Search

Tissue cross-linking represents an important and often used technique to enhance the mechanical properties of biomaterials. For the first time, we investigated biochemical and structural properties of genipin (GE) cross-linked equine pericardium (EP) using optical imaging techniques in tandem with quantitative atomic force microscopy (AFM). EP was cross-linked with GE at 37 °C, and its biochemical and biomechanical properties were observed at various time points up to 24 h. GE cross-linked EP was monitored by the normalized ratio between its second-harmonic generation (SHG) and two-photon autofluorescence emissions and remained unchanged for untreated EP; however, a decreasing ratio due to depleted SHG and elevated autofluorescence and a fluorescence band at 625 nm were found for GE cross-linked EP. The mean autofluorescence lifetime of GE cross-linked EP also decreased. The biochemical signature of GE cross-linker and shift in collagen bands were detected and quantified using shifted excitation Raman difference spectroscopy as an innovative approach for tackling artifacts with high fluorescence backgrounds. AFM images indicated a higher and increasing Young's modulus correlated with cross-linking, as well as collagen structural changes in GE cross-linked EP, qualitatively explaining the observed decrease in the second-harmonic signal. In conclusion, we obtained detailed information about the biochemical, structural, and biomechanical effects of GE cross-linked EP using a unique combination of optical and force microscopy techniques in a nondestructive and label-free manner.

Plasmon-induced catalysis of the thiols 4-aminothiophenol (ATP) and 4-nitrothiophenol (NTP) has been investigated in numerous studies. Currently, two reaction pathways are discussed in the literature, one leading to dimerization to 4,4’-dimercaptoazobenzene (DMAB), and the other, depending on experimental conditions, resulting in a monomer commonly assigned to ATP. In this joint experimental-theoretical study, we disentangle the involved photo-/plasmon-mediated reaction mechanisms by thorough control of the reaction conditions, particularly the involved surface-enhanced Raman scattering (SERS) substrates. The Raman spectra experimentally and strongly suggest that the formation of a new stable intermediate plays a crucial role. Tracking the reaction with time-dependent SERS experiments allows us to build the connection between the dimer (DMAB) and monomer pathways and to propose potential reaction pathways for different environmental conditions. Furthermore, theoretical modelling addressing the excited-states properties of key intermediates involved in both reaction pathways and the respective thermodynamics allows to investigate the underlying reaction mechanism in more detail – complementing the spectroscopic results.

A 60-membered library of vitamin A-functionalized P(MMA-stat-DMAEMA)-b-PPEGMA block copolymers was synthesized by RAFT polymerization. The retinoic acid was coupled to hydroxyl groups present in the hydrophilic PPEGMA block. The polymers were investigated for their ability to encapsulate ribonucleic acids through nanoparticle (NP) formulation using the emulsion/solvent evaporation method. The localization of retinyl in surface-near regions of the NPs was indicated by surface enhanced Raman spectroscopy, and the interaction of the NPs with a retinol binding protein was investigated by an analytical ultracentrifuge. The systematic analysis of the NP library in terms of the encapsulation efficiency of the ribonucleic acids, the toxicity of the NPs, and the cellular uptake helped identifying suitable candidates for cellular internalization studies. The cell uptake was investigated by flow cytometry and fluorescence microscopy and reveals structure dependent uptake behavior of the examined particles.

Sample degradation, in particular of biomolecules, frequently occurs in surface-enhanced Raman spectroscopy (SERS) utilizing supported silver SERS substrates. Currently, thermal and/or photocatalytic effects are considered to cause sample degradation. This paper establishes the efficient inhibition of sample degradation using iodide which is demonstrated by a systematic SERS study of a small peptide in aqueous solution. Remarkably, a distinct charge separation-induced surface potential difference is observed for SERS substrates under laser irradiation using Kelvin probe force microscopy. This directly unveils the photocatalytic effect of Ag-SERS substrates. Based on the presented results, it is proposed that plasmonic photocatalysis dominates sample degradation in SERS experiments and the suppression of typical SERS sample degradation by iodide is discussed by means of the energy levels of the substrate under mild irradiation conditions. This approach paves the way toward more reliable and reproducible SERS studies of biomolecules under physiological conditions.

The stability of surface-enhanced Raman spectroscopy (SERS) substrates in different organic solvents and different buffer solutions was investigated. SERS substrates were fabricated by a microwave-assisted synthesis approach and the morphological as well as chemical changes of the SERS substrates were studied. It was demonstrated that the SERS substrates treated with methanol, ethanol, or N,N-dimethylformamide (DMF) were comparable and showed overall good stability and did not show severe morphological changes or a strong decrease in their Raman activity. Toluene treatment resulted in a strong decrease in the Raman activity whereas dimethyl sulfoxide (DMSO) treatment completely preserved or even slightly improved the Raman enhancement capabilities. SERS substrates immersed into phosphate-buffered saline (PBS) solutions were observed to be rather instable in low and neutral pH buffer solutions. Other buffer systems showed less severe influences on the SERS activity of the substrates and a carbonate buffer at pH 10 was found to even improve SERS performance. This study represents a guideline on the stability of microwave-fabricated SERS substrates or other SERS substrates consisting of non-stabilized silver nanoparticles for the application of different organic solvents and buffer solutions.

A 60-membered library of vitamin A-functionalized P(MMA-stat-DMAEMA)-b-PPEGMA block copolymers was synthesized by RAFT polymerization. Subsequently, retinoic acid was coupled to hydroxyl groups present in the hydrophilic PPEGMA block. The polymers were investigated for their ability to encapsulate ribonucleic acids through nanoparticle (NP) formulation using the emulsion/solvent evaporation method. The localization of vitamin A in surface-near regions of the NPs was indicated by surface enhanced Raman spectroscopy, and the interaction of the NPs with a retinol binding protein was investigated by analytical ultracentrifugation. The systematic analysis of the NP library in terms of the encapsulation efficiency of the ribonucleic acids, the toxicity of the NPs, and the cellular uptake helped identifying suitable candidates for cellular internalization studies. The cell uptake was investigated by flow cytometry and fluorescence microscopy and reveals structure dependent uptake behavior of the examined particles.

Understanding the property-function relation of nanoparticles in various application fields involves determining their physicochemical properties, which is still a remaining challenge to date. While a multitude of different characterization tools can be applied, these methods by themselves can only provide an incomplete picture. Therefore, novel analytical techniques are required, which can address both chemical functionality and provide structural information at the same time with high spatial resolution. This is possible by using tip-enhanced Raman spectroscopy (TERS), but due to its limited depth information, TERS is usually restricted to investigations of the nanoparticle surface. Here, TERS experiments are established on polystyrene nanoparticles (PS NPs) after resin embedding and microtome slicing. With that, unique access to their internal morphological features is gained, and thus, enables differentiation between information obtained for core- and shell-regions. Complementary information is obtained by means of transmission electron microscopy (TEM) and from force-distance curve based atomic force microscopy (FD-AFM). This multimodal approach achieves a high degree of discrimination between the resin and the polymers used for nanoparticle formulation. The high potential of TERS combined with advanced AFM spectroscopy tools to probe the mechanical properties is applied for quality control of the resin embedding procedure.

Plasmonic nanoantennas have found broad applications in the fields of photovoltaics, electroluminescence, non-linear optics and for plasmon enhanced spectroscopy and microscopy. Of particular interest are fundamental limitations beyond the dipolar approximation limit. We introduce asymmetric gold nanoparticle antennas (AuNPs) with improved optical near-field properties based on the formation of sub-nanometer size gaps, which are suitable for studying matter with high-resolution and single molecule sensitivity. These dumbbell antennas are characterized in regard to their far-field and near-field properties and are compared to similar dimer and trimer antennas with larger gap sizes. The tailoring of the gap size down to sub-nanometer length scales is based on the integration of rigid macrocyclic cucurbituril molecules. Stable dimer antennas are formed with an improved ratio of the electromagnetic field enhancement and confinement. This ratio, taken as a measure of the performance of an antenna, can even exceed that exhibited by trimer AuNP antennas composed of comparable building blocks with larger gap sizes. Fluctuations in the far-field and near-field properties are observed, which are likely caused by distinct deviations of the gap geometry arising from the faceted structure of the applied colloidal AuNPs.

The ability of supramolecular transition metal coordination complexes to form stabilized, long-living, radiative charge-separated states has drawn interest to employ these triplet-state emitters for the design of photonic devices. Their applicability as photosensitizers of electron transfer in molecular photonic systems is directly coupled to fundamental studies of their rich and highly versatile photochemical and photophysical properties. Here, we demonstrate that the properties of individual dual-luminescent Ru2+-bis-terpyridine complexes can be addressed with excellent sensitivity in single-complex antenna-enhanced phosphorescence investigations. This sensitivity enables studying environmentally imposed alterations of their photophysical properties, e.g., in thin film applications. In contrast to ensemble averaging investigations in solution, single-complex antenna-enhanced phosphorescence investigations corroborate the existence of Ru2+-bis-terpyridine complexes with spectrally shifted emission peaks ...

The potential of supramolecular transition metal coordination complexes to form robust, long-living, radiative charge transfer states makes this class of triplet state emitters ideal candidates for application as photosensitizes or in photonic devices. Antenna-enhanced phosphorescence experiments on single Ru2+-bis-terpyridine complexes incorporated into a thin PMMA film show that phosphorescence emission spectra can exhibit shifts depending on the local environment [J. F. Herrmann, P. S. Popp, A. Winter, U. S. Schubert and C. Hoppener, ACS Photonics, 2016, 3, 1897-1906]. Here, we demonstrate that the environmentally altered spectral properties of individual dual-luminescent Ru2+-bis-terpyridine complexes in PMMA and acetonitrile can be reproduced by DFT-based vibrationally resolved Franck-Condon spectra, if the phosphorescent emission of different molecular conformations is taken into account. Furthermore, we demonstrate that the triplet emission of these complexes occurs from a metal-to-ligand charge transfer (MLCT) state.

Gold–polymer hybrid nanoparticles attract wide interest as building blocks for the engineering of photonic materials and plasmonic (active) metamaterials with unique optical properties. In particular, the coupling of the localized surface plasmon resonances of individual metal nanostructures in the presence of nanometric gaps can generate highly enhanced and confined electromagnetic fields, which are frequently exploited for metal-enhanced light–matter interactions. The optical properties of plasmonic structures can be tuned over a wide range of properties by means of their geometry and the size of the inserted nanoparticles as well as by the degree of order upon assembly into 1D, 2D, or 3D structures. Here, the synthesis of silica-stabilized gold–poly(N-isopropylacrylamide) (SiO2-Au-PNIPAM) core–satellite superclusters with a narrow size distribution and their incorporation into ordered self-organized 3D assemblies are reported. Significant alterations of the plasmon resonance are found for different assembled structures as well as strongly enhanced Raman signatures are observed. In a series of experiments, the origin of the highly enhanced signals can be assigned to the interlock areas of adjacent SiO2-Au-PNIPAM core–satellite clusters and their application for highly sensitive nanoparticle-enhanced Raman spectroscopy is demonstrated.

A fully controllable process for the fabrication of carbon nanotube assemblies is presented on the basis of a sequential electrochemical oxidation lithography process. This approach utilizes the local chemical conversion of a n-octadecyltrichlorosilane self-assembled monolayer into a template featuring polar acid groups. The capability to utilize such chemically active templates for the site-selective assembly of individual carbon nanotubes was demonstrated, and a hierarchical, sequential structuring routine to obtain crossed CNT configurations, formed by preselected carbon nanotubes, was implemented. The introduced process allows the reliable and well-controlled fabrication of tailor-made nanoscopic assemblies of nanomaterials toward their integration into complex device frameworks and could provide control over the electrical properties of the fabricated assemblies.

The ability of metal surfaces and nanostructures to localize and enhance optical fields is the primary reason for their application in biosensing and imaging. Local field enhancement boosts the signal-to-noise ratio in measurements and provides the possibility of imaging with resolutions significantly better than the diffraction limit. In fluorescence imaging, local field enhancement leads to improved brightness of molecular emission and to higher detection sensitivity and better discrimination. We review the principles of plasmonic fluorescence enhancement and discuss applications ranging from biosensing to bioimaging.

Coupling of plasmon resonances in metallic gap antennas is of interest for a wide range of applications due to the highly localized strong electric fields supported by these structures, and their high sensitivity to alterations of their structure, geometry, and environment. Morphological alterations of asymmetric nanoparticle dimer antennas with (sub)-nanometer size gaps are assigned to changes of their optical response in correlative dark-field spectroscopy and high-resolution transmission electron microscopy (HR-TEM) investigations. This multimodal approach to investigate individual dimer structures clearly demonstrates that the coupling of the plasmon modes, in addition to well-known parameters such as the particle geometry and the gap size, is also affected by the relative alignment of both nanoparticles. The investigations corroborate that the alignment of the gap forming facets, and with that the gap area, is crucial for their scattering properties. The impact of a flat versus a rounded gap structure on the optical properties of equivalent dimers becomes stronger with decreasing gap size. These results hint at a higher confinement of the electric field in the gap and possibly a different onset of quantum transport effects for flat and rounded gap antennas in corresponding structures for very narrow gaps.

We introduce a method for a simultaneous topographical and optical characterization of aperture probes for scanning near-field optical microscopy which is based on imaging of small sized fluorescent nanospheres (∼20 nm). The near-field optical fluorescence image of a nanosphere maps the intensity distribution of light at the end face of the probe whereas the simultaneously taken height image contains information about the aperture–sample distance. We used this method to control a mechanical modification of a near-field probe. By squeezing a probe repeatedly against a smooth glass substrate and thereby removing obstructing protrusions the aperture was brought as close as possible to the sample surface which resulted in a strongly improved optical resolution.

We experimentally demonstrate cascaded field enhancement by means of gold nanoparticle dimer and trimer antennas. The local field enhancement is probed by single-molecule fluorescence using fluorophores with high intrinsic quantum efficiency (Q(0)>80%). Using a self-similar trimer antenna consisting of 80, 40, and 20 nm gold nanoparticles, we demonstrate a fluorescence enhancement of 40 and a spatial confinement of 15 nm. Compared with a single gold nanoparticle, the self-similar trimer antenna improves the enhancement-confinement ratio by more than an order of magnitude. Self-similar antennas hold promise for high-resolution imaging and spectroscopy, ultrasensitive detection, and efficient single-photon sources.

High-Resolution Imaging with Single Protein Sensitivity holds promise for studies of the chemical organization of cellular structures. Such investigations are important to reveal abnormalities from the regular spatial distribution of specific membrane proteins.

Understanding the diversity of biological processes requires methods that can address single proteins in their natural environment and provide insights into structural and functional properties, as well as the local distribution of each individual protein. We use an optical antenna in the form of a single gold nanoparticle to localize incident laser radiation to 50 nm, significantly smaller than the diffraction limit of light. Our approach enables us to optically resolve individual plasma-membrane-bound Ca2+ pumps (PMCA4) immersed in aqueous environments and to determine the distribution of interprotein distances. We are able to correlate the protein maps with local topology. Improved antenna geometries will make it possible to resolve, identify, and probe single membrane proteins in live cells with true protein resolution of 5−10 nm.

Antenna-based near-field microscopy uses nanoscopic metal particles to localize incident laser radiation down to 50 nm. Imaging erythrocyte plasma-membranes with this technique identifies individual calcium ion pumps and reveals their inhomogeneous distribution within the plasma-membrane.

The applications of scanning near-field optical microscopy to biological specimens under physiological conditions have so far been very rare since common techniques for a probe-sample distance control are not as well suited for operation in liquid as under ambient conditions. We have shown previously that our own approach for a distance control, based on a short aperture fibre probe and a tuning fork as force sensor in a tapping mode, works well even on soft material in water. By means of an electronic self-excitation circuit, which compensates for changes of the resonance frequency due to evaporation of liquid, the stability of the force feedback has now been further improved. We present further evidence for the excellent suitability of the tapping-mode-like distance control to an operation in liquid, for example, by force-imaging of double-stranded DNA. Moreover, we demonstrate that a nuclear envelope in liquid can be imaged with a high optical resolution of approximately 70 nm without affecting its structural integrity. Thereby, single nuclear pores in the nuclear envelope with a nearest neighbour distance of approximately 120 nm have been optically resolved for the first time.

We present a probe concept for scanning near-field optical microscopy combining the excellent background suppression of aperture probes with the superior light confinement of apertureless probes. A triangular aperture at the tip of a tetrahedral waveguide (full taper angle $\ensuremath{\sim}90\ifmmode^\circ\else\textdegree\fi{}$) shows a strong field enhancement at only one rim when illuminated with light of suitable polarization. Compared to a circular aperture of equivalent size, the resolution capability is doubled without loss of brightness. For a $\ensuremath{\sim}60\text{ }\text{ }\mathrm{n}\mathrm{m}$ sized triangular aperture, we measured an optical resolution $l40\text{ }\text{ }\mathrm{n}\mathrm{m}$ and a transmission of $\ensuremath{\sim}{10}^{\ensuremath{-}4}$.

The seeded growth of poly(ethylene imine) – gold nanoparticle clusters enables the formation of particle assemblies with tunable optical properties. Clusters with increasing particle sizes, filling factors and assemblies consisting of PEI–gold–silver core shell particles can be synthesized in this way. Profound structural characterization is carried out via TEM imaging and FIB milling which allows visualizing the cross-section of the clusters. Determination of the optical properties was performed via UV-Vis spectroscopy and spectral dark field microscopy of individual particles. Additionally, numerical calculations were carried out based on the Mie theory. The results are in good agreement with the experimental findings and reveal the contribution of different multipoles to the spectra which cannot be resolved by UV-Vis spectroscopy in solution. The isotropic nature and adjustable properties of these clusters could render them versatile building blocks for metamaterials.

Scanning near-field optical microscopy (SNOM) circumvents the diffraction limit of conventional light microscopy and is able to achieve optical resolutions substantially below 100 nm. However, in the field of cell biology SNOM has been rarely applied, probably because previous techniques for sample-distance control are less sensitive in liquid than in air. Recently we developed a distance control based on a tuning fork in tapping mode, which is also well-suited for imaging in solution. Here we show that this approach can be used to visualize single membrane protein complexes kept in physiological media throughout. Nuclear envelopes were isolated from Xenopus laevis oocytes at conditions shown recently to conserve the transport functions of the nuclear pore complex (NPC). Isolated nuclear envelopes were fluorescently labeled by antibodies against specific proteins of the NPC (NUP153 and p62) and imaged at a resolution of approximately 60 nm. The lateral distribution of epitopes within the supramolecular NPC could be inferred from an analysis of the intensity distribution of the fluorescence spots. The different number densities of p62- and NUP153-labeled NPCs are determined and discussed. Thus we show that SNOM opens up new possibilities for directly visualizing the transport of single particles through single NPCs and other transporters.