Investigating the kinetics of layer development during the color etching of low-carbon steel with in-situ spectroscopic ellipsometry.

Color etching is a useful corrosive process, widely applied in metallography to study the microstructure of metals. To prove the existence of the previously hypothesized steady-state etching rate, in-situ investigations were performed with spectroscopic ellipsometry during the color etching of ferritic materials. Kinetic information regarding the refractive index, extinction coefficient, and layer thickness were used to calculate the steady-state layer buildup rate, which was 1.90 ± 0.15 nm/s for low-carbon steel and 0.99 ± 0.06 nm/s for cast iron owing to its better corrosion resistance. The presented methodology and findings could help understanding other processes that involve the development of layers on metallic surfaces.

Hydrodynamic function and spring constant calibration of FluidFM micropipette cantilevers.

Fluidic force microscopy (FluidFM) fuses the force sensitivity of atomic force microscopy with the manipulation capabilities of microfluidics by using microfabricated cantilevers with embedded fluidic channels. This innovation initiated new research and development directions in biology, biophysics, and material science. To acquire reliable and reproducible data, the calibration of the force sensor is crucial. Importantly, the hollow FluidFM cantilevers contain a row of parallel pillars inside a rectangular beam. The precise spring constant calibration of the internally structured cantilever is far from trivial, and existing methods generally assume simplifications that are not applicable to these special types of cantilevers. In addition, the Sader method, which is currently implemented by the FluidFM community, relies on the precise measurement of the quality factor, which renders the calibration of the spring constant sensitive to noise. In this study, the hydrodynamic function of these special types of hollow cantilevers was experimentally determined with different instruments. Based on the hydrodynamic function, a novel spring constant calibration method was adapted, which relied only on the two resonance frequencies of the cantilever, measured in air and in a liquid. Based on these results, our proposed method can be successfully used for the reliable, noise-free calibration of hollow FluidFM cantilevers.

Plasmonic Effect of Gold-Patchy Silica Nanoparticles on Green Light-Photopolymerizable Dental Resin.

A low ratio of polymerization is a major problem in resin-based composites. In this paper, the plasmonic effect of gold-covered silica nanoparticles on the physicochemical and mechanical properties of bisphenol A diglycidyl dimethacrylate (Bis-GMA), triethylene glycol dimethacrylate (TEGDMA) and urethane dimethacrylate (UDMA) green light-photopolymerizable dental resin was investigated at an intensity of 1.4 mW/cm2 for 40 s. Transmission electron microscopy (TEM) showed silica of about 350 nm covered with 12-15 nm gold nanoparticles (Au NPs) at 100% nominal coverage. Five different concentrations of bare and patchy silica particles were used; in the latter composite, the calculated Au wt% were 0.0052 wt%, 0.0104 wt%, 0.0208 wt%, 0.04160 wt%, and 0.0823 wt%. The plasmon peak of patchy silica-filled nanocomposite overlapped with the absorption of Irgacure 784 photoinitiator and green LED light emission peak. The effect of plasmon-enhanced polymerization achieved with green light illumination was analyzed using diametral tensile strength (DTS), differential scanning calorimetry (DSC), surface plasmon resonance imaging (SPRi), and degree of conversion (DC) based on Raman spectroscopy. The values of the Au NP with 0.0208 wt% was found to be maximum in all the measured data. Based on our result, it can be concluded that the application of patchy silica particles in dental resin can improve the polymerization ratio and the mechanical parameters of the composite.

Towards Digital Twins of Plasmonic Sensors: Constructing the Complex Numerical Model of a Plasmonic Sensor Based on Hexagonally Arranged Gold Nanoparticles.

In this work, we aim to design the digital twin of a plasmonic sensor based on hexagonally arranged ellipsoidal gold nanoparticles fixed to a glass substrate. Based on electron microscopy images of three experimentally realized nanoparticle arrangement types, we constructed numerical models in environments based on finite element method (FEM) and boundary element method (BEM), namely COMSOL Multiphysics for FEM and the MNPBEM Matlab Toolbox for BEM. Models with nonperiodic and periodic boundary conditions with different unit cells were constructed to study the plasmonic behavior of both the single ellipsoidal particles and the hexagonal nanoparticle arrangements. The effect of the geometrical parameters, namely the interparticle distance, the nanoparticle diameter and thickness, on the resulting LSPR peak positions and bulk refractive index sensitivities were studied in detail, also taking into account the effect of the SiO2 substrate (pillars) under the ellipsoidal particles. We have demonstrated that by optimizing the models, the LSPR peak positions (and sensitivities) can match the experimentally measured values within 1 nm (nm/RIU) precision. The comparison of simulation conditions and the detailed discussion of the effect of the geometrical parameters and used gold dielectric functions on the obtained sensitivities can be very beneficial for the optimization of plasmonic sensor constructions through numerical simulations.

Microstructured soft devices for the growth and analysis of populations of homogenous multicellular tumor spheroids.

Multicellular tumor spheroids are rapidly emerging as an improved in vitro model with respect to more traditional 2D culturing. Microwell culturing is a simple and accessible method for generating a large number of uniformly sized spheroids, but commercially available systems often do not enable researchers to perform complete culturing and analysis pipelines and the mechanical properties of their culture environment are not commonly matching those of the target tissue. We herein report a simple method to obtain custom-designed self-built microwell arrays made of polydimethylsiloxane or agarose for uniform 3D cell structure generation. Such materials can provide an environment of tunable mechanical flexibility. We developed protocols to culture a variety of cancer and non-cancer cell lines in such devices and to perform molecular and imaging characterizations of the spheroid growth, viability, and response to pharmacological treatments. Hundreds of tumor spheroids grow (in scaffolded or scaffold-free conditions) at homogeneous rates and can be harvested at will. Microscopy imaging can be performed in situ during or at the end of the culture. Fluorescence (confocal) microscopy can be performed after in situ staining while retaining the geographic arrangement of spheroids in the plate wells. This platform can enable statistically robust investigations on cancer biology and screening of drug treatments.

The Effect of Femtosecond Laser Irradiation and Plasmon Field on the Degree of Conversion of a UDMA-TEGDMA Copolymer Nanocomposite Doped with Gold Nanorods.

In this work, the effects of femtosecond laser irradiation and doping with plasmonic gold nanorods on the degree of conversion (DC) of a urethane dimethacrylate (UDMA)-triethylene glycol dimethacrylate (TEGDMA) nanocomposite were investigated. The UDMA-TEGDMA photopolymer was prepared in a 3:1 weight ratio and doped with dodecanethiol- (DDT) capped gold nanorods of 25 × 75 or 25 × 85 nm nominal diameter and length. It was found that the presence of the gold nanorods alone (without direct plasmonic excitation) can increase the DC of the photopolymer by 6-15%. This increase was found to be similar to what could be achieved with a control heat treatment of 30 min at 180 °C. It was also shown that femtosecond laser impulses (795 nm, 5 mJ pulse energy, 50 fs pulse length, 2.83 Jcm-2 fluence), applied after the photopolymerization under a standard dental curing lamp, can cause a 2-7% increase in the DC of undoped samples, even after thermal pre-treatment. The best DC values (12-15% increase) were obtained with combined nanorod doping and subsequent laser irradiation close to the plasmon resonance peak of the nanorods (760-800 nm), which proves that the excited plasmon field can directly facilitate double bond breakage (without thermoplasmonic effects due to the short pulse length) and increase the crosslink density independently from the initial photopolymerization process.

Cell-substratum and cell-cell adhesion forces and single-cell mechanical properties in mono- and multilayer assemblies from robotic fluidic force microscopy.

The epithelium covers, protects, and actively regulates various formations and cavities of the human body. During embryonic development the assembly of the epithelium is crucial to the organoid formation, and the invasion of the epithelium is an essential step in cancer metastasis. Live cell mechanical properties and associated forces presumably play an important role in these biological processes. However, the direct measurement of cellular forces in a precise and high-throughput manner is still challenging. We studied the cellular adhesion maturation of epithelial Vero monolayers by measuring single-cell force-spectra with high-throughput fluidic force microscopy (robotic FluidFM). Vero cells were grown on gelatin-covered plates in different seeding concentrations, and cell detachment forces were recorded from the single-cell state, through clustered island formation, to their complete assembly into a sparse and then into a tight monolayer. A methodology was proposed to separate cell-substratum and cell-cell adhesion force and energy (work of adhesion) contributions based on the recorded force-distance curves. For comparison, cancerous HeLa cells were also measured in the same settings. During Vero monolayer formation, a significantly strengthening adhesive tendency was found, showing the development of cell-cell contacts. Interestingly, this type of step-by-step maturation was absent in HeLa cells. The attachment of cancerous HeLa cells to the assembled epithelial monolayers was also measured, proposing a new high-throughput method to investigate the biomechanics of cancer cell invasion. We found that HeLa cells adhere significantly stronger to the tight Vero monolayer than cells of the same origin. Moreover, the mechanical characteristics of Vero monolayers upon cancerous HeLa cell influence were recorded and analyzed. All these results provide insight into the qualitative assessment of cell-substratum and cell-cell mechanical contacts in mono- and multilayered assemblies and demonstrate the robustness and speed of the robotic FluidFM technology to reveal biomechanical properties of live cell assemblies with statistical significances.

Correction: Bonyár, A. Maximizing the Surface Sensitivity of LSPR Biosensors through Plasmon Coupling-Interparticle Gap Optimization for Dimers Using Computational Simulations. Biosensors 2021, 11, 527.

In the original publication [...].

Simple and automatic monitoring of cancer cell invasion into an epithelial monolayer using label-free holographic microscopy.

The invasiveness of cancer cells describes the metastasizing capability of a primary tumor. The straightforward detection and quantification of cancer cell invasion are important to predict the survival rate of a cancer patient and to test how anti-cancer compounds influence cancer progression. Digital holographic microscopy based M4 Holomonitor (HM) is a technique that allows the label-free monitoring of cell morphological and kinetical parameters in real-time. Here, a fully confluent epithelial monolayer derived from the African green monkey kidney (Vero) on a gelatin-coated surface was established, then HeLa cells were seeded on top of the monolayer, and their behavior was monitored for 24 h using HM. Several cancer cells showing invasiveness were detected during this period, while other HeLa cells did not show any signs of aggressivity. It was demonstrated that the invasion of single cancer cells is soundly observable and also quantifiable through monitoring parameters such as phase shift, optical volume, area, and motility, which parameters can easily be obtained and processed automatically. Based on the experimental data, the invasion speed of cancer cells entering the epithelial layer can be defined as the shrinking of detected single-cell volume per unit time. The invasion speed and its correlation with cell migration parameters were analyzed in depth. A clear linear relationship between migration and invasion speed was found, cancer cells with stronger migration have slower invasion speed. These results not only describe the effect of how cancer cells invade the underlying monolayer in contrast to non-invasive HeLa cells, but could help in future research to optimize drugs affecting cell invasibility in a fully automated, label-free and high-throughput manner.

Population distributions of single-cell adhesion parameters during the cell cycle from high-throughput robotic fluidic force microscopy.

Single-cell adhesion plays an essential role in biological and biomedical sciences, but its precise measurement for a large number of cells is still a challenging task. At present, typical force measuring techniques usually offer low throughput, a few cells per day, and therefore are unable to uncover phenomena emerging at the population level. In this work, robotic fluidic force microscopy (FluidFM) was utilized to measure the adhesion parameters of cells in a high-throughput manner to study their population distributions in-depth. The investigated cell type was the genetically engineered HeLa Fucci construct with cell cycle-dependent expression of fluorescent proteins. This feature, combined with the high-throughput measurement made it possible for the first time to characterize the single-cell adhesion distributions at various stages of the cell cycle. It was found that parameters such as single-cell adhesion force and energy follow a lognormal population distribution. Therefore, conclusions based on adhesion data of a low number of cells or treating the population as normally distributed can be misleading. Moreover, we found that the cell area was significantly the smallest, and the area normalized maximal adhesion force was significantly the largest for the colorless cells (the mitotic (M) and early G1 phases). Notably, the parameter characterizing the elongation of the cells until the maximum level of force between the cell and its substratum was also dependent on the cell cycle, which quantity was the smallest for the colorless cells. A novel parameter, named the spring coefficient of the cell, was introduced as the fraction of maximal adhesion force and maximal cell elongation during the mechanical detachment, which was found to be significantly the largest for the colorless cells. Cells in the M phase adhere in atypical way, with so-called reticular adhesions, which are different from canonical focal adhesions. We first revealed that reticular adhesion can exert a higher force per unit area than canonical focal adhesions, and cells in this phase are significantly stiffer. The possible biological consequences of these findings were also discussed, together with the practical relevance of the observed population-level adhesion phenomena.

Development and In-Depth Characterization of Bacteria Repellent and Bacteria Adhesive Antibody-Coated Surfaces Using Optical Waveguide Biosensing.

Bacteria repellent surfaces and antibody-based coatings for bacterial assays have shown a growing demand in the field of biosensors, and have crucial importance in the design of biomedical devices. However, in-depth investigations and comparisons of possible solutions are still missing. The optical waveguide lightmode spectroscopy (OWLS) technique offers label-free, non-invasive, in situ characterization of protein and bacterial adsorption. Moreover, it has excellent flexibility for testing various surface coatings. Here, we describe an OWLS-based method supporting the development of bacteria repellent surfaces and characterize the layer structures and affinities of different antibody-based coatings for bacterial assays. In order to test nonspecific binding blocking agents against bacteria, OWLS chips were coated with bovine serum albumin (BSA), I-block, PAcrAM-g-(PMOXA, NH2, Si), (PAcrAM-P) and PLL-g-PEG (PP) (with different coating temperatures), and subsequent Escherichia coli adhesion was monitored. We found that the best performing blocking agents could inhibit bacterial adhesion from samples with bacteria concentrations of up to 107 cells/mL. Various immobilization methods were applied to graft a wide range of selected antibodies onto the biosensor's surface. Simple physisorption, Mix&Go (AnteoBind) (MG) films, covalently immobilized protein A and avidin-biotin based surface chemistries were all fabricated and tested. The surface adsorbed mass densities of deposited antibodies were determined, and the biosensor;s kinetic data were evaluated to divine the possible orientations of the bacteria-capturing antibodies and determine the rate constants and footprints of the binding events. The development of affinity layers was supported by enzyme-linked immunosorbent assay (ELISA) measurements in order to test the bacteria binding capabilities of the antibodies. The best performance in the biosensor measurements was achieved by employing a polyclonal antibody in combination with protein A-based immobilization and PAcrAM-P blocking of nonspecific binding. Using this setting, a surface sensitivity of 70 cells/mm2 was demonstrated.

Maximizing the Surface Sensitivity of LSPR Biosensors through Plasmon Coupling-Interparticle Gap Optimization for Dimers Using Computational Simulations.

The bulk and surface refractive index sensitivities of LSPR biosensors, consisting of coupled plasmonic nanosphere and nano-ellipsoid dimers, were investigated by simulations using the boundary element method (BEM). The enhancement factor, defined as the ratio of plasmon extinction peak shift of multi-particle and single-particle arrangements caused by changes in the refractive index of the environment, was used to quantify the effect of coupling on the increased sensitivity of the dimers. The bulk refractive index sensitivity (RIS) was obtained by changing the dielectric medium surrounding the nanoparticles, while the surface sensitivity was modeled by depositing dielectric layers on the nanoparticle in an increasing thickness. The results show that by optimizing the interparticle gaps for a given layer thickness, up to ~80% of the optical response range of the nanoparticles can be utilized by confining the plasmon field between the particles, which translates into an enhancement of ~3-4 times compared to uncoupled, single particles with the same shape and size. The results also show that in these cases, the surface sensitivity enhancement is significantly higher than the bulk RI sensitivity enhancement (e.g., 3.2 times vs. 1.8 times for nanospheres with a 70 nm diameter), and thus the sensors' response for molecular interactions is higher than their RIS would indicate. These results underline the importance of plasmonic coupling in the optimization of nanoparticle arrangements for biosensor applications. The interparticle gap should be tailored with respect to the size of the used receptor/target molecules to maximize the molecular sensitivity, and the presented methodology can effectively aid the optimization of fabrication technologies.

PDMS Bonding Technologies for Microfluidic Applications: A Review.

This review summarizes and compares the available surface treatment and bonding techniques (e.g., corona triggered surface activation, oxygen plasma surface activation, chemical gluing, and mixed techniques) and quality/bond-strength testing methods (e.g., pulling test, shear test, peel test, leakage test) for bonding PDMS (polydimethylsiloxane) with other materials, such as PDMS, glass, silicon, PET (polyethylene terephthalate), PI (polyimide), PMMA (poly(methyl methacrylate)), PVC (polyvinyl chloride), PC (polycarbonate), COC (cyclic olefin copolymer), PS (polystyrene) and PEN (polyethylene naphthalate). The optimized process parameters for the best achievable bond strengths are collected for each substrate, and the advantages and disadvantages of each method are discussed in detail.

Label-free tracking of whole-cell response on RGD functionalized surfaces to varied flow velocities generated by fluidic rotation.

Fluidic flow plays important roles in colloid and interface sciences. Measuring adsorption, aggregation processes and living cell behavior under a fluidic environment with varied flow velocities in a parallel and high-throughput manner remains to be a challenging task. Here a method is introduced to monitor cell response to well-defined flow with varied velocities over an array of label-free resonant waveguide grating (RWG) based optical biosensors. The arrangement consists of a circular well with an array of biosensors at the bottom surface. By rotating the liquid over the biosensor array using a magnetic stirrer bar, flow velocities from zero to a predefined maximum can be easily established over different locations within the biosensor array as characterized in detail by numerical simulations. Cell adhesion and detachment measurements on an Arg-Gly-Asp (RGD) peptide functionalized surface were performed to demonstrate i) measurements at a wide range of simultaneous flow velocities over the same interface; ii) the possibility of parallel measurements at the same flow conditions in one run; and iii) the simple tuning of the employed range of flow velocities. Our setup made it possible to analyze the magnitude and rate of cell detachment at various flow velocities in parallel and determine the critical velocity and force where cells start to detach from the RGD motif displaying biomimetic surface. Furthermore, cellular response to simultaneous mechanical (flow) and chemical stimulation was also investigated using trypsin as a model. This study opens a new possibility to investigate interface phenomena under predefined and conveniently varied flow conditions.

Dissociation Constant of Integrin-RGD Binding in Live Cells from Automated Micropipette and Label-Free Optical Data.

The binding of integrin proteins to peptide sequences such as arginine-glycine-aspartic acid (RGD) is a crucial step in the adhesion process of mammalian cells. While these bonds can be examined between purified proteins and their ligands, live-cell assays are better suited to gain biologically relevant information. Here we apply a computer-controlled micropipette (CCMP) to measure the dissociation constant (Kd) of integrin-RGD-binding. Surface coatings with varying RGD densities were prepared, and the detachment of single cells from these surfaces was measured by applying a local flow inducing hydrodynamic lifting force on the targeted cells in discrete steps. The average behavior of the populations was then fit according to the chemical law of mass action. To verify the resulting value of Kd2d = (4503 ± 1673) 1/µm2, a resonant waveguide grating based biosensor was used, characterizing and fitting the adhesion kinetics of the cell populations. Both methods yielded a Kd within the same range. Furthermore, an analysis of subpopulations was presented, confirming the ability of CCMP to characterize cell adhesion both on single cell and whole population levels. The introduced methodologies offer convenient and automated routes to quantify the adhesivity of living cells before their further processing.

Optimization of Plasmonic Gold Nanoparticle Concentration in Green LED Light Active Dental Photopolymer.

Gold nanoparticles (AuNPs) display surface plasmon resonance (SPR) as a result of their irradiation at a targeted light frequency. SPR also results in heat production that increases the temperature of the surrounding environment, affecting polymerization. The aim was to investigate the SPR effect of AuNPs on a dimethacrylate-based photopolymer system. The tested composites were designed to overlap the illumination required for the polymerization and the plasmon effect. The 5 nm-sized dodecanethiol capped AuNPs were applied in different concentrations in the matrix that were irradiated with green light (λ = 532 nm), where the Irgacure 784 photoinitiator also absorbs the light. The plasmonic effect was investigated for the refractive index change by surface plasmon resonance imaging (SPRi) supplemented by ellipsometry. Moreover, optical transmission and transmission electron micrographs (TEM), diametral tensile stress (DTS), and confocal Raman spectroscopy was performed to determine the degree of conversion (DC) at 1.0, 1.4, and 2.0 mW/cm2 light intensities. It was found that the optimal conditions were at 0.0208 wt% AuNPs concentration and 1.4 mW/cm2 light intensity at which the refractive index change, DTS, and DC data were all maximal. The study confirmed that AuNPs are applicable to improve the polymerization efficiency of dental composite resin.

Large Scale Fabrication of Ordered Gold Nanoparticle-Epoxy Surface Nanocomposites and Their Application as Label-Free Plasmonic DNA Biosensors.

A robust and scalable technology to fabricate ordered gold nanoparticle arrangements on epoxy substrates is presented. The nanoparticles are synthesized by solid-state dewetting on nanobowled aluminum templates, which are prepared by the selective chemical etching of porous anodic alumina (PAA) grown on an aluminum sheet with controlled anodic oxidation. This flexible fabrication technology provides proper control over the nanoparticle size, shape, and interparticle distance over a large surface area (several cm2), which enables the fine-tuning and optimization of their plasmonic absorption spectra for LSPR and SERS applications between 535 and 625 nm. The nanoparticles are transferred to the surface of epoxy substrates, which are subsequently selectively etched. The resulting nanomushrooms arrangements consist of ordered epoxy nanopillars with flat, disk-shaped nanoparticles on top, and their bulk refractive index sensitivity is between 83 and 108 nm RIU-1. Label-free DNA detection is successfully demonstrated with the sensors by using a 20 base pair long specific DNA sequence from the parasite Giardia lamblia. A red-shift of 6.6 nm in the LSPR absorbance spectrum was detected after the 2 h hybridization with 1 µM target DNA, and the achievable LOD was around 5 nM. The reported plasmonic sensor is one of the first surface AuNP/polymer nanocomposites ever reported for the successful label-free detection of DNA.

Publisher Correction: Spring constant and sensitivity calibration of FluidFM micropipette cantilevers for force spectroscopy measurements.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Towards more reliable AFM force-curve evaluation: A method for spring constant selection, adaptive lever sensitivity calibration and fitting boundary identification.

This work discusses key issues regarding the atomic force microscopy (AFM) force-curve evaluation practice, which can affect the determined Young's modulus of the investigated sample. These issues are 1) the proper calibration of lever sensitivity and the effect of its variation between the measurements; 2) the selection of proper cantilever spring constant for the investigated sample; and 3) the selection of the fitting boundaries for the contact mechanics model-based force-curve evaluation. A method is proposed, which solves the above mentioned issues, namely, categorizes the obtained force-curves based on the relation between the elastic properties of the sample and the spring constant of the cantilever, and thus helps in the selection of the proper spring constant for the given surface; helps in the identification of the optimal model-fitting boundaries, and also, provides a way of adaptive lever sensitivity calibration. The method is demonstrated on PDMS (polydimethylsiloxane) samples, which were irradiated with various fluences of ion beams to control their elastic properties in the 4 MPa - 22 GPa range. Our proposed method, if applied correctly can significantly increase the reliability of AFM force-curve evaluation.

Spring constant and sensitivity calibration of FluidFM micropipette cantilevers for force spectroscopy measurements.

The fluidic force microscope (FluidFM) can be considered as the nanofluidic extension of the atomic force microscope (AFM). This novel instrument facilitates the experimental procedure and data acquisition of force spectroscopy (FS) and is also used for the determination of single-cell adhesion forces (SCFS) and elasticity. FluidFM uses special probes with an integrated nanochannel inside the cantilevers supported by parallel rows of pillars. However, little is known about how the properties of these hollow cantilevers affect the most important parameters which directly scale the obtained spectroscopic data: the inverse optical lever sensitivity (InvOLS) and the spring constant (k). The precise determination of these parameters during calibration is essential in order to gain reliable, comparable and consistent results with SCFS. Demonstrated by our literature survey, the standard error of previously published SCFS results obtained with FluidFM ranges from 11.8% to 50%. The question arises whether this can be accounted for biological diversity or may be the consequence of improper calibration. Thus the aim of our work was to investigate the calibration accuracy of these parameters and their dependence on: (1) the aperture size (2, 4 and 8 µm) of the hollow micropipette type cantilever; (2) the position of the laser spot on the back of the cantilever; (3) the substrate used for calibration (silicon or polystyrene). It was found that both the obtained InvOLS and spring constant values depend significantly on the position of the laser spot. Apart from the theoretically expectable monotonous increase in InvOLS (from the tip to the base of the cantilever, as functions of the laser spot's position), we discerned a well-defined and reproducible fluctuation, which can be as high as ±30%, regardless of the used aperture size or substrate. The calibration of spring constant also showed an error in the range of -13/+20%, measured at the first 40 µm of the cantilever. Based on our results a calibration strategy is proposed and the optimal laser position which yields the most reliable spring constant values was determined and found to be on the first pair of pillars. Our proposed method helps in reducing the error introduced via improper calibration and thus increases the reliability of subsequent cell adhesion force or elasticity measurements with FluidFM.