Efeito de diferentes protocolos de acabamento/polimento na resistência à fadiga e módulo de Weibull de diferentes tipos de zircônia / Effect of different finishing/polishing protocols on the fatigue strength and Weibull modulus of different types of zirconia

Objetivo: Avaliar o efeito de diferentes protocolos de acabamento/polimento na resistência à fadiga das novas gerações de zircônia. Materiais e Métodos: Foram confeccionados noventa (N=90) discos cerâmicos (Ø:12mm; 1,2 mm-ISO 6872), sendo 45 em zircônia ultratranslúcida (UT- VITA, Vita Zahnfabrik) e 45 de uma cerâmica de zircônia híbrida 3Y-TZP e 5Y-PSZ com gradiente de translucidez (GT- e.max Zircad prime GT, Ivoclar). Após a sinterização dos discos cerâmicos, estes foram divididos em 6 grupos (n=15), de acordo com fatores "cerâmica (zircônia UT e zircônia GT)" e "Protocolo de acabamento e polimento" (Pontas Diamantadas + Borrachas; Borrachas; Controle). Os discos foram submetidos ao ensaio de resistência à fadiga pelo método stepwise stress (5Hz para 10.000 ciclos), com um step de 57 MPa e 80 MPa, começando em 170 e 240 MPa, para a zircônia UT e GT, respectivamente, e prosseguindo até 100.000 ciclos ou a detecção da fratura. Foram realizadas também análises extras de Difração de raios X (DRX), microscopia de força atômica (AFM) e rugosidade superficial. Os dados de resistência à fadiga (MPa) e rugosidade (µm) foram avaliados estatisticamente através de ANOVA 2 fatores e teste de Tukey (5%). Foi realizada a análise de "Kaplan-Meier" seguida pelo teste de Mantel-Cox (Log Rank test) e pela comparação múltipla aos pares, todos com nível de significância de 5%. Além disso, também foi utilizada a análise de Weibull. Resultados: ANOVA (2 fatores) revelou que o fator "Protocolo de Acabamento e Polimento" (p=0,0006), "tipo de zircônia" (p=0,000) e a interação dos dois (p=0,0000) apresentaram significância estatística para a resistência à fadiga. A zircônia GT (761,47 MPa) obteve valores médios de resistência superiores a zircônia UT (385,87 MPa), independentemente do tipo de acabamento e polimento. Para zircônia UT o acabamento com pontas diamantadas reduziu os valores de resistência à fadiga (273,44DMPa) comparados ao grupo controle (503,96CMPa) e estatisticamente semelhante ao grupo polidos apenas com borracha (308,22DMPa). Na zircônia GT o polimento com borrachas melhorou a resistência do material (871,35A MPa) quando comparado ao grupo controle (664,29B MPa) que, por sua vez, foi semelhante ao grupo com acabamento com pontas diamantadas (748,78B MPa). Foi possível observar que o tipo de protocolo de acabamento e polimento influenciou a fratura em fadiga das duas cerâmicas, porém, o número de ciclo de sobrevida só foi significativo para a zircônia UT. Conclusão: A resistência à fadiga das duas zircônias foi influenciada pelos diferentes protocolos de acabamento e polimento. Para zircônia UT o polimento com borrachas e pontas diamantadas reduziu a resistência mecânica, já para a zircônia GT, todos os protocolos de acabamento e polimento melhoraram as propriedades mecânicas do material (AU).

Objective: To evaluate the effect of different finishing/polishing protocols on the fatigue strength of new generations of zirconia. Materials and Methods: Ninety (N=90) ceramic discs (Ø:12mm; 1.5mm-ISO 6872) were made, 45 of ultra-translucent zirconia (UT-VITA, Vita Zahnfabrik) and 45 of a hybrid zirconia ceramic 3Y-TZP and 5Y-PSZ with translucency gradient (GT-e.max Zircad prime GT, Ivoclar). After sintering, the ceramic discs were divided into 6 groups (n=15), according to the factors "ceramic (UT and GT)" and "Finishing and polishing protocol" (Diamond burs + Rubbers; Rubbers and Control). The discs were subjected to the fatigue resistance test by the stepwise stress method (5Hz for 10,000 cycles) with a step increment of 57 MPa and 80 MPa, starting at 170 and 240 MPa, for UT and GT zirconia, respectively, and continuing up to 100,000 cycles or failure detection. Complementary analyzes of X-ray Diffraction (XRD), atomic force microscopy (AFM) and surface roughness were also carried out. Results were statistically evaluated using 2-way ANOVA, Tukey test (5%) and Weibull analysis. The results of fatigue resistance and roughness were statistically evaluated using 2-way ANOVA and Tukey test (5%). The "Kaplan-Meier" analysis was performed followed by the Mantel-Cox test (Log Rank test) and the multiple comparison in pairs, all with a significance level of 5%. In addition, Weibull analysis was also carried out. Results: ANOVA (2-way) revealed that the factor "Finishing and Polishing Protocol" (p=0.0006), "type of zirconia" (p=0.000) and the interaction between them (p=0.0000) were statistically significant for resistance to fatigue. GT zirconia (761.47 MPa) had higher resistance values than UT zirconia (385.87 MPa), regardless of the type of finishing and polishing. For UT zirconia, finishing with diamond burs reduced the values of resistance to fatigue (273.44DMPa) compared to the control group (503.96CMPa) and statistically similar to the group polished only with rubber (308.22DMPa). In GT zirconia, polishing with rubbers improved the resistance of the material (871.35A MPa) when compared to the control group (664.29B MPa), which, in turn, was similar to the group finished with diamond burs (748.78B MPa). The Weibull modulus did not show statistical significance between the groups (p=0.300) but the characteristic strength showed statistically significant differences (p=0.0000). It was possible to observe that the type of finishing and polishing protocol influenced the fatigue fracture of the two ceramics, however, the number of survival cycles was only significant for the UT zirconia. Conclusion: The fatigue strength of the two zirconia was influenced by the different finishing and polishing protocols. For UT zirconia, polishing with rubbers and diamond burs reduced the mechanical resistance, whereas for GT zirconia, all finishing and polishing protocols improved the mechanical properties of the material (AU).

Cyclic Fatigue Resistance and Surface Roughness of Rotary NiTi Instruments after Simulated Clinical Use in Curved Root Canals - An Atomic Force Microscopy Study

Abstract Objective: To examine the cyclic fatigue resistance and surface topography of TruNatomy and ProTaper Gold nickel-titanium rotary files and evaluate the presence of alterations to surface topography following instrumentation in simulated curved canals. Material and Methods: Twenty-four nickel-titanium instruments, twelve each of TN and PTG file systems, were evaluated for cyclic fatigue resistance. The rotary files were rotated in a simulated root canal with standardized diameter, angle of curvature, and radius of curvature in a custom-made cyclic fatigue testing device until the instrument fracture occurred. The time to fracture for each instrument was recorded with a stopwatch; in seconds in each group. Fractured instruments were subjected to atomic force microscopy analysis measuring the average roughness and the root mean square values to investigate surface features of endodontic files. Mean values and standard deviation were calculated. Data were analyzed using the Mann-Whitney U test. Results: Time to fracture was marginally higher in PTG instruments than in the TN file systems. PTG files exhibited higher surface roughness when compared with TN files (p<0.05). Conclusion: TN file system had a higher cyclic fatigue resistance than PTG. Cyclic fatigue causing file breakage did affect the surface topography of the files. PTG files showed a higher surface porosity value than the TN files (AU).

Detection of Circulating Serum microRNA/Protein Complexes in ASD Using Functionalized Chips for an Atomic Force Microscope.

MicroRNAs, which circulate in blood, are characterized by high diagnostic value; in biomedical research, they can be considered as candidate markers of various diseases. Mature microRNAs of glial cells and neurons can cross the blood-brain barrier and can be detected in the serum of patients with autism spectrum disorders (ASD) as components of macrovesicles, macromolecular protein and low-density lipoprotein particles. In our present study, we have proposed an approach, in which microRNAs in protein complexes can be concentrated on the surface of AFM chips with oligonucleotide molecular probes, specific against the target microRNAs. MicroRNAs, associated with the development of ASD in children, were selected as targets. The chips with immobilized molecular probes were incubated in serum samples of ASD patients and healthy volunteers. By atomic force microscopy (AFM), objects on the AFM chip surface have been revealed after incubation in the serum samples. The height of these objects amounted to 10 nm and 6 nm in the case of samples of ASD patients and healthy volunteers, respectively. MALDI-TOF-MS analysis of protein components on the chip surface allowed us to identify several cell proteins. These proteins are involved in the binding of nucleic acids (GBG10, RT24, RALYL), in the organization of proteasomes and nucleosomes (PSA4, NP1L4), and participate in the functioning of the channel of active potassium transport (KCNE5, KCNV2).

Direct-Write Patterning of Biomimetic Lipid Membranes In Situ with FluidFM.

The creation of biologically inspired artificial membranes on substrates with custom size and in close proximity to each other not only provides a platform to study biological processes in a simplified manner, but they also constitute building blocks for chemical or biological sensors integrated in microfluidic devices. Scanning probe lithography tools such as dip-pen nanolithography (DPN) have opened a new paradigm in this regard, although they possess some inherent drawbacks like the need to operate in air environment or the limited choice of lipids that can be patterned. In this work, we propose the use of the fluid force microscopy (FluidFM) technology to fabricate biomimetic membranes without losing the multiplexing capability of DPN but gaining flexibility in lipid inks and patterning environment. We shed light on the driving mechanisms of the FluidFM-mediated lithography processes in air and liquid. The obtained results should prompt the creation of more realistic biomimetic membranes with arbitrary complex phospholipid mixtures, cholesterol, and potential functional membrane proteins directly patterned in physiological environment.

Rigid-body fitting to atomic force microscopy images for inferring probe shape and biomolecular structure.

Atomic force microscopy (AFM) can visualize functional biomolecules near the physiological condition, but the observed data are limited to the surface height of specimens. Since the AFM images highly depend on the probe tip shape, for successful inference of molecular structures from the measurement, the knowledge of the probe shape is required, but is often missing. Here, we developed a method of the rigid-body fitting to AFM images, which simultaneously finds the shape of the probe tip and the placement of the molecular structure via an exhaustive search. First, we examined four similarity scores via twin-experiments for four test proteins, finding that the cosine similarity score generally worked best, whereas the pixel-RMSD and the correlation coefficient were also useful. We then applied the method to two experimental high-speed-AFM images inferring the probe shape and the molecular placement. The results suggest that the appropriate similarity score can differ between target systems. For an actin filament image, the cosine similarity apparently worked best. For an image of the flagellar protein FlhAC, we found the correlation coefficient gave better results. This difference may partly be attributed to the flexibility in the target molecule, ignored in the rigid-body fitting. The inferred tip shape and placement results can be further refined by other methods, such as the flexible fitting molecular dynamics simulations. The developed software is publicly available.

Extended mechanical force measurements using structured illumination microscopy.

Quantifying cell generated mechanical forces is key to furthering our understanding of mechanobiology. Traction force microscopy (TFM) is one of the most broadly applied force probing technologies, but its sensitivity is strictly dependent on the spatio-temporal resolution of the underlying imaging system. In previous works, it was demonstrated that increased sampling densities of cell derived forces permitted by super-resolution fluorescence imaging enhanced the sensitivity of the TFM method. However, these recent advances to TFM based on super-resolution techniques were limited to slow acquisition speeds and high illumination powers. Here, we present three novel TFM approaches that, in combination with total internal reflection, structured illumination microscopy and astigmatism, improve the spatial and temporal performance in either two-dimensional or three-dimensional mechanical force quantification, while maintaining low illumination powers. These three techniques can be straightforwardly implemented on a single optical set-up offering a powerful platform to provide new insights into the physiological force generation in a wide range of biological studies. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations.

Single-molecule force spectroscopy is a powerful tool for studying protein folding. Over the last decade, a key question has emerged: how are changes in intrinsic biomolecular dynamics altered by attachment to µm-scale force probes via flexible linkers? Here, we studied the folding/unfolding of α3D using atomic force microscopy (AFM)-based force spectroscopy. α3D offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed α3D's configurational diffusion constant within the context of Kramers theory varies with pH. The resulting pH dependence provides a test for AFM-based force spectroscopy's ability to track intrinsic changes in protein folding dynamics. Experimentally, however, α3D is challenging. It unfolds at low force (<15 pN) and exhibits fast-folding kinetics. We therefore used focused ion beam-modified cantilevers that combine exceptional force precision, stability, and temporal resolution to detect state occupancies as brief as 1 ms. Notably, equilibrium and nonequilibrium force spectroscopy data recapitulated the pH dependence measured using smFRET, despite differences in destabilization mechanism. We reconstructed a one-dimensional free-energy landscape from dynamic data via an inverse Weierstrass transform. At both neutral and low pH, the resulting constant-force landscapes showed minimal differences (∼0.2 to 0.5 kBT) in transition state height. These landscapes were essentially equal to the predicted entropic barrier and symmetric. In contrast, force-dependent rates showed that the distance to the unfolding transition state increased as pH decreased and thereby contributed to the accelerated kinetics at low pH. More broadly, this precise characterization of a fast-folding, mechanically labile protein enables future AFM-based studies of subtle transitions in mechanoresponsive proteins.

Robust scan synchronized force-fluorescence imaging.

Simultaneous atomic force microscope (AFM) and sample scanning confocal fluorescence microscope measurements are widely used to obtain mechanistic and structural insights into protein dynamics in live cells. However, the absence of a robust technique to synchronously scan both AFM and confocal microscope piezo stages makes it difficult to visualize force-induced changes in fluorescent protein distribution in cells.  To address this challenge, we have built an integrated AFM-confocal fluorescence microscope platform that implements a synchronous scanning method which eliminates image artifacts from piezo motion ramping, produces accurate pixel binning and enables the collection of a scanned image of a sample while applying force to a single point on the sample. As proof of principle, we use this instrument to monitor the redistribution of fluorescent E-cadherin, an essential transmembrane protein, in live cells, upon application of mechanical force.

Comparative Evaluation of Surface Roughness of Resin- Modified Glass Ionomer and Glass Hybrid Restorative Materials Simulated by Tooth Brushing: An in-Vitro Study

ABSTRACT Objective: Tocompare the effect of tooth brushing on surface roughness of Resin-Modified Glass Ionomer Cement (RMGIC; GC Gold label 2LC Light Cured Universal Restorative) and Glass Hybrid (GH; GC EQUIA SYSTEM- EQUIA Forte™ Fil and EQUIA Forte™ Coat) restorative material at 1- and 3-months interval simulated by tooth brushing. Material and Methods: RMGIC and GH material specimens (20 each) were prepared according to manufacturer instructions in 10mm × 2 mm dimensions using a mylar strip. A specially designed toothbrush simulator was used along with Oral B Pro 2 2000N powered toothbrush and Colgate Total dentifrice (Colgate-Palmolive India limited; Relative dentin abrasivity - RDA:70- Low abrasive) to perform brushing strokes. Specimens were subjected to surface roughness analysis before and after simulated tooth brushing at baseline, 1, and 3 months. Results: The intragroup comparison was done using repeated-measures ANOVA. Intergroup comparisons were done using an independent sample t-test and General Linear Model (ANCOVA). Surface roughness increased from baseline through 3 months in both RMGIC and GH groups. The mean surface roughness in RMGIC group was significantly higher than GH group at baseline 1 and 3-months, respectively (p<0.001, <0.001, and <0.001). Interaction between group and baseline surface roughness was not significant (p=0.466). The estimated marginal means were significantly higher in RMGIC than GH group (p=0.008). Conclusion: The surface roughness of both RMGIC and GH restorative increased from baseline to 1 month and 3 months after the simulated toothbrushing protocol. GH exhibited significantly lower surface roughness than RMGIC at all the tested intervals.

In-Situ Investigation on Nanoscopic Biomechanics of Streptococcus mutans at Low pH Citric Acid Environments Using an AFM Fluid Cell.

Streptococcus mutans (S. mutans) is widely regarded as the main cause of human dental caries via three main virulence factors: adhesion, acidogenicity, and aciduricity. Citric acid is one of the antibiotic agents that can inhibit the virulence capabilities of S. mutans. A full understanding of the acidic resistance mechanisms (ARMs) causing bacteria to thrive in citrate transport is still elusive. We propose atomic force microscopy (AFM) equipped with a fluid cell to study the S. mutans ARMs via surface nanomechanical properties at citric acid pH 3.3, 2.3, and 1.8. Among these treatments, at pH 1.8, the effect of the citric acid shock in cells is demonstrated through a significantly low number of high adhesion zones, and a noticeable reduction in adhesion forces. Consequently, this study paves the way to understand that S. mutans ARMs are associated with the variation of the number of adhesion zones on the cell surface, which is influenced by citrate and proton transport. The results are expected to be useful in developing antibiotics or drugs involving citric acid for dental plaque treatment.

Laser spectroscopic technique for direct identification of a single virus I: FASTER CARS.

From the famous 1918 H1N1 influenza to the present COVID-19 pandemic, the need for improved viral detection techniques is all too apparent. The aim of the present paper is to show that identification of individual virus particles in clinical sample materials quickly and reliably is near at hand. First of all, our team has developed techniques for identification of virions based on a modular atomic force microscopy (AFM). Furthermore, femtosecond adaptive spectroscopic techniques with enhanced resolution via coherent anti-Stokes Raman scattering (FASTER CARS) using tip-enhanced techniques markedly improves the sensitivity [M. O. Scully, et al, Proc. Natl. Acad. Sci. U.S.A. 99, 10994-11001 (2002)].

Biological physics by high-speed atomic force microscopy.

While many fields have contributed to biological physics, nanotechnology offers a new scale of observation. High-speed atomic force microscopy (HS-AFM) provides nanometre structural information and dynamics with subsecond resolution of biological systems. Moreover, HS-AFM allows us to measure piconewton forces within microseconds giving access to unexplored, fast biophysical processes. Thus, HS-AFM provides a tool to nourish biological physics through the observation of emergent physical phenomena in biological systems. In this review, we present an overview of the contribution of HS-AFM, both in imaging and force spectroscopy modes, to the field of biological physics. We focus on examples in which HS-AFM observations on membrane remodelling, molecular motors or the unfolding of proteins have stimulated the development of novel theories or the emergence of new concepts. We finally provide expected applications and developments of HS-AFM that we believe will continue contributing to our understanding of nature, by serving to the dialogue between biology and physics. This article is part of a discussion meeting issue 'Dynamic in situ microscopy relating structure and function'.

Type V Collagen in Scar Tissue Regulates the Size of Scar after Heart Injury.

Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.

Nanoscale investigation of human skin and study of skin penetration of Janus nanoparticles.

Janus nanoparticles (JNP) are innovative nanocarriers with an interesting pharmaceutical and cosmetic potential. They are characterized by the presence of a lipid compartment associated with an aqueous compartment delimited by a phospholipid bilayer containing phospholipids and non-ionic surfactants. The hydrodynamic diameter of JNP varies between 150 and 300 nm. The purpose of this study was to answer the following questions: after cutaneous application, are JNP penetrating? If so, how deep? And in which state, intact or degraded? It was essential to understand these phenomena in order to control the rate and kinetics of diffusion of active ingredients, which can be encapsulated in this vehicle for pharmaceutical or cosmetic purposes. An innovative technique called AFM-IR, was used to elucidate the behavior of JNP after cutaneous application. This instrument, coupling atomic force microscopy and IR spectroscopy, allowing to perform chemical analysis at the nanometer scale thanks to local absorption measurements. The identification of organic molecules at the nanoscale is possible without any labelling. Before cutaneous application of JNP, the nano-structure of untreated human skin was investigated with AFM-IR. Then, in vitro human skin penetration of JNP was studied using Franz cells, and AFM-IR allowed us to perform ultra-local information investigations.

Fluctuation-Based Super-Resolution Traction Force Microscopy.

Cellular mechanics play a crucial role in tissue homeostasis and are often misregulated in disease. Traction force microscopy is one of the key methods that has enabled researchers to study fundamental aspects of mechanobiology; however, traction force microscopy is limited by poor resolution. Here, we propose a simplified protocol and imaging strategy that enhances the output of traction force microscopy by increasing i) achievable bead density and ii) the accuracy of bead tracking. Our approach relies on super-resolution microscopy, enabled by fluorescence fluctuation analysis. Our pipeline can be used on spinning-disk confocal or widefield microscopes and is compatible with available analysis software. In addition, we demonstrate that our workflow can be used to gain biologically relevant information and is suitable for fast long-term live measurement of traction forces even in light-sensitive cells. Finally, using fluctuation-based traction force microscopy, we observe that filopodia align to the force field generated by focal adhesions.

Graphene-based sensing of oxygen transport through pulmonary membranes.

Lipid-protein complexes are the basis of pulmonary surfactants covering the respiratory surface and mediating gas exchange in lungs. Cardiolipin is a mitochondrial lipid overexpressed in mammalian lungs infected by bacterial pneumonia. In addition, increased oxygen supply (hyperoxia) is a pathological factor also critical in bacterial pneumonia. In this paper we fabricate a micrometer-size graphene-based sensor to measure oxygen permeation through pulmonary membranes. Combining oxygen sensing, X-ray scattering, and Atomic Force Microscopy, we show that mammalian pulmonary membranes suffer a structural transformation induced by cardiolipin. We observe that cardiolipin promotes the formation of periodic protein-free inter-membrane contacts with rhombohedral symmetry. Membrane contacts, or stalks, promote a significant increase in oxygen gas permeation which may bear significance for alveoli gas exchange imbalance in pneumonia.

Tribo-Induced Interfacial Material Transfer of an Atomic Force Microscopy Probe Assisting Superlubricity in a WS2/Graphene Heterojunction.

Robust superlubricity of 2D materials could be obtained by transferring graphene on the tip surface for the formation of interlayer friction of heterojunction, owing to the availability of stable interfacial incommensurate contact. Nevertheless, the material transfer mechanisms assisting superlubricity via atomic force microscopy (AFM) probe are still hardly comprehended. In this work, we reported a superlow friction coefficient (0.003) of the WS2/graphene heterojunction governed by graphene flake-transferred AFM tips and achieved a superlubricity state of velocity independence. Both low adhesion of the heterojunction and excellent wear-resistance for tip were also observed, which were attributed to the extremely low interface interaction during the incommensurate contact. The in-depth investigation on the frictional contact zones of probes was performed through high-resolution transmission electron microscopy. The observations emphasize the prevailing mechanisms of tribo-induced interfacial material transfer when AFM probes scan on the surface of 2D materials. The evolution of the superlubricity state principally depends on the establishment of interfacial nanostructures in the self-adaptive running-in period, by different contact mechanics and tribo-reconstructing pathways. These results stimulate a technical route to develop superlubricious tribopairs of 2D materials and guide a promising perspective in the engineering system.

Fabrication of a Biocompatible Mica/Gold Surface for Tip-Enhanced Raman Spectroscopy.

Tip-enhanced Raman spectroscopy (TERS) is a promising technique for structural studies of biological systems and biomolecules, owing to its ability to provide a chemical fingerprint with sub-diffraction-limit spatial resolution. This application of TERS has thus far been limited, due to difficulties in generating high field enhancements while maintaining biocompatibility. The high sensitivity achievable through TERS arises from the excitation of a localized surface plasmon resonance in a noble metal atomic force microscope (AFM) tip, which in combination with a metallic surface can produce huge enhancements in the local optical field. However, metals have poor biocompatibility, potentially introducing difficulties in characterizing native structure and conformation in biomolecules, whereas biocompatible surfaces have weak optical field enhancements. Herein, a novel, biocompatible, highly enhancing surface is designed and fabricated based on few-monolayer mica flakes, mechanically exfoliated on a metal surface. These surfaces allow the formation of coupled plasmon enhancements for TERS imaging, while maintaining the biocompatibility and atomic flatness of the mica surface for high resolution AFM. The capability of these substrates for TERS is confirmed numerically and experimentally. We demonstrate up to five orders of magnitude improvement in TERS signals over conventional mica surfaces, expanding the sensitivity of TERS to a wide range of non-resonant biomolecules with weak Raman cross-sections. The increase in sensitivity obtained through this approach also enables the collection of nanoscale spectra with short integration times, improving hyperspectral mapping for these applications. These mica/metal surfaces therefore have the potential to revolutionize spectromicroscopy of complex, heterogeneous biological systems such as DNA and protein complexes.

Examining Cell-Cell Interactions in the Kidney Using AFM Single-Cell Force Spectroscopy.

The ability of individual cells to synchronize activity is a basic feature of efficient and appropriate tissue function. Central to this is the physicochemical binding between cells through multiprotein complexes that functionally mediate adhesion. Importantly, the direct connection of physical properties and intercellular signaling is of great importance to certain pathologies including diabetes. Atomic force microscopy (AFM) single-cell force spectroscopy (SCFS) is a high-resolution technique that provides a statistically reliable measurement of the minute forces involved in cell tethering and membrane dynamics. Detection of altered nanoscale forces underlying the loss of adhesion in early tubular injury is pivotal for the development of novel therapeutic strategies for diabetic nephropathy. Here we describe the step-by-step use of an integrated AFM-SCFS system designed to measure functional force-displacement in separating renal tubular epithelial cells. Parameters such as unbinding forces, detachment energy, and distance to complete separation can be obtained from force-displacement (F-d) curves and are critical in assessing how physical changes of cellular adhesion contribute to cell contact, coupling, and communication in the diabetic kidney.