pumping in SERS.

Single-molecule vibrational pumping in surface-enhanced Raman scattering (SERS) is demonstrated rigorously using the bi-analyte SERS method at low temperatures. These experiments reveal a systematic difference between the radiative SERS cross section estimated from the Stokes intensity and that obtained by pumping itself (from the anti-Stokes-to-Stokes ratio), the latter being always larger. This difference can only be reliably demonstrated in the single-molecule SERS regime, for it is otherwise affected by complications of the averaging (over the enhancement distribution) of the signals of several molecules.