Contact Mode Atomic Force Microscopy Research Articles

Study on particle size and field effect with sp2/sp3 ratio of hydrogenated diamond-like carbon

Hydrogenated diamond-like carbon (HDLC) films were synthesized through a reactive gas-plasma process employing methane (CH4) and hydrogen (H2) as precursor gases on a silicon (100) wafer substrate, conducted at room temperature. The deposition process utilized a biased enhanced nucleation technique, varying the flow rate ratio of hydrogen and methane. The investigations revealed that increasing the methane flow rate led to a reduction in grain size and an augmented nucleation density of HDLC, as evidenced by contact-mode atomic force microscopy (AFM) images. This study demonstrated the effective control of diamond grain growth by introducing high-methane-concentration pulses during deposition. The field emission characteristics of HDLC samples were analyzed, revealing threshold fields of 12.2 V/μm for nanocrystalline films, 8.5 V/μm for subcrystalline films and 4.1 V/μm for microcrystalline films, corroborated by Raman spectra. Surface energy measurements indicated hydrophobic behavior in the samples. Notably, a decrease in the hydrogen/methane ratio was found to increase the sp2 character, which correlated with the emission field. AFM analysis of HDLC samples yielded surface roughness values ranging from 0.2 nm to approximately 0.01 nm, confirming the continuous, non-porous and smooth nature of the surfaces.

Contact Mode Atomic Force Microscopy as a Rapid Technique for Morphological Observation and Bacterial Cell Damage Analysis.

Electron microscopy is one of the tools required to characterize cellular structures. However, the procedure is complicated and expensive due to the sample preparation for observation. Atomic force microscopy (AFM) is a very useful characterization technique due to its high resolution in three dimensions and because of the absence of any requirement for vacuum and sample conductivity. AFM can image a wide variety of samples with different topographies and different types of materials. AFM provides high-resolution 3D topography information from the angstrom level to the micron scale. Unlike traditional microscopy, AFM uses a probe to generate an image of the surface topography of a sample. In this protocol, the use of this type of microscopy is suggested for the morphological and cell damage characterization of bacteria fixed on a support. Strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Pseudomonas hunanensis (isolated from garlic bulb samples) were used. In this work, bacterial cells were grown in specific culture media. To observe cell damage, Staphylococcus aureus and Escherichia coli were incubated with different concentrations of nanoparticles (NPs). A drop of bacterial suspension was fixed on a glass support, and images were taken with AFM at different scales. The images obtained showed the morphological characteristics of the bacteria. Further, employing AFM, it was possible to observe the damage to the cellular structure caused by the effect of NPs. Based on the images obtained, contact AFM can be used to characterize the morphology of bacterial cells fixed on a support. AFM is also a suitable tool for the investigation of the effects of NPs on bacteria. Compared to electron microscopy, AFM is an inexpensive and easy-to-use technique.

Novel type of whisker-tip cantilever based on GaN microrods for atomic force microscopy

High-resolution scanning probe microscopy (SPM) is a fundamental and efficient technology for surface characterization of modern materials at the subnanometre scale. The bottleneck of SPM is the probe and scanning tip. Materials with stable electrical, thermal, and mechanical properties for high-aspect-ratio (AR) tips are continuously being developed to improve their accuracy. Among these, GaN is emerging as a significant contender that serves as a replacement for standard Si probes. In this paper, for the first time, we present an approach that demonstrates the application of GaN microrods (MRs) as high-AR SPM probes. GaN MRs were grown using molecular beam epitaxy, transferred and mounted on a cantilever using focused electron beam-induced deposition and milled in a whisker tip using a focused ion beam in a scanning electron/ion microscope. The presence of a native oxide layer covering the GaN MR surface was confirmed by X-ray photoelectron spectroscopy. Current-voltage map measurements are also presented to indicate the elimination of the native oxide layer from the tip surface. The utility of the designed probes was tested using conductive atomic force microscopy and a 24-hour durability test in contact mode atomic force microscopy. Subsequently, the graphene stacks were imaged.

Probing the morphological and mechanical changes in a C. elegans model of Huntington's disease.

Huntington's Disease (HD) is a fatal neurodegenerative disease caused by a mutation in the huntingtin gene, in which CAG nucleotides are excessively repeated. In turn, translated huntingtin protein (htt) contains an expanded polyglutamine (polyQ) domain, which triggers toxic protein aggregation, the hallmark feature of this disease. Common symptoms of HD, such as chorea, muscle atrophy, and behavioral and neurological decline, continue to worsen as the disease progresses. This study aims to investigate the relationship between polyQ domain length and age-dependent morphological and mechanical changes in a Caenorhabditis elegans model of HD. C. elegans are an established model species to investigate aging, as morphological and mechanical changes—such as epidermal roughening and whole body decrease in Young's modulus, respectively—occur as a function of age. Moreover, worm mobility exhibits a deficit as a result of such changes. We expect to observe age-dependent morphological changes much earlier in the pathogenic C. elegans (Q128), as opposed to age-matched, non-pathogenic C. elegans (Q15). Morphological analysis was performed using tapping mode atomic force microscopy (AFM), and images were processed using custom MATLAB scripts to determine RMS roughness. Further studies will be conducted to investigate the role of age-dependent changes in mechanical properties, such as Young's modulus, as they relate to polyQ domain length. In-situ contact mode AFM will be used to achieve this. We expect to observe age-dependent mechanical changes much earlier in the Q128 worms, in comparison to the Q15 worms. Pending results, additional studies may be conducted to determine if these changes serve as a peripheral biomarker of HD, as well as the role of such a biomarker in diagnostic, prognostic, and therapeutic strategies.

Nanoscale topotactic phase transformation modulated by triboelectrification for high memory storage

The applications of triboelectrification have attracted much attention due to the direct regulation of charges with external mechanical stimuli. Atomic force microscopy (AFM) is an effective method to achieve controllable nanoscale friction. Herein, we demonstrated a phase transformation from the brownmillerite SrCoO2.5 (BM-SCO) to perovskite SrCoO3-δ (PV-SCO) as modulated by nanoscale friction with the contact mode AFM. Besides, such phase transformation can be reversible by applying a negative voltage. More importantly, when defining the surface potential of different phases as binary bits of “1” and “0”, we could reach high-density data storage as the surface charge is constantly written and erased in nanoscale regions. This work opens a new way for the regulation of phase transformation by triboelectrification, and realizes the surface charge patterning based on different phases at the nanoscale by AFM tip friction, which is of great significance for the application of nano devices in high-density data storage.

Exploring electromechanical utility of GaAs interdigitated transducers; using finite-element-method-based parametric analysis and experimental comparison

Analysis of interdigitated transducers often relies on phenomenological models to approximate device electrical performance. While these approaches prove essential for signal processing applications, phenomenological models provide limited information on the device’s mechanical response and physical characteristics of the generated acoustic field. Finite element method modeling, in comparison, offers a robust platform to study the effects of the full device geometry on critical performance parameters of interdigitated transducer devices. In this study, we fabricate a surface acoustic wave resonator on semi-insulating GaAs (100), which consists of an interdigitated transducer and acoustic mirror assembly. The device is subsequently modeled using fem software. A vector network analyzer is used to measure the experimental device scattering response, which compares well with the simulated results. The wave characteristics of the experimental device are measured by contact-mode atomic force microscopy, which validates the simulation’s mechanical response predictions. We further show that a computational parametric analysis can be used to optimize device designs for series resonance frequency, effective coupling coefficient, quality factor, and maximum acoustic surface displacement.

Nanotopography Evaluation of NiTi Alloy Exposed to Artificial Saliva and Different Mouthwashes.

Nitinol (NiTi) alloy is a widely used material for the production of orthodontic archwires. Its corrosion behavior in conditions that exist in the oral cavity still remains a great characterization challenge. The motivation behind this work is to reveal the influence of commercially available mouthwashes on NiTi orthodontic archwires by performing non-electrochemical corrosion tests and quantifying the changes in the nanotopography of commercially available NiTi orthodontic wires. In this study, we examined the behavior of NiTi alloy archwires exposed for 21.5 days to different corrosive media: artificial saliva, Eludril®, Aquafresh®, and Listerine®. The corrosion was characterized by contact mode atomic force microscopy (AFM) before and after the corrosion tests. A novel analysis methodology was developed to obtain insight into locations of material gain or material loss based on standard surface roughness parameters Sa, Sdr, Ssk, and S10z. The developed methodology revealed that fluoride-containing mouthwashes (Aquafresh® and Listerine®) dominantly cause material loss, while chloride-containing mouthwash (Eludril®) can cause both material loss and material gain. The sample exposed to artificial saliva did not display significant changes in any parameter.

Control of interaction force in constant-height contact mode atomic force microscopy

Contact mode is a versatile and widely used technique for imaging samples using the Atomic Force Microscope (AFM). When contact mode imaging is performed in constant-height mode, it enables linear and faster response but leads to uncontrolled tip-sample forces. Here, a control strategy based on magnetic actuation is proposed to achieve high-bandwidth control of the tip-sample forces in constant-height contact mode AFM. A magnetic particle attached to the AFM probe is actuated by an external solenoid and employed for force regulation. A quasi-static model has been proposed and employed to develop the control strategy. Likewise, the contact natural-frequency, which decides the limit of achievable speed, has been shown to be significantly higher relative to the free probe and to be relatively insensitive to the particle size. Subsequently, a setup is developed to validate the control strategy and demonstrate reduction of tip-sample force variation by over a factor of 12 compared to conventional constant-height mode operation. Likewise, in comparison with conventional contact mode AFM, an improvement of linearity by over a factor of 9 and improvement in response speed by a factor of 100 have been demonstrated while imaging hard samples. The system has been shown to image topography at speeds of 2.44 frames per second while regulating the interaction force. Finally, the stiffness of a sample has also been characterized using the developed system and simultaneous estimation of topography has also been demonstrated. They are shown to agree well with theoretical expectations.

(Digital Presentation) Investigation of Microstructure Dependent Inhibitor Adsorption Mechanism on Carbon Steel By in Situ Atomic Force Microscopy

In the petroleum industry, organic surfactant type inhibitors have been widely applied in the oil and gas industry considering their high efficiency. Carbon steels with various carbide contents have also been used extensively as pipeline materials during the transport and storage of crude oil due to their excellent mechanical properties and low cost. The previous investigation of inhibition mechanism on carbon steel is limited by lack of surface characterization techniques at a molecular level. Atomic force microscopy (AFM) can not only provide localized visual observation, but can also achieve the detection of mechanical properties of an inhibitor film. In this study, in situ AFM contact mode friction imaging and in situ AFM tapping mode phase imaging techniques have been applied to investigate the influence of the ferritic-pearlitic microstructure of carbon steel on inhibitor adsorption mechanisms during CO2 corrosion under different inhibitor concentrations. At concentration above surface saturation, AFM contact mode friction images show a large friction contrast between inhibitor covered cementite structures and ferrite structures, while in the absence of inhibitor, this friction contrast almost disappears, indicating the inhibitor adsorption induced this difference. AFM tapping mode phase images indicate uniform adsorption of inhibitor on both cementite and ferrite structures.The differences observed in these AFM analyses indicate that the adhesion force of inhibitor molecules on cementite could be smaller than on ferrite, or the molecular orientations of inhibitor molecules adsorbed on cementite and ferrite structures could be different. The special adsorption behavior of inhibitor molecules on cementite means the carbide component of the microstructure could directly influence inhibitor adsorption and may decrease inhibitor efficiency. Simultaneous linear polarization resistance tests in electrochemical AFM (EC-AFM) cell indicates a maximum inhibition efficiency at concentration above saturation.Keywords : Contact mode AFM, friction contrast, tapping mode AFM, phase contrast, cementite, ferrite, organic inhibitor, adhesion force.Statement of importance:Studies related to inhibitor adsorption mechanisms on ferritc-pearlitic carbon steels can be connected with the practical issues that iron carbide can impair the corrosion inhibitor performance on carbon steels. This AFM work connects the corrosion inhibition effect with the carbon steel components at a molecular level for the first time. Physical explanations regarding the interactions between inhibitor molecules and carbon steel microstructures can also be provided with the assistance of molecular simulations.Figure caption: Contact and tapping mode AFM images of inhibitor film formed on UNS G1018 steel at 2 CMC (100ppm) tetradecyldimethylbenzylammonium inhibitor + 1 wt% NaCl. The contact mode friction image indicates a large friction contrast between inhibitor covered cementite and ferrite structure, while the tapping mode phase image indicates a negligible softness contrast between cementite and ferrite structure. Figure 1

Dipolar Noise in Fluorinated Molecular Wires.

We demonstrate a strategy to directly map and quantify the effects of dipole formation on electrical transports and noises in the self-assembled monolayers (SAMs) of molecular wires. In this method, the SAM patterns of fluorinated molecules with dipole moments were prepared on conducting substrates, and a conducting probe in contact-mode atomic force microscopy was utilized to map currents and noises through the probe on the molecular patterns. The maps were analyzed to extract the characteristic parameters of dipolar noises in SAMs, and the results were compared with those of hydrogenated molecular patterns without dipole moments. At rather low bias conditions, the fluorinated molecular junctions exhibited a tunneling conduction and a resistance value comparable to that of the hydrogenated molecules with a six-times-longer length, which was attributed to stronger dipoles formation in fluorinated molecules. Interestingly, conductance (G) in different regions of fluorinated molecular patterns exhibited a strong correlation with a noise power spectral density of SI/I2 like SI/I2 ∝ G−2, which can be explained by enhanced barrier fluctuations produced by the dipoles of fluorinated molecules. Furthermore, we observed that the noise power spectral density of fluorinated molecules showed an anomalous frequency (f) dependence like SI/I2 ∝ 1/f1.7, possibly due to the slowing down of the tunneling of carriers from increased barrier fluctuations. In rather high bias conditions, conductions in both hydrogenated and fluorinated molecules showed a transition from tunneling to thermionic charge transports. Our results provide important insights into the effects of dipoles on mesoscopic transport and resistance-fluctuation in molecules and could have a significant impact on the fundamental understanding and applications in this area.

Boxcar Averaging Scanning Nonlinear Dielectric Microscopy.

Scanning nonlinear dielectric microscopy (SNDM) is a near-field microwave-based scanning probe microscopy method with a wide variety of applications, especially in the fields of dielectrics and semiconductors. This microscopy method has often been combined with contact-mode atomic force microscopy (AFM) for simultaneous topography imaging and contact force regulation. The combination SNDM with intermittent contact AFM is also beneficial for imaging a sample prone to damage and using a sharp microscopy tip for improving spatial resolution. However, SNDM with intermittent contact AFM can suffer from a lower signal-to-noise (S/N) ratio than that with contact-mode AFM because of the shorter contact time for a given measurement time. In order to improve the S/N ratio, we apply boxcar averaging based signal acquisition suitable for SNDM with intermittent contact AFM. We develop a theory for the S/N ratio of SNDM and experimentally demonstrate the enhancement of the S/N ratio in SNDM combined with peak-force tapping (a trademark of Bruker) AFM. In addition, we apply the proposed method to the carrier concentration distribution imaging of atomically thin van der Waals semiconductors. The proposed method clearly visualizes an anomalous electron doping effect on few-layer Nb-doped MoS2. The proposed method is also applicable to other scanning near-field microwave microscopes combined with peak-force tapping AFM such as scanning microwave impedance microscopy. Our results indicate the possibility of simultaneous nanoscale topographic, electrical, and mechanical imaging even on delicate samples.

Size dependence of charge retention in gold-nanoparticles sandwiched between thin layers of titanium oxide and silicon oxide

Nonvolatile memory technology is a necessary component in many electronic devices. With the scaling down of memory devices to achieve high density and low power consumption, floating gate devices encounter various challenges like high leakage current, which leads to reliability issues and a decrease in charge density. Therefore, the use of metal nanoparticles (NPs) as charge storage centers is becoming a promising candidate due to their excellent scalability and favorable reliability. In this work, we demonstrate the charge storage dependency on the size of a gold-nanoparticle (Au-NP) by using a contact mode atomic force microscope. The individually dispersed Au-NPs are sandwiched between a thin layer (3 nm) of TiO2 blocking layer and SiO2 (2 nm) tunneling layer thin films. The consecutive I–V sweeps on a pristine device of stacking TiO2/Au-NP/SiO2/n-Si show that the threshold voltage (ΔV) increases with the increase in the Au-NP size, whereas the retention shows much more stability time with smaller size NPs, in the range of 10 nm.

Linescan Lattice Microscopy: A Technique for the Accurate Measurement and Mapping of Lattice Spacing and Strain with Atomic Force Microscopy.

Lateral resolution and accuracy in scanning probe microscopies are limited by the nonideality of piezoelectric scanning elements due to phenomena including nonlinearity, hysteresis, and creep. By taking advantage of the well-established atomic-scale stick-slip phenomenon in contact-mode atomic force microscopy, we have developed a method for simultaneously indexing and measuring the spacing of surface atomic lattices using only Fourier analysis of unidirectional linescan data. The first step of the technique is to calibrate the X-piezo response using the stick-slip behavior itself. This permits lateral calibration to better than 1% error between 2.5 nm and 9 μm, without the use of calibration gratings. Lattice indexing and lattice constant determination are demonstrated in this way on the NaCl(001) crystal surface. After piezo calibration, lattice constant measurement on a natural bulk MoS2(0001) surface is demonstrated with better than 0.2% error. This is used to measure nonuniform thermal mismatch strain for chemical vapor deposition (CVD)-grown monolayer MoS2 as small as 0.5%. A spatial mapping technique for the lattice spacing is developed and demonstrated, with absolute accuracy better than 0.2% and relative accuracy better than 0.1%, within a map of 12.5 × 12.5 nm2 pixels using bulk highly oriented pyrolytic graphite (HOPG) and MoS2 as reference materials.

Corneal stromal roughness after VisuMax and Intralase femtosecond laser photodisruption: An atomic force microscopy study.

PurposeTo compare the induced corneal stromal bed roughness measured with atomic force microscopy (AFM) after LASIK flap creation with the IntraLase 60 kHz and the VisuMax femtosecond laser platforms.MethodsThree freshly enucleated porcine eyes were operated with each femtosecond laser in this experimental study. Standard LASIK treatment parameters were used for the experiment. After LASIK flap creation, the corneal stromal roughness was assessed using a JPK NanoWizard II® AFM in contact mode immersed in liquid. Olympus OMCL-RC800PSA commercial silicon nitride cantilever tips were used. Surface measurements were made in 10 regions of the central cornea of each sample measuring 20 x 20 microns, at 512 x 512 point resolution. Roughness was measured using the root-mean-square (RMS) value within the given regions.ResultsMeasurements from 30 regions of the 3 eyes (10 measurements per eye) in the Intralase (FS1) group, and 30 regions of the 3 eyes (10 measurements per eye) in the VisuMax (FS2) group were analyzed. There was a statistically significant difference in mean ± standard deviation RMS values between the FS1 and the FS2 groups (360 ± 120 versus 230 ± 100 nm respectively; P< 0.00001).ConclusionThis AFM study indicates that the surface of the stromal bed after LASIK flap creation is smoother in the FS2 group than the FS1 group.

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.

Single frequency vertical piezoresponse force microscopy

Piezoresponse force microscopy (PFM) uses a cantilever arm to track the electromechanical motion of the electric dipole moment to visualize the ferroelectric domain structure, which provides an important insight into the physics of switchable electric polarization—especially for memory devices and integrated microelectronics. Here, I provide a tutorial on single frequency vertical PFM, the most basic mode of PFM. I will start with the basic components of atomic force microscopy (AFM), including tip, cantilever, X–Y stage, Z actuator, and lock-in amplifier. Contact mode AFM will be briefly explained and discussed, where you can find two modes: constant deflection and constant height modes. Single-frequency vertical PFM splits the frequency domain of tip vibration into low and high frequencies and uses a low-pass filter to nullify any motion caused by topography (constant deflection). In contrast, the lock-in amplifier will pinpoint the vibration induced by the vertical piezoelectric strain along the sample’s surface normal (constant height). This tutorial will provide an overall and detailed step by step instruction to conduct PFM imaging and piezoresponse hysteresis loop measurement using atomic force microscopy and a lock-in amplifier and teach how to interpret the PFM images and the piezoresponse hysteresis loops for various applications.

Observation of stress corrosion cracking using real-time in situ high-speed atomic force microscopy and correlative techniques

Contact-mode high-speed atomic force microscopy (HS-AFM) has been utilised to measure in situ stress corrosion cracking (SCC) with nanometre resolution on AISI Type 304 stainless steel in an aggressive salt solution. SCC is an important failure mode in many metal systems but has a complicated mechanism that makes failure difficult to predict. Prior to the in situ experiments, the contributions of microstructure, environment and stress to SCC were independently studied using HS-AFM. During SCC measurements, uplift of grain boundaries before cracking was observed, indicating a subsurface contribution to the cracking mechanism. Focussed ion beam milling revealed a network of intergranular cracks below the surface lined with a thin oxide, indicating that the SCC process is dominated by local stress at oxide-weakened boundaries. Subsequent analysis by atom probe tomography of a crack tip showed a layered oxide composition at the surface of the crack walls. Oxide formation is posited to be mechanistically linked to grain boundary uplift. This study shows how in situ HS-AFM observations in combination with complementary techniques can give important insights into the mechanisms of SCC.

Mechanical removal of surface residues on graphene for TEM characterizations

Contamination on two-dimensional (2D) crystal surfaces poses serious limitations on fundamental studies and applications of 2D crystals. Surface residues induce uncontrolled doping and charge carrier scattering in 2D crystals, and trapped residues in mechanically assembled 2D vertical heterostructures often hinder coupling between stacked layers. Developing a process that can reduce the surface residues on 2D crystals is important. In this study, we explored the use of atomic force microscopy (AFM) to remove surface residues from 2D crystals. Using various transmission electron microscopy (TEM) investigations, we confirmed that surface residues on graphene samples can be effectively removed via contact-mode AFM scanning. The mechanical cleaning process dramatically increases the residue-free areas, where high-resolution imaging of graphene layers can be obtained. We believe that our mechanical cleaning process can be utilized to prepare high-quality 2D crystal samples with minimum surface residues.

Nonlinear vibrations control of a contact-mode AFM model via a time-delayed positive position feedback

In this work, the nonlinear vibrations of a contact-mode atomic force microscopy (AFM) model subjected to multi excitations are controlled via a time-delayed positive position feedback (PPF) controller. A perturbation method is implemented to obtain the approximate nonlinear dynamical behavior of the AFM system. Different response curves, before and after control, are included utilizing frequency response equations. At multiple resonance cases (primary, sub-harmonic, and internal), the gain effects on the vibration amplitude and stability behavior of the controlled system are studied. The acquired results revealed that PPF controller is efficient in reducing the studied system oscillations. The efficiency of the controller Ea was about 193, means that the controller reduced the vibration by about 99.5%. For increasing feedback signal gain α2, the stability region in τ1-τ2 plane was shrinking. Finally, verification curves are plotted to depict the degree of closeness between analytical predictions and numerical simulation.

Characterizing liver sinusoidal endothelial cell fenestrae on soft substrates upon AFM imaging and deep learning

BackgroundLiver sinusoidal endothelial cells (LSECs) display unique fenestrated morphology. Alterations in the size and number of fenestrae play a crucial role in the progression of various liver diseases. While their features have been visualized using atomic force microscopy (AFM), the in situ imaging methods and off-line analyses are further required for fenestra quantification. MethodsPrimary mouse LSECs were cultured on a collagen-I-coated culture dish, or a polydimethylsiloxane (PDMS) or polyacrylamide (PA) hydrogel substrate. An AFM contact mode was applied to visualize fenestrae on individual fixed LSECs. Collected images were analyzed using an in-house developed image recognition program based on fully convolutional networks (FCN). ResultsKey scanning parameters were first optimized for visualizing the fenestrae on LSECs on culture dish, which was also applicable for the LSECs cultured on various hydrogels. The intermediate-magnification morphology images of LSECs were used for developing the FCN-based, fenestra recognition program. This program enabled us to recognize the vast majority of fenestrae from AFM images after twice trainings at a typical accuracy of 81.6% on soft substrate and also quantify the statistics of porosity, number of fenestrae and distribution of fenestra diameter. ConclusionsCombining AFM imaging with FCN training is able to quantify the morphological distributions of LSEC fenestrae on various substrates. SignificanceAFM images acquired and analyzed here provided the global information of surface ultramicroscopic structures over an entire cell, which is fundamental in understanding their regulatory mechanisms and pathophysiological relevance in fenestra-like evolution of individual cells on stiffness-varied substrates.