Atomic Force Microscopy Methods Research Articles

Effect of F-Actin Organization in Lamellipodium on Viscoelasticity and Migration of Huh-7 Cells Under pH Microenvironments Using AM-FM Atomic Force Microscopy

Cytoskeleton is responsible for fundamental cellular processes and functions. The filamentous actin (F-actin) is a key constituent of the cytoskeleton system which is intrinsically viscoelastic and greatly determines the mechanical properties of cells. The organization and polymerization of F-actin are relevant to the viscoelasticity distribution and the migration of living cells responding to pH microenvironments. Recently, progression in various diseases such as cancers have been found that cellular migration is related to the alterations in the viscoelasticity of lamellipodium. However, the correlation among F-actin organization, viscoelastic properties and cellular migration of living cancer cells under different pH microenvironments are still poorly understood. Conventional experimental methods of optical microscopy and atomic force microscopy (AFM) can neither break the trade-off between resolution and rate in cytoskeleton imaging, nor achieve the structural characterization and the mechanical measurement simultaneously. Although multifrequency AFM with amplitude modulation-frequency modulation (AM–FM) enables us to probe both the surface topography and the viscoelasticity distribution of cells, it is difficult to image the cytoskeletal filaments with the diameter down to the scale of tens of nanometers. Here, we have improved the AM-FM AFM by employing the high damping of cell culture medium to increase the signal-to-noise ratio and achieve a stable imaging of F-actin with the resolution down to 50 nm underin situmicroenvironment. The approach that can successfully visualize the structures of cytoskeletal filaments and measure the distribution of mechanical properties simultaneously enable us to understand the relationship between the organization of F-actin and the viscoelasticity of living Huh-7 cancer cells under different pH values. Our experimental results have demonstrated that, unlike the randomly distributed F-actin and the homogeneous viscoelasticity at the normal pH level of 7.4, the living Huh-7 cancer cells with the reduced pH level of 6.5 show highly oriented and organized F-actin along the lamellipodium direction associated with the significant gradient increase both in elasticity and viscosity, which are confirmed by immunofluorescence confocal microscopy. The F-actin organization and the gradient viscoelasticity of lamellipodium provide structural and mechanical understanding on the adhesion and migration of living cancer cells that undergo metastasis and malignant transformation.

Bioinformatics methods for identification of amyloidogenic peptides show robustness to misannotated training data

Several disorders are related to amyloid aggregation of proteins, for example Alzheimer’s or Parkinson’s diseases. Amyloid proteins form fibrils of aggregated beta structures. This is preceded by formation of oligomers—the most cytotoxic species. Determining amyloidogenicity is tedious and costly. The most reliable identification of amyloids is obtained with high resolution microscopies, such as electron microscopy or atomic force microscopy (AFM). More frequently, less expensive and faster methods are used, especially infrared (IR) spectroscopy or Thioflavin T staining. Different experimental methods are not always concurrent, especially when amyloid peptides do not readily form fibrils but oligomers. This may lead to peptide misclassification and mislabeling. Several bioinformatics methods have been proposed for in-silico identification of amyloids, many of them based on machine learning. The effectiveness of these methods heavily depends on accurate annotation of the reference training data obtained from in-vitro experiments. We study how robust are bioinformatics methods to weak supervision, encountering imperfect training data. AmyloGram and three other amyloid predictors were applied. The results proved that a certain degree of misannotation in the reference data can be eliminated by the bioinformatics tools, even if they belonged to their training set. The computational results are supported by new experiments with IR and AFM methods.

C subunit of the ATP synthase is an amyloidogenic calcium dependent channel-forming peptide with possible implications in mitochondrial permeability transition

The c subunit is an inner mitochondrial membrane (IMM) protein encoded by three nuclear genes. Best known as an integral part of the F0 complex of the ATP synthase, the c subunit is also present in other cytoplasmic compartments in ceroid lipofuscinoses. Under physiological conditions, this 75 residue-long peptide folds into an α-helical hairpin and forms oligomers spanning the lipid bilayer. In addition to its physiological role, the c subunit has been proposed as a key participant in stress-induced IMM permeabilization by the mechanism of calcium-induced permeability transition. However, the molecular mechanism of the c subunit participation in IMM permeabilization is not completely understood. Here we used fluorescence spectroscopy, atomic force microscopy and black lipid membrane methods to gain insights into the structural and functional properties of unmodified c subunit protein that might make it relevant to mitochondrial toxicity. We discovered that c subunit is an amyloidogenic peptide that can spontaneously fold into β-sheets and self-assemble into fibrils and oligomers in a Ca2+-dependent manner. C subunit oligomers exhibited ion channel activity in lipid membranes. We propose that the toxic effects of c subunit might be linked to its amyloidogenic properties and are driven by mechanisms similar to those of neurodegenerative polypeptides such as Aβ and α-synuclein.

Electroanalytical determination of Allura Red in beverage samples using an anodically pretreated graphite electrode

ABSTRACT In this study, an anodically pretreated pencil graphite electrode (Ptre/PGE) which has oxygen-containing groups was used for the electrochemical determination of Allura Red (AR) by differential pulse voltammetry (DPV) method for the first time in the literature. In order to be more selective and sensitive the pencil graphite electrode, the anodic pretreatment process was performed by applying a constant +1.8 V potential for 250 s in pH 9 phosphate buffer solution using chronoamperometry method. The characterisation studies of the sensor were performed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and atomic force microscopy (AFM) methods. Interference effects of sunset yellow, tartrazine, amaranth, aspartame, glucose, gelatin, and ascorbic acid on AR determination with Ptre/PGE were investigated. The limit of detection and quantitation values was found as 75 and 251 nmol L−1, respectively. The Ptre/PGE has been used for the determination of AR in cherry juice and energy drink samples, where it provides recovery ranging from 98.40% to 100.8%.

Optical properties of native (anodic) layer on the InAlAs surface of different morphology

The influence of InAlAs/InP(001) heterostructures surface morphology on the optical properties (dispersive refractive index and extinction coefficient) of the anodic layer, formed by the oxidation in low-energy Townsend gas-discharge O2-Ar-containing plasma at room temperature, was studied using atomic force microscopy and ellipsometry methods. High resolution transmission electron microscopy and X-ray photoelectron spectroscopy were used to determine the anodic layer morphology and chemical composition. It is shown that the growth structural defects (pits) on the InAlAs surface, with a density 106–107 cm−2, do not significantly influence the optical properties of the amorphous anodic layers, mainly consisting of In2O3, Al2O3, As2O3 and elemental arsenic quite uniformly distributed over their depth and area. The thickness of thin (≤10 nm) oxide layers on InAlAs is measured at a high accuracy by the nondestructive ellipsometry method using the optical model of a single-layer isotropic film on an absorbing substrate.

Cost‐effective and Facile Production of a Phosphorus‐doped Graphite Electrode for the Electrochemical Determination of Pyridoxine

AbstractIn this study; a sensitive, selective, and simple electrochemical sensor was developed to determine low concentration pyridoxine (Py) using a phosphorus‐doped pencil graphite electrode (P‐doped/PGE). Electrode modification was implemented using the chronoamperometry method at +2.0 V constant potential and 100 seconds in 0.1 mol L−1 H3PO4 supporting electrolyte solution. The characterization processes of the P‐doped/PGE were carried out using electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), X‐ray photoelectron spectroscopy (XPS), field emission scanning electron microscopy (FESEM), energy‐dispersive X‐ray spectroscopy (EDX), and atomic force microscope (AFM) methods. In the concentration study, using the differential pulse voltammetry (DPV) method, a linear calibration plot was acquired in the concentration range of 0.5 to 300 μmol L−1 Py. The limit of quantification (LOQ) and limit of detection (LOD) of the developed method were calculated as 0.219 μmol L−1 and 0.0656 μmol L−1, respectively. Detection of Py has been successfully performed on the P‐doped/PGE in the beverage samples. As a result, the method developed has been shown to have fast, low cost, and simple for the sensitive and selective detection of Py as an effective electrode.

Research of Structure of Elastic Self-Adhesive Radiation Shielding Coatings

An algorithm for studying the structure of radiation shielding materials using the atomic force microscopy (AFM) method has been developed and described. Using the proposed method, the structure of tungsten-containing radiation shielding materials was studied and the difference in the microstructure of the samples and the nature of the distribution of the filler were revealed.

Sub-nano to nanometer wear and tribocorrosion of titanium oxide-metal surfaces by in situ atomic force microscopy

Wear and tribocorrosion of passive oxide film covered metals have been studied at the micro and macroscopic scales. Recent advances in nanotechnology have contributed to breakthroughs in understanding of fundamental friction and wear mechanisms of atomically thin 2D materials at the nanoscale. However, for metals and materials without ultra-flat surfaces, a gap in knowledge exists at or below a few nanometers, which is too small for continuum mechanics theories and experiments including conventional atomic force microscopy (AFM) methods, due to resolution limits arising from surface roughness. Here, we report the near-atomic-scale wear of titanium in air and physiological solution from a single atomic layer to beyond the full oxide thickness using an AFM-based tribology method. Sub-nano to nanometer wear of titanium was revealed with different stages of contact pressure dependent wear regions identified as wear depth increased, featured by a transition from atomic wear (below 2.4 GPa) to elasto-plastic driven wear (above 3.6 GPa) at its oxide thickness (3.8 nm) in air. Higher stress was required to generate a similar wear penetration process in PBS compared to air. Tribocorrosion at this scale was grain orientation and voltage-dependent. Our study opens up a new method to achieve reliable angstrom-level resolution wear quantification to advance the understanding of wear and tribocorrosion of metals at the nanoscale. Statement of significanceExperimental tests of wear for metallic biomaterials at the nanoscale are difficult because engineered metal surfacesare never perfectly atomically flat, limiting the resolution of precise wear measurements to a few nanometers scale or more. To systematically address this problem, we have introduced the AFM ‘image-wear-image’ tribology method and obtained quantitative stress dependent measurement of the near-atomic-scale wear of titanium surfaces in air and tribocorrosion in physiological solution from a single atomic layer to beyond the full oxide film thickness. This allowedto measure sub-nano scale wear by partial removal of oxide. Nanoscale wear has been found to be grain orientation-dependent above the ‘atomic scale’ wear region. The nano-tribocorrosion of CP-Ti across scales and voltage effects on oxides in physiological solution was studied. Our study opens up a new method for future studies to advance the understanding of sub-nanoscale and nanoscale wear and tribocorrosion phenomenon as well as oxide growth mechanism of metallic biomaterials.

High performance, self-powered and thermally stable 200–750 nm spectral responsive gallium nitride (GaN) based broadband photodetectors

We have developed and first reported Au/Ni/Pr2O3/GaN broadband photodetector device with photoresponsivity from 205 nm to 750 nm under self-powered mode. Using the Tauc's plot, the direct optical bandgap was found to be 4.8, 2.45 and 2.35 eV for the as-deposited, 550 °C and 650 °C post annealed samples, respectively. XRD study revealed that the phase of the as-deposited and post-annealed Pr2O3 films on GaN was monoclinic in nature. Atomic force microscopy method was engaged in non-contact mode to investigate the surface morphology of Pr2O3 films on GaN and the rms surface roughness was improved for the 650 °C post-annealed sample as 1.1 nm compared to other samples. In XPS study, two main Pr3d well separated XPS peaks were noticed at 933.5 eV (m) and 953.9 eV (m′) which corresponds to spin-orbit doublets of 3d5/2 and 3d3/2, respectively. In addition, the transient response measurements of the fabricated 650 °C post annealed device confirmed good stability even for the applied bias voltage of 20 V in the entire UV region. Further, the 650 °C post annealed device at 20 V provided best rise and fall times of 0.57 s and 0.41 s at 360 nm, respectively. The responsivity of the fabricated BB PD device at 650 °C revealed the evidence of bias dependency because of photoconductive gain. The attained progress in the fabricated PD device performance was due to the improved light absorption as evidenced from the optical absorption study. This results the transport of photogenerated electron-hole pairs across the bandgap by limiting recombination/trap states and intentionally deposited LT GaN buffer layer at the interface of sapphire/GaN. These results suggest that the developed BB PD device could also have the potential usage in optoelectronic device applications.

Dynamic friction energy dissipation and enhanced contrast in high frequency bimodal atomic force microscopy

Dynamic friction occurs not only between two contact objects sliding against each other, but also between two relative sliding surfaces several nanometres apart. Many emerging micro- and nano-mechanical systems that promise new applications in sensors or information technology may suffer or benefit from noncontact friction. Herein we demonstrate the distance-dependent friction energy dissipation between the tip and the heterogeneous polymers by the bimodal atomic force microscopy (AFM) method driving the second order flexural and the first order torsional vibration simultaneously. The pull-in problem caused by the attractive force is avoided, and the friction dissipation can be imaged near the surface. The friction dissipation coefficient concept is proposed and three different contact states are determined from phase and energy dissipation curves. Image contrast is enhanced in the intermediate setpoint region. The work offers an effective method for directly detecting the friction dissipation and high resolution images, which overcomes the disadvantages of existing methods such as contact mode AFM or other contact friction and wear measuring instruments.

Surface structure of Gd20Co80 alloy

Gd 20 Co 80 alloy was studied applying experimental methods of spectral ellipsometry, atomic force and scanning tunneling microscopy. The experimental results exhibit the eutectic two-phase structure of this alloy. Clusters of a phase with a lower content had a lesser concentration of free carriers, which resulted in smaller tunnel currents during the measurements. To analyze the experimental data, a theoretical approach was developed, which was based both on the quantum-mechanical methods of configurational interaction and on three-diagonal Toeplitz matrices formalism. This approach allowed us to describe in detail the energy bands formation process in solid clusters with a relatively small quantity of atoms, which as a consequence enabled to describe the Shockley surface states as well as the existence of a surface layer with partially formed energy bands. Spectral-ellipsometric measurements of Gd 20 Co 80 alloy thin films confirmed a significant difference between the measured optical constants for 20-nm thick films and larger films. Quantum-mechanical molecular calculus allowed to obtain optical constants for several supercells of Gd-Co chemical compounds and confirmed the acquired experimental and theoretical results.

Preparation and growth mechanism of the flower-like whiskers of γ-, θ-, and α-Al2O3 phases by homogeneous precipitation/calcination method

The different alumina whiskers phases are extensively used to reinforce high-performance composite materials. The understanding of the mutual relationship of the different phases is key for simulating Al2O3 whiskers containing systems and is of general interest for the metal oxide and ceramic communities. In this paper, we used homogeneous precipitation to synthesized flower-like boehmite whisker precursors from aluminum sulfate and urea. The synthesized precursors were calcined at 600, 900, and 1200 °C to obtain flower-like whiskers of γ-, θ-, and α-Al2O3 phases, respectively. The precursor boehmite crystals prepared in closed, semi-closed, and open reactor systems were qualitatively analyzed using SEM. The results showed that the usea hydrolysis, which serves as a source of OH− ions, is slow under closed-reactor conditions, which favors the growth of the boehmite crystal nuclei into whiskers. We adopted the L16 (43) orthogonal design of experiment to analyze the influence of reaction temperature, time, and the amount of urea (relative to the amount of Al2(SO4)3) in the closed system. Synthesis temperature of 160 °C and the processing time of 8 h yielded optimal boehmite flower-like whiskers. Physicochemical properties of the precursor and series of alumina phases were characterized using thermogravimetric/differential thermal analysis (TG/DTA), X-ray diffraction (XRD), scanning electron microscopy (SEM), and atomic force microscopy (AFM) methods. Finally, the obtained results and the thorough analysis of the growth mechanism suggested the step-growth model of boehmite flower-like whiskers.

Structural Features of Textured Zinc-Oxide Films Obtained by the Ion-Beam Sputtering Method

Textured ZnO films obtained on amorphous substrates using the ion-beam sputtering method are studied. X-ray diffraction and atomic-force microscopy methods show that the resulting films have a polycrystalline structure immediately after deposition. It is established that further annealing of the samples in the temperature range from 200°C to 500°C results in recrystallization, which leads to changes in the grain size and surface roughness. A dependence of the crystallization intensity on the deposition conditions is found, which is related to the number of defects in the unannealed films. In films with an initially more perfect structure, heat treatment at 500°C results in the growth of grains by more than 2 times and a decrease in the roughness by ∼40%.

Optical diffraction from Ge2Sb2Te5 fishnet metasurfaces

We report the creation of Ge2Sb2Te5 metasurfaces on sapphire substrates by the ablation method and the study of their structural properties by scanning electron microscopy (SEM), atomic force microscopy (AFM), and optical diffraction. The main emphasis is on the optical technique, which boils down to obtaining bright Laue diffraction patterns on a screen, observing them with the naked eye, and analyzing the fine structure of diffraction reflections. It has been demonstrated that in one simple optical experiment it is possible to assess the quality of fabricated metasurfaces, determine the structure symmetry, and, moreover, determine the number of structural elements and lattice constants of the micron-sized metasurface. The accuracy of the optical technique is confirmed by comparison with the results of studies by SEM and AFM methods.

Surface Tension Measurements of Aqueous Liquid-Air Interfaces Probed with Microscopic Indentation.

To elucidate the intricate role that the sea surface microlayer (SML) and sea spray aerosols (SSAs) play in climate, understanding the chemical complexity of the SML and how it affects the physical-chemical properties of the microlayer and SSA are important to investigate. While the surface tension of the SML has been studied previously using conventional experimental tools, accurate measurements must be localized to the thickness of the air-liquid interface of the SML. Here we explore the atomic force microscopy (AFM) capabilities to quantify the surface tension of aqueous solution droplets with (sub)micrometer indentation depths into the interface. Sample droplets of hexanoic acid at molar concentrations ranging from 0.1 to 80 mM and SML from a recent wave flume study were investigated. A constant-radius AFM nanoneedle was used to probe ca. 200 μL droplets with 0.3-1.2 μm indentation depths. As a comparison, the surface tension of bulk samples was also measured using a conventional force tensiometer. The data for the hexanoic acid show an excellent overlap between the AFM and force tensiometer surface tension measurements. For the surface tension measurements of the SML, however, the measured values from the AFM were 2.5 mN/m lower than that from the force tensiometer, which was attributed to the structural and chemical complexity of the SML, differences in the probing depth for each method, and the time scale required for the surface film to restructure as the needle is retracted away from the liquid surface. Overall, the study confirmed the accuracy of the AFM method in quantifying the surface tension of aqueous solutions over a wide range of concentrations for surface-active organic compounds. The methodology can be further used to reveal small, yet important, differences in the surface tension of complex air-liquid interfaces such as liquid systems where the type and concentration of surfactants vary with the distance from the air-liquid interface. For such complex systems, AFM measurements of the surface tension as a function of the probing depth and pulling rate may reveal a sublayer film structure of the liquid interface.

Влияние пуринергической сигнальной системы на свойства клеток крови человека при старении организма

Adenosine triphosphate (ATP) molecules and nucleotide derivatives contribute to a wide range of cellular reactions through the activation of A2 and P2 purinergic receptors located on the cell surface. Almost any type of cells expresses such receptors, including red blood cells and white blood cells. The study of purinergic transmission of signals mediating calcium mobilization, actin polymerization, chemotaxis, release of mediators, tissue oxygenation, cell maturation, cytotoxicity, cell death and cell–cell interactions in dynamics at different stages of ontogenesis is an important issue in biology and medicine today. This paper aimed to explore the biophysical properties of blood cells during the activation of the purinergic signalling system in middle-aged people and older adults. To activate the elements of the purinergic signalling system, an in vitro model of mechanical stress was used. Blood samples were analysed using the methods of atomic force microscopy, namely, force spectroscopy and the Kelvin probe. When the purinergic signalling system is activated, an increase in ATP blood concentration is identified in the blood of both middle-aged people and older adults. In the blood of older adults, a smaller amount of ATP was recorded compared with the blood of middle-aged people, which can be due to changes in the expression of purinoreceptors on the surface of red blood cells at ageing. Erythrocytes and granulocytes in older adults are characterized by increased surface stiffness and potential upon activation of the purinergic signalling system, compared with cells of middle-aged people. In response to mechanical stress, lymphocyte membrane stiffness in older adults increases more significantly than in middle-aged people. Thus, the results indicate an important role of the ATP molecule and its derivatives in the regulation of functioning of blood cells through binding to receptors of the P2 family. Research on the changes in the biophysical properties of red blood cells and white blood cells at activation of the purinergic signalling system will allow us to identify new effects of purine compounds on intercellular interaction during ageing.

Antimicrobial Activity of Antifungal Drugs on Candida Parapsilosis Studied by Scanning Ionconductance Microscopy (SICM)

Candida parapsilosis infections are predominantly opportunistic with high mortality rates, affecting elderly or immunocompromised patients. Such infections are usually difficult to treat, since the repertoire of antifungal drugs is limited. Candida is highly resistant to the most common and widely used antifungal drugs, which lead to lysis of the fungal cell wall. Several imaging techniques are currently available to study the effects of antifungal drugs. Methods of atomic force microscopy are widely used to visualize the shell and measure the stiffness of yeast, as well as electron microscopy, which allows obtaining high-resolution images. For these methods, sample preparation of biological samples is expensive or involves drying the cells, which negatively effects on the cell wall, and makes it difficult to assess the drug effect. That problem can be solved by using an innovative non-invasive method of scanning ion-conductance microscopy (SICM). SICM allows mapping a sample in saline, excluding direct contact between the biomaterial and the probe. Thus, sample preparation is greatly simplified and mechanical impact on the sample is minimized. In this work, SICM was used as a tool for visualizing the surface of living Candida parapsilosis cells treated with new antifungal drugs (hybrid of triazole and thiazolidine) at various concentrations and exposure times. Using the latest developments of SICM technology, quantitatively measured the size and depth/height of cavities and bulges on the cell wall. Lysis of the cell wall of the Candida parapsilosis strain resistant to mycosodin and fluconazole was discovered. The process of biofilm formation was also considered. This research was funded by Ministry of Education and Science of the Russian Federation implemented by a governmental decree dated 16 March 2013, no. 211, The Russian Science Foundation, grant number No 19-19-00626.

Investigation of anticorrosive behaviour of novel tert-butyl 4-[(4-methyl phenyl) carbonyl] piperazine-1-carboxylate for carbon steel in 1M HCl

The main focus of current research is on the synthesis and anticorrosive activity of novel heterocyclic compounds tert-butyl 4-[(4-methyl phenyl) carbonyl] piperazine-1-carboxylate [TBMPCPC]. Electrochemical, quantum chemical, and surface characterization studies at elevated temperatures (303–333 K) for carbon steel in 1M HCl solution studied this molecule's corrosion inhibition property. It is observed from the results of electrochemical studies that the TBMPCPC may be able to effectively protect the steel plate surface with an inhibition efficiency of 91.5 % at 25 ppm in corrosive media. The corrosion inhibition depends on concentration, as concentration also increases inhibition efficiency due to the strong and spontaneous adsorption on the metal's surface. The Tafel polarization measurements concluded that the inhibitor works as a mixed form to protect the carbon steel in the bulk solution from corrosion. The adsorption of the TBMPCPC onto the metal surface was in reliable with the isothermal model of the Langmuir adsorption. The scanning electron microscopy clearly showed that the inhibitor was substantially deposited on the metal surface, indicating substantial inhibition. The surface morphology of carbon steel in the absence and existence of an inhibitor in 1 M HCl is also studied using the Atomic Force Microscopic method.

Investigation of Radiation Erosion in a Gas Discharge Detector by Atomic-Force Microscopy

Atomic-force microscopy methods were used to study specimens of cathodes of multiwire proportional chambers subjected to a long-term exposure to a β-source 90Sr in the course of laboratory longevity tests. The changes in the surface morphology of the copper foil at the detector cathode that have occurred as a result of action of an electron beam have been described. The quantitative estimate and analysis of the evolution of radiation defects depending on the irradiation dose are presented. The similarity of the radiation-induced defects in the laboratory prototypes and in the full-scale proportional chambers after their use for 10 years at the Large Hadron Collider is demonstrated.

Temperature-Dependent Nanomechanical Properties of Adsorbed Poly-NIPAm Microgel Particles Immersed in Water.

The temperature dependence of nanomechanical properties of adsorbed poly-NIPAm microgel particles prepared by a semibatch polymerization process was investigated in an aqueous environment via indentation-based atomic force microscopy (AFM) methods. Poly-NIPAm microgel particles prepared by the classical batch process were also characterized for comparison. The local mechanical properties were measured between 26 and 35 °C, i.e., in the temperature range of the volume transition. Two different AFM tips with different shapes and end radii were utilized. The nanomechanical properties measured by the two kinds of tips showed a similar temperature dependence of the nanomechanical properties, but the actual values were found to depend on the size of the tip. The results suggest that the semibatch synthesis process results in the formation of more homogeneous microgel particles than the classical batch method. The methodological approach reported in this work is generally applicable to soft surface characterization in situ.