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)'.

BioAFMviewer: An interactive interface for simulated AFM scanning of biomolecular structures and dynamics.

We provide a stand-alone software, the BioAFMviewer, which transforms biomolecular structures into the graphical representation corresponding to the outcome of atomic force microscopy (AFM) experiments. The AFM graphics is obtained by performing simulated scanning over the molecular structure encoded in the corresponding PDB file. A versatile molecular viewer integrates the visualization of PDB structures and control over their orientation, while synchronized simulated scanning with variable spatial resolution and tip-shape geometry produces the corresponding AFM graphics. We demonstrate the applicability of the BioAFMviewer by comparing simulated AFM graphics to high-speed AFM observations of proteins. The software can furthermore process molecular movies of conformational motions, e.g. those obtained from servers which model functional transitions within a protein, and produce the corresponding simulated AFM movie. The BioAFMviewer software provides the platform to employ the plethora of structural and dynamical data of proteins in order to help in the interpretation of biomolecular AFM experiments.

Resolution-Free Accurate DNA Contour Length Estimation from Atomic Force Microscopy Images.

This research presented an accurate and efficient contour length estimation method developed for DNA digital curves acquired from Atomic Force Microscopy (AFM) images. This automation method is calibrated against different AFM resolutions and ideal to be extended to all different kinds of biopolymer samples, encompassing all different sample stiffnesses. The methodology considers the digital curve local geometric relationship, as these digital shape segments and pixel connections represent the actual morphology of the biopolymer sample as it is being imaged from the AFM scanning. In order to incorporate the true local geometry relationship that is embedded in the continuous form of the original sample, one needs to find this geometry counterpart in the digitized image. This counterpart is realized by taking the skeleton backbone of the sample contour and by using these digitized pixels' connection relationship to find its local shape representation. In this research, one uses the 8-connect Freeman Chain Code (CC) to describe the directional connection between DNA image pixels, in order to account for the local shapes of four connected pixels. The result is a novel shape number (SN) system derived from CC, which is a fully automated algorithm that can be applied to DNA samples of any length for accurate estimation, with efficient computational cost. This shape-wise consideration is weighted to modify the local length with great precision, accounting for all the different morphologies of the biopolymer sample, and resulted with accurate length estimation, as the error falls below 0.07%, an order of magnitude improvement compared to previous findings.

Inverse tissue mechanics of cell monolayer expansion.

Living tissues undergo deformation during morphogenesis. In this process, cells generate mechanical forces that drive the coordinated cell motion and shape changes. Recent advances in experimental and theoretical techniques have enabled in situ measurement of the mechanical forces, but the characterization of mechanical properties that determine how these forces quantitatively affect tissue deformation remains challenging, and this represents a major obstacle for the complete understanding of morphogenesis. Here, we proposed a non-invasive reverse-engineering approach for the estimation of the mechanical properties, by combining tissue mechanics modeling and statistical machine learning. Our strategy is to model the tissue as a continuum mechanical system and to use passive observations of spontaneous tissue deformation and force fields to statistically estimate the model parameters. This method was applied to the analysis of the collective migration of Madin-Darby canine kidney cells, and the tissue flow and force were simultaneously observed by the phase contrast imaging and traction force microscopy. We found that our monolayer elastic model, whose elastic moduli were reverse-engineered, enabled a long-term forecast of the traction force fields when given the tissue flow fields, indicating that the elasticity contributes to the evolution of the tissue stress. Furthermore, we investigated the tissues in which myosin was inhibited by blebbistatin treatment, and observed a several-fold reduction in the elastic moduli. The obtained results validate our framework, which paves the way to the estimation of mechanical properties of living tissues during morphogenesis.

Klebsiella pneumoniae antibiotic resistance identified by atomic force microscopy.

In the last decade the detection of the resistance of bacteria to antibiotics treatment, developed by different kind of bacteria, is becoming a huge problem. We hereby present a different approach to the current problem of detection of bacteria resistance to antibiotics. Our aims were to use the atomic force microscopy (AFM) to investigate bacteria morphological changes in response to antibiotics treatment and explore the possibility of reducing the time required to obtain information on their resistance. In particular, we studied Klebsiella pneumoniae bacteria provided by the Lavagna Hospital ASL4 Liguria (Italy), where there are cases linked with antibiotics resistance of the Klebsiella pneumoniae. By comparing AFM images of bacteria strains treated with different antibiotics is possible to identify unambiguously the Klebsiella pneumoniae strains resistant to antibiotics. In fact, the analysis of the AFM images of the antibiotic-sensitive bacteria shows clearly the presence of morphological alterations in the cell wall. While in the case of the antibiotic-resistant bacteria morphological alterations are not present. This approach is based on an easy and potentially rapid AFM analysis.

Avaliação in vitro e in vivo de superfícies de titânio revestidas com vidro bioativo / In vitro and in vivo evaluation of a bioactive titanium surface modified by bioglass coating

Objetivo: Esse estudo experimental in vitro e in vivo testou a capacidade de osseoindução de uma nova superfície de titânio nanoestruturada revestida com vidro bioativo contendo fosfato de cálcio. Metodologia: A rugosidade superficial foi avaliada por microscopia de força atômica utilizando 9 corpos de prova dos três diferentes grupos: titânio microtexturizado (Ticp) e revestidos com vidro bioativo e secos nas temperaturas de 370 C (BGTi37) ou 6000 C (BGTi600). Células osteoblásticas primárias obtidas das calvárias de ratos neonatos foram cultivadas in vitro em meio α-MEM suplementado em contato ou não (controle) com discos de titânio microtexturizado (Ticp) e revestidos (BGTi37 e BGTi600). A viabilidade celular e produção de fosfatase alcalina foram avaliadas após 7 dias de cultura e a mineralização após 14 dias de cultura. Os dados foram submetidos a análise de variância (ANOVA) seguido pelo teste de Tukey, com nível de significância de 5%. A morfologia dos osteoblastos em contato com as três superfícies foi avaliado por microscopia eletrônica de varredura após 7 e 14 dias. Quatorze parafusos de titânio microtexturizados (Ticp -controle) e quatorze parafusos experimentais revestidos com vidro bioativo e secos à 370 C (BGTi37) foram instalados aleatoriamente nas tíbias de 14 ratos Wistar. Os animais foram eutanasiados após 14 e 28 dias e suas tíbias preparadas e analisadas por microtomografia computadorizada. Resultados: O grupo Ticp apresentou a maior rugosidade média(129,6 nm), seguido do grupo BGTi600 (91,85 nm), que foram estatisticamente semelhantes. O grupo BGTi37 apresentou a menor rugosidade(74,51 nm), sendo significativamente menor do que os outros dois grupos. A proporção de células viáveis, a produção de fosfatase alcalina e a mineralização do grupo BGTi600 foi significativamente menor do que as do grupo controle e do Ticp. Para os demais grupos (BGTi37, Ticp e controle),a proporção de células viáveis, produção de fosfatase alcalina e mineralização foram semelhantes. O número de osteoblastos em contato com todas as superfícies foi maior no período de 14 dias comparado ao período de 7 dias. A maior quantidade de osteoblastos foi observada em contato com a superfície de Ticp e a menor quantidade em contato com a superfície de BGTi600. Os osteoblastos em contato com a superfície Ticp apresentaram-se com morfologia poligonal e maiores do que os dos grupos BGTi37 e BGTi600, que apresentam-se com morfologia mais alongada, mais notadamente no grupo BGTi600. A quantidade de prolongamentos citoplasmáticos, junções intercelulares e vesículas observadas nos espécimes do grupo BGTi600 foi notadamente menor do que nos grupos Ticp e BGTi37. Os parâmetros avaliados por microtomografia computadorizada da cortical e da medular ósseas em torno dos parafusos experimentais (BGTi37) e controles (Ticp) foram estatisticamente semelhantes. Conclusões: A superfície BGTi37 apresentou comportamento biológico semelhante à uma superfície de titânio microtexturizada (Ticp), com ótimos resultados de longo prazo já consolidados na literatura. Fato bastante promissor, considerando as possibilidades de aprimoramento dessa superfície experimental em futuros estudos.

Objectives: This experimental in vitro and in vivo study tested the osteoinduction ability of a new nanostructured titanium surface coated with bioglass with calcium phosphate. Methods: Surface roughness was evaluated by atomic force microscopy using 9 specimens of three groups: sandblasting and acid etching commercially pure titanium (cpTi) and bioglass coated dried at temperatures of 370 C (BGTi37) or 6000 C (BGTi600). Rat calvarial osteogenic cells were cultured in supplemented α-MEM medium in contact or not (control) with sandblasting and acid etching (SLA) commercially pure titanium discs (cpTi) and bioglass coated (BGTi37 and BGTi600). Cell viability and alkaline phosphatase (ALP) activity were measured after 7 days of culture. The mineralization was assessed after 14 days of culture. The data were compared by analysis of variance (ANOVA) complemented by Tukey test. The level of significance was 5%. Scanning electron microscopy after 7 and 14 days assessed osteoblasts morphology in contact with the three surfaces. Fourteen SLA commercially pure titanium screws (cpTi -control) and fourteen experimental screws bioglass coated dried at temperatures of 370 C (BGTi37) were randomly placed into 14 male Wistar rats' tibiae. The animals were sacrificed after 14 and 28 days and their tibias processed for micro-CT analysis. Results: The cpTi group (129.6 nm) showed the highest average roughness, followed by BGTi600 group (91.85 nm), which were statistically similar. The BGTi37 group (74.51 nm) showed the lowest surface roughness compared to the other two groups. Cell viability, ALP activity and mineralization of BGTi600 group were significantly lower than the control and cpTi groups. BGTi37, cpTi and control groups showed no significant differences in cell viability, ALP activity and mineralization. The number of cells in contact with all surfaces was higher in 14 days compared to 7 days. Higher amount of osteoblasts was observed in contact with the cpTi surface and the smaller amount in contact with the BGTi600 surface. Osteoblasts in contact to cpTi surface showed a flat polygonal shape and were larger than the BGTi37 and BGTi600 groups, which presented with a sharper morphology, most notably in the BGTi600 group. The number of cytoplasmic processes, intercellular junctions and vesicles observed in specimens of BGTi600 group was markedly lower than in cpTi and BGTi37 groups. The micro-CT parameters of the cortical and trabecular bone around the experimental (BGTi37) and controls (Ticp) screws presented no statistical differences. Conclusions: The BGTi37 surface showed biological behavior similar to a SLA titanium surface (cpTi), with excellent long-term results already established in the literature. A very promising fact, considering the improvement possibilities of this experimental surface in future studies

Desenvolvimento e avaliação de eficácia de nanoemulsão catiônica bioativa na proteção capilar aos danos foto-oxidativos / Development and evaluation of the effectiveness of bioactive cationic nanoemulsion in protecting hair photo-oxidative damage

A radiação solar, composta por radiação ultravioleta (UV), visível (Vis) e infravermelho, é responsável por acelerar os processos de alteração de cor e do conteúdo proteico da fibra capilar. Visando contornar este problema, este trabalho propõe a incorporação do flavonoide quercetina, de reconhecida atividade antioxidante, em uma nanoemulsão catiônica de aplicação capilar. Para tanto, foram desenvolvidas formulações contendo quercetina a 0,5% (p/p) pelo método de baixa energia sub-PIT. A formulação de menor índice de polidispersão (IPD) foi selecionada e submetida à Avaliação de Estabilidade Normal. Neste ensaio, a nanoemulsão foi armazenada em diferentes condições de temperatura por 90 dias, sendo analisados: características organolépticas, valor de pH, atividade antioxidante, conteúdo de quercetina, diâmetro médio de gotícula e potencial zeta. A fotoestabilidade da nanoemulsão envolveu a determinação do perfil de absorção e da sua atividade antioxidante após períodos de exposição à radiação UV/Vis. Posteriormente, a nanoemulsão foi caracterizada quanto aos seguintes parâmetros: eficiência de encapsulamento, perfil reológico, morfologia das gotículas por Microscopia Eletrônica de Transmissão Criogênica e Microscopia de Força Atômica (AFM). A possível interação entre a quercetina e os demais tensoativos presentes na nanoemulsão foi avaliada por Microscopia Confocal de Fluorescência e Análise térmica. A segurança da nanoemulsão foi determinada pelo método in vitro HET-CAM e por biocompatibilidade cutânea, em voluntários. A eficácia da nanoemulsão catiônica na fotoproteção das características da fibra capilar descolorida tratada com tintura cores loiro (12.0) ou ruivo (6.66) foi determinada avaliando-se os parâmetros cor, tração à ruptura, penteabilidade, fricção, perda proteica, morfologia das cutículas e nível de melanina radical por Espectroscopia de Ressonância Paramagnética Eletrônica (EPR), sendo calculado o Fator de Proteção Radicalar (FPR). As mechas de cabelo tingidas foram expostas à radiação UV/Vis artificial (500 W/m2) por até 180 h, sendo os parâmetros analisados antes e após o período de exposição. A nanoemulsão selecionada pelo reduzido IPD apresentava diâmetro médio de gotícula e potencial zeta iguais a 24,97±0,30 nm e 19,6±2,19 mV, respectivamente. Na Avaliação de Estabilidade Normal, a nanoemulsão armazenada a 45,0±2,0° C apresentou alterações significativas de todos os parâmetros avaliados, exceto potencial zeta, sendo que a elevação do diâmetro médio de gotícula acarretou em perda da transparência. A oxidação da quercetina e a instabilidade do tipo Ostwald ripening (ω3) foram as responsáveis pelas modificações observadas. No armazenamento a 5,0±2,0° C, a nanoemulsão manteve todos os parâmetros inalterados, mas a 25±2,0° C houve elevação discreta do diâmetro médio de gotícula, sem perda da funcionalidade. A nanoemulsão apresentou elevada fotoestabilidade, sem alteração da atividade antioxidante após exposição ao UV/Vis. A caracterização da nanoemulsão mostrou que a eficiência de encapsulamento foi de 99,8%, no mínimo, a formulação apresentou típico comportamento newtoniano e as gotículas apresentavam formato esférico. As imagens obtidas por Microscopia Confocal de Fluorescência e o ensaio de Análise térmica mostraram que a quercetina se encontra dentro das gotícula atuando, também, como co-tensoativo, por interagir com os tensoativos, além de exercer sua função antioxidante. A nanoemulsão foi classificada como levemente irritante (método HET-CAM), sendo esse baixo potencial de irritação corroborado pelo teste de biocompatibilidade cutânea. Na avaliação de eficácia, observou-se que a nanoemulsão protegeu a cor total (dE*) do cabelo tingido de loiro em 54%, e elevou a alteração da cor do cabelo tingido de ruivo em 47% (t = 180 h) em comparação à mecha controle. Além disso, a nanoemulsão melhorou a penteabilidade e reduziu os coeficientes de fricção. A radiação UV/Vis provocou elevada perda proteica e redução da espessura das cutículas em aproximadamente 50%. Concluiu-se, pelos resultados obtidos, que as moléculas que compoem a tintura capilar, principalmente os pigmentos mais escuros, atuaram como filtros solares, pois elas protegeram as estruturas proteicas da fibra. A nanoemulsão apresentou FPR igual a 3,31 e 4,14, para as mechas tingidas de loiro e ruivo, respectivamente. O FPR indica a capacidade de uma formulação em reduzir o nível de radicais livres formados por indução da radiação UV/Vis, um dos fatores que induzem alterações na fibra capilar tingida. Assim, considerando que a radiação UV/Vis atua tanto por mecanismos diretos quanto indiretos, e que alterações significativas de cor foram observadas mesmo quando o nível de radicais livres foi reduzido pela ação da quercetina, deve ser incorporada à formulação fotoprotetora capilar filtros solares associados a antioxidantes nanoestruturados. Tais filtros devem ficar aderidos à cutícula, de modo a protegê-la da degradação proteica e reduzir a entrada de radiação para o interior da fibra capilar, local onde os antioxidantes nanoestruturados devem atuar como uma segunda linha de defesa

The solar radiation, comprising ultraviolet (UV), visible (VIS) and infrared, is responsible for accelerating color and protein content changes in the hair fiber. In order to avoid this problem, this work proposes the incorporation of the flavonoid quercetin, a recognized antioxidant molecule, in a cationic nanoemulsion for hair application. For this, formulations containing quercetin 0.5% (w/w) were developed by the low-energy sub-PIT method. The formulation with a lower polydispersity index (PDI), which had HLB value (Hydrophilic-Lipophilic Balance) equal to 12.5 was selected and subjected to the Normal Stability Test. In this assay, the nanoemulsion was stored under different temperature conditions for 90 days, and the following parameters were analyzed: organoleptic properties, pH, antioxidant activity, quercetin content, average droplet diameter and zeta potential. The photostability of the nanoemulsion involved the determination of the absorption profile and its antioxidant activity after periods of exposure to UV/Vis radiation. Subsequently, the nanoemulsion was characterized according to the following parameters: encapsulation efficiency, rheological profile, morphology of the droplets by Cryogenic Transmission Electron Microscopy and Atomic Force Microscopy (AFM). The possible interaction between quercetin and other surfactants present in the nanoemulsion was evaluated by Confocal Fluorescence Microscopy and thermal analysis. The safety of the nanoemulsion was assessed by the in vitro HET-CAM method and by cutaneous biocompatibility in volunteers. The photoprotection effectiveness of the bioactive cationic nanoemulsion was evaluated in blond (color 12.0) and auburn (color 6.66) dyed hair fibers by assessing the parameters: color, tensile break, combing, friction, protein loss, morphology of cuticles and level of melanin radical by Electron Paramagnetic Resonance Spectroscopy (EPR). The Radical Hair Protection Factor (RHF) was calculated. Dyed hair tresses were exposed to UV/Vis artificial radiation (500 W/m2) for 180 h. The parameters were analyzed before and after the exposure period. The nanoemulsion selected due to its reduced PDI had an average droplet diameter and zeta potential equal to 24.97±0.30 nm and 19.6±2.19 mV, respectively. In the Normal Stability Test, the nanoemulsion stored at 45.0 ± 2.0º C showed significant changes in all parameters except zeta potential, and the increase in the average droplet diameter resulted in the loss of transparency. Oxidation of quercetin and Ostwald ripening instability (ω3) were responsible for the changes. At 5.0 ± 2.0º C, the nanoemulsion kept all parameters unchanged, but at 25.0±2.0º C there was a slight increase in the average droplet diameter without loss of functionality. The nanoemulsion showed high photostability since antioxidant activity was not altered after UV/Vis exposure. The characterization of the nanoemulsion showed that the encapsulation efficiency was 99.8% at least, the formulation showed typical Newtonian behavior and droplets were spherical. The images obtained by Confocal Fluorescence Microscopy and thermal analysis showed that quercetin was within the droplet acting, also, as a cosurfactant, due to the interaction with the surfactants. The nanoemulsion was classified as slightly irritating (HET-CAM method), and this low irritation potential was supported by the cutaneous biocompatibility assay. The photoprotective effectiveness evaluation showed that the nanoemulsion protected the total color (dE*) of blond dyed hair in 54%, but raised the color change of auburn dyed hair in 47% (t = 180 h). In addition, the nanoemulsion improved combability and reduced coefficients of friction. UV/Vis radiation caused high protein loss and reduced cuticle thickness by approximately 50%. It was concluded that the molecules that compose hair dye, especially the darker pigments, acted as sun filters because they protected the protein structures of the hair fiber. The nanoemulsion showed RHF equal to 3.31 and 4.14 for blond and auburn dyed hair, respectively. The RHF indicates the ability of a formulation to reduce the level of free radicals formed by UV/Vis induction, one of the factors that induce changes in the dyed hair fibers. Thus, considering that the UV/Vis radiation acts by direct and indirect mechanisms and that significant changes in color were observed even when the level of free radicals has been reduced by the quercetin, chemical filters should be incorporated into hair formulations associated with nanostructured antioxidants in order to fully protect hair fiber. Such filters must be attached to the cuticle, protecting it from protein degradation and reducing the radiation input into the hair fiber, where the nanostructured antioxidants must act as a second line of defense

Functional extension of high-speed AFM for wider biological applications.

High-speed atomic force microscopy (HS-AFM) has been established and used, which can visualize biomolecules in dynamic action at high spatiotemporal resolution without disturbing their function. Various studies conducted in the past few years have demonstrated that the dynamic structure and action of biomolecules revealed with HS-AFM can provide greater insights than ever before into how the molecules function. However, this microscopy has still limitations in some regards. Recently, efforts have been carried out to overcome some of the limitations. As a result, it has now become possible to visualize dynamic processes occurring even on live cells and perform simultaneous observations of topographic and fluorescent images at a high rate. In this review, we focus on technical developments for expanding the range of objects and phenomena observable by HS-AFM as well as for granting multiple functionalities to HS-AFM.

Quantitative analysis of dynamic adhesion properties in human hepatocellular carcinoma cells with fullerenol.

In this study, the effect of fullerenol (C60(OH)24) on the cellular dynamic biomechanical behaviors of living human hepatocellular carcinoma (SMCC-7721) cancer cells were investigated by atomic force microscope (AFM) nanoindentation. As an important biomarker of cellular information, the cell adhesion is essential to maintain proper functioning as well as links with the pathogenesis and canceration. Nonetheless, it is challenging to properly evaluate the complex adhesion properties as all the biomechanical parameters interfere with each other. To investigate the dynamic adhesion changes, especially in the case of the fullerenol treatment, the detachment force and work, adhesion events, and membrane tether properties were measured and analyzed systematically with the proposed quantitative method. The statistical analyses suggest that, under the same operating parameters of AFM, the dependence of adhesion energy on the tip-cell contact area is weakened after the fullerenol treatment and the probability of adhesion decreases significantly from 30.6% to 4.2%. In addition, the disruption of the cytoskeleton resulted in a 34% decrease of the average membrane tether force and a 21% increase of the average tether length. Benefiting from the quantitative method, this work contributes to revealing the effects of fullerenol on the cellular biomechanical properties of the living SMCC-7721 cells in a precise and rigorous way and additionally is further instructive to interpret the interaction mechanism of other potential nanomedicines with living cells.

Single molecule binding dynamics measured with atomic force microscopy.

We present a new method to analyse simultaneous Topography and RECognition Atomic Force Microscopy data such that it becomes possible to measure single molecule binding rates of surface bound proteins. We have validated this method on a model system comprising a S-layer surface modified with Strep-tagII for binding sites and strep-tactin bound to an Atomic Force Microscope tip through a flexible Poly-Ethylene-Glycol linker. At larger distances, the binding rate is limited by the linker, which limits the diffusion of the strep-tactin molecule, but at lateral distances below 3 nm, the binding rate is solely determined by the intrinsic molecular characteristics and the surface geometry and chemistry of the system. In this regime, Kon as determined from single molecule TREC data is in agreement with Kon determined using traditional biochemical methods.

Magnetic force microscopy of superparamagnetic nanoparticles.

The use of magnetic force microscopy (MFM) to detect probe-sample interactions from superparamagnetic nanoparticles in vitro in ambient atmospheric conditions is reported here. By using both magnetic and nonmagnetic probes in dynamic lift-mode imaging and by controlling the direction and magnitude of the external magnetic field applied to the samples, it is possible to detect and identify the presence of superparamagnetic nanoparticles. The experimental results shown here are in agreement with the estimated sensitivity of the MFM technique. The potential and challenges for localizing nanoscale magnetic domains in biological samples is discussed.

Sensitivity of surface roughness parameters to changes in the density of scanning points in multi-scale AFM studies. Application to a biomaterial surface.

In the field of biomaterials surfaces, the ability of the atomic force microscope (AFM) to access the surface structure at unprecedented spatial (vertical and lateral) resolution, is helping in a better understanding on how topography affects the overall interaction of biological cells with the material surface. Since cells in a wide range of sizes are in contact with the biomaterial surface, a quantification of the surface structure in such a wide range of dimensional scales is needed. With the advent of the AFM, this can be routinely done in the lab. In this work, we show that even when it is clear that such a scale-dependent study is needed, AFM maps of the biomaterial surface taken at different scanning lengths are not completely consistent when they are taken at the same scanning resolution, as it is usually done: AFM images of different scanning areas have different point-to-point physical distances. We show that this effect influences the quantification of the average (R(a)) and rms (R(q)) roughness parameters determined at different length scales. This is the first time this inconsistency is reported and should be taken into account when roughness is measured in this way. Since differences will be in general in the range of nanometres, this is especially interesting for those processes involving the interaction of the biomaterial surface with small biocolloids as bacteria, while this effect should not represent any problems for larger animal cells.

Imaging nanoscale spatio-temporal thermal fluctuations.

Spatial and temporal fluctuations of the electric polarization were imaged in polymer thin films near the glass transition using electric force microscopy. Below the glass transition the fluctuations are quasi-static, and spatial fluctuations were found to quantitatively agree with predictions for thermal fluctuations. Temporal fluctuations appear near the glass transition. Images of the space-time nanoscale dynamics near the glass transition are produced and analyzed.

Atomic force microscopy measurements of protein-ligand interactions on living cells.

Cell adhesion receptors are expressed on the surface of cells and can mediate binding to other cells and to the extracellular matrix. Here, we describe in detail the use of atomic force microscopy (AFM)-based force spectroscopy for studying cell detachment forces on living leukocytes. With this technique it is now possible to measure force with resolution down to the level of individual molecules. AFM force spectroscopy is particularly well suited for research in cell adhesion, which has relevance in both the medical and life sciences including immunology, cancer and stem cell research, and human pharmacology. Along with its limitations, we herein, describe how the rupture force of a single complex formed between the integrin receptor leukocyte function-associated antigen (LFA)-1, expressed on the surface of a living leukocyte, and immobilized intercellular adhesion molecule-1 (ICAM-1) was measured. With only minor modifications this protocol can be used to study other adhesion receptors on almost any mammalian cell or bacterial system. This protocol is also suitable for studying single-molecule de-adhesion events in cell-free systems as well as between two living cells.

Accurate height and volume measurements on soft samples with the atomic force microscope.

The suitability of three common atomic force microscope (AFM) imaging modes for quantitative height and volume measurements on soft samples was investigated. The height and volume of rehydrated human metaphase chromosomes in liquid were measured using the contact mode, the tapping mode, and the force mapping mode. In both the contact and tapping modes, the measured height and volume strongly depended on the imaging setpoint that sets the imaging force. Measurement deviations up to 50% were observed. The force mapping mode, on the other hand, yielded reproducible height and volume measurements independent of the imaging force. It is therefore suggested that the force mapping mode should be used whenever the height or volume of soft samples need to be accurately determined.

Combined nanomanipulation by atomic force microscopy and UV-laser ablation for chromosomal dissection.

Nanomanipulation and nanoextraction on a scale close to and beyond the resolution limit of light microscopy is needed for many modern applications in biological research. For the manipulation of biological specimens a combined microscope allowing for ultraviolet (UV) microbeam laser manipulation together with manipulation by an atomic force microscope (AFM) was used. In a one-step procedure, human metaphase chromosomes were dissected optically by the UV-laser ablation and mechanically by AFM manipulation. With both methods, sub-400-nm cuts could be achieved routinely. Thus, the AFM is an indispensable tool for in situ quality control of nanomanipulation. However, already on this scale the dilation of the topographic AFM image due to the tip geometry can become significant. Therefore the AFM images were restored using a tip geometry obtained by a blind tip-reconstruction algorithm. Cross-sectional analysis of the restored image reveals a 380-nm-wide UV-laser cut and AFM cuts between 70 nm and 280 nm.

Atomic force microscopy imaging of novel type of polymeric colloidal nanostructures.

Polymeric nanoparticles were prepared by the interfacial poly-condensation of the lipophilic monomer, phtaloyldichloride and the hydrophilic monomer, diethylenetriamine, in the presence and absence of the surfactant Pluronic F68. The colloidal systems were analysed by dynamic light scattering and atomic force microscopy, the structures formed have two populations (150 and 350 nm) in the presence of the surfactant and one population (450 nm) in the absence of the surfactant. The results can be interpreted in terms of the formation of hollow nanocapsules that collapse on deposition and drying.

Effect of experimental fluorosis on the surface topography of developing enamel crystals.

Dental fluorosis is an increasing problem, yet the precise mechanism by which fluoride exerts its effects remains obscure. In the present study, we have used atomic force microscopy to image and quantitate surface features of enamel crystals isolated from specific developmental stages of fluorotic and control rat incisors. The results showed a significant decrease in crystal surface roughness with development in control tissue. Crystals from fluorotic tissue were significantly rougher than controls at all stages of development, did not decrease in roughness during the later stages of their development and had many morphological abnormalities. These data clearly demonstrate an effect for fluoride on enamel crystal surfaces which could reflect changes in the nature and distribution of growth sites and/or in mineral-matrix interactions. These would be expected to affect crystal growth during maturation, resulting in the characteristic porous appearance of fluorotic lesions in mature teeth.