Scanning Probe Microscopy Techniques for Studying the Cell Glycocalyx.

The glycocalyx is a brush-like layer that covers the surfaces of the membranes of most cell types. It consists of a mixture of carbohydrates, mainly glycoproteins and proteoglycans. Due to its structure and sensitivity to environmental conditions, it represents a complicated object to investigate. Here, we review studies of the glycocalyx conducted using scanning probe microscopy approaches. This includes imaging techniques as well as the measurement of nanomechanical properties. The nanomechanics of the glycocalyx is particularly important since it is widely present on the surfaces of mechanosensitive cells such as endothelial cells. An overview of problems with the interpretation of indirect data via the use of analytical models is presented. Special insight is given into changes in glycocalyx properties during pathological processes. The biological background and alternative research methods are briefly covered.

A Deep-Learning Framework for the Automated Recognition of Molecules in Scanning-Probe-Microscopy Images.

Computer vision as a subcategory of deep learning tackles complex vision tasks by dealing with data of images. Molecular images with exceptionally high resolution have been achieved thanks to the development of techniques like scanning probe microscopy (SPM). However, extracting useful information from SPM image data requires careful analysis which heavily relies on human supervision. In this work, we develop a deep learning framework using an advanced computer vision algorithm, Mask R-CNN, to address the challenge of molecule detection, classification and instance segmentation in binary molecular nanostructures. We employ the framework to determine two triangular-shaped molecules of similar STM appearance. Our framework could accurately differentiate two molecules and label their positions. We foresee that the application of computer vision in SPM images will become an indispensable part in the field, accelerating data mining and the discovery of new materials.

Scanning Probe Microscopy Bone Marrow Determination of Steogenic Differentiation of Mesenchymal Stem Cells.

To address the question of determining the osteogenic differentiation of mesenchymal stem cells, the bone marrow studies were performed using probe microscopy. All adherent bone marrow was used to isolate the bone marrow mesenchymal stem cells and expanded and purified in vitro. Its morphology under an inverted microscope was observed. We used Zuogui Pills to differentiate the separation methods. Alcian blue staining, modified calcium cobalt alkaline phosphatase staining, and neuron-specific enolase immunohistochemical staining were performed. The experimental results are shown below. The morphology of the isolated and purified cells was analyzed with an inverted microscope, and the isolated and purified cells were analyzed with Zuogui Pill. Alcian blue staining, modified calcium cobalt alkaline phosphatase staining, and neuron-specific enolase immunohistochemical staining confirmed that the cells differentiated into cartilage and osteoblasts, and the cell structure and morphology were similar to those of the bone marrow mesenchymal stem cells. The results showed that the adherent mode of cells obtained from the whole bone marrow was the rat bone marrow mesenchymal stem cells, and the Zuogui Pills could induce multidirectional differences in the bone marrow mesenchymal stem cells.

An Assessment of MXenes through Scanning Probe Microscopy.

Recently, exploring the unique properties of 2D materials has constituted a new wave of research, which lead these materials to enormous applications ranging from optoelectronics to healthcare systems. Due to the profusion of surface terminated functionalities, MXenes have become an emerging class of 2D materials that can be easily integrated with other materials. The versatility of MXenes allows to tune their finest material properties for further device applications. This review initiates with the classification of preparation methods of MXenes, where the authors elaborate on the significance of top-down approaches including the exfoliation of solid layers. Next, the focus is diverted toward the materials analysis of MXenes including their terminations analysis as well as their intriguing electrical and mechanical behaviors through scanning probe microscopy. Finally, critical challenges and perspectives for MXenes analysis and applications are explored and discussed. Therefore, this comprehensive review can encourage researchers, and offer a precise direction to employ MXenes in various applications.

Raman and scanning probe microscopy for differentiating surface imprints of E. coli and B. cereus.

Molecularly imprinted polymers (MIPs) are artificial recognition materials mimicking biological recognition entities such as antibodies. The general model of imprinting assumes that functional monomers interact with functional groups present on the target species which leads to cavities complementing the template in surface chemistry and shape thus ensuring recognition. However, to date there is little independent experimental evidence supporting that the surface chemistry in the imprints is tailored to analyte recognition and thus differs from the surface chemistry of the surrounding polymer. Herein, we investigate such chemical differences between imprints of Escherichia coli and Bacillus cereus in poly(styrene-co-DVB) and a commercial acrylate-based polymer by the means of confocal Raman microscopy and PLS-DA. The MIPs were generated using a stamping approach. Peak-force QNM measurements were conducted to rule out residues of bacterial cells in the imprints. While imprints of E. coli and B. cereus could be distinguished based on their Raman spectra in the acrylate-based polymer, differentiation in poly(styrene-co-DVB) was not significant. This could be a result of a higher potential of acrylate functional groups for interacting with lipopolysaccharides and peptidoglycans on bacteria surfaces compared to the phenyl groups of poly(styrene-co-DVB) and emphasizes the importance of the right choice of functional monomers for a specific target analyte.

Binary-state scanning probe microscopy for parallel imaging.

Scanning probe microscopy techniques, such as atomic force microscopy and scanning tunnelling microscopy, are harnessed to image nanoscale structures with an exquisite resolution, which has been of significant value in a variety of areas of nanotechnology. These scanning probe techniques, however, are not generally suitable for high-throughput imaging, which has, from the outset, been a primary challenge. Traditional approaches to increasing the scalability have involved developing multiple probes for imaging, but complex probe design and electronics are required to carry out the detection method. Here, we report a probe-based imaging method that utilizes scalable cantilever-free elastomeric probe design and hierarchical measurement architecture, which readily reconstructs high-resolution and high-throughput topography images. In a single scan, we demonstrate imaging with a 100-tip array to obtain 100 images over a 1-mm2 area with 106 pixels in less than 10 min. The potential for large-scale tip integration and the advantage of a simple probe array suggest substantial promise for our approach to high-throughput imaging far beyond what is currently possible.

High resolution imaging of viruses: Scanning probe microscopy and related techniques.

Scanning probe microscopy is a group of measurements that provides 3D visualization of viruses in different environmental conditions including liquids and air. Besides 3D topography it is possible to measure the properties like mechanical rigidity and stability, adhesion, tendency to crystallization, surface charge, etc. Choosing the right substrate and scanning parameters makes it much easier to obtain reliable data. Rational interpretation of experimental results should take into account possible artifacts, proper filtering and data presentation using specially designed software packages. Animal and human virus characterization is in the focus of many intensive studies because of their potential harm to higher organisms. The article focuses on high-resolution visualization of plant viruses. Tobacco mosaic virus, potato viruses X and B and others are not dangerous for the human being and are widely used in different applications such as vaccine preparation, construction of building units in nanotechnology and material science applications, nanoparticle production and delivery, and even metrology. The methods of virus's deposition, visualization, and consequent image processing and interpretation are described in details. Specific examples of viruses imaging are illustrated using the FemtoScan Online software, which has typical and all the necessary built-in functions for constructing three-dimensional images, their processing and analysis. Despite visible progress in visualizing the viruses using probe microscopy, many unresolved problems still remain. At present time the probe microscopy data on viruses is not systemized. There is no descriptive atlas of the images and morphology as revealed by this type of high resolution microscopy. It is worth emphasizing that new virus investigation methods will appear due to the progress of science.

Time-Resolved Scanning Ion Conductance Microscopy for Three-Dimensional Tracking of Nanoscale Cell Surface Dynamics.

Nanocharacterization plays a vital role in understanding the complex nanoscale organization of cells and organelles. Understanding cellular function requires high-resolution information about how the cellular structures evolve over time. A number of techniques exist to resolve static nanoscale structure of cells in great detail (super-resolution optical microscopy, EM, AFM). However, time-resolved imaging techniques tend to either have a lower resolution, are limited to small areas, or cause damage to the cells, thereby preventing long-term time-lapse studies. Scanning probe microscopy methods such as atomic force microscopy (AFM) combine high-resolution imaging with the ability to image living cells in physiological conditions. The mechanical contact between the tip and the sample, however, deforms the cell surface, disturbs the native state, and prohibits long-term time-lapse imaging. Here, we develop a scanning ion conductance microscope (SICM) for high-speed and long-term nanoscale imaging of eukaryotic cells. By utilizing advances in nanopositioning, nanopore fabrication, microelectronics, and controls engineering, we developed a microscopy method that can resolve spatiotemporally diverse three-dimensional (3D) processes on the cell membrane at sub-5-nm axial resolution. We tracked dynamic changes in live cell morphology with nanometer details and temporal ranges of subsecond to days, imaging diverse processes ranging from endocytosis, micropinocytosis, and mitosis to bacterial infection and cell differentiation in cancer cells. This technique enables a detailed look at membrane events and may offer insights into cell-cell interactions for infection, immunology, and cancer research.

Topography and Permeability Analyses of Vasculature-on-a-Chip Using Scanning Probe Microscopies.

Microphysiological systems (MPS) or organs-on-chips (OoC) can emulate the physiological functions of organs in vitro and are effective tools for determining human drug responses in preclinical studies. However, the analysis of MPS has relied heavily on optical tools, resulting in difficulties in real-time and high spatial resolution imaging of the target cell functions. In this study, the role of scanning probe microscopy (SPM) as an analytical tool for MPS is evaluated. An access hole is made in a typical MPS system with stacked microchannels to insert SPM probes into the system. For the first study, a simple vascular model composed of only endothelial cells is prepared for SPM analysis. Changes in permeability and local chemical flux are quantitatively evaluated during the construction of the vascular system. The morphological changes in the endothelial cells after flow stimulation are imaged at the single-cell level for topographical analysis. Finally, the possibility of adapting the permeability and topographical analysis using SPM for the intestinal vascular system is further evaluated. It is believed that this study will pave the way for an in situ permeability assay and structural analysis of MPS using SPM.

Automated and Autonomous Experiments in Electron and Scanning Probe Microscopy.

Machine learning and artificial intelligence (ML/AI) are rapidly becoming an indispensable part of physics research, with domain applications ranging from theory and materials prediction to high-throughput data analysis. In parallel, the recent successes in applying ML/AI methods for autonomous systems from robotics to self-driving cars to organic and inorganic synthesis are generating enthusiasm for the potential of these techniques to enable automated and autonomous experiments (AE) in imaging. Here, we aim to analyze the major pathways toward AE in imaging methods with sequential image formation mechanisms, focusing on scanning probe microscopy (SPM) and (scanning) transmission electron microscopy ((S)TEM). We argue that automated experiments should necessarily be discussed in a broader context of the general domain knowledge that both informs the experiment and is increased as the result of the experiment. As such, this analysis should explore the human and ML/AI roles prior to and during the experiment and consider the latencies, biases, and prior knowledge of the decision-making process. Similarly, such discussion should include the limitations of the existing imaging systems, including intrinsic latencies, non-idealities, and drifts comprising both correctable and stochastic components. We further pose that the role of the AE in microscopy is not the exclusion of human operators (as is the case for autonomous driving), but rather automation of routine operations such as microscope tuning, etc., prior to the experiment, and conversion of low latency decision making processes on the time scale spanning from image acquisition to human-level high-order experiment planning. Overall, we argue that ML/AI can dramatically alter the (S)TEM and SPM fields; however, this process is likely to be highly nontrivial and initiated by combined human-ML workflows and will bring challenges both from the microscope and ML/AI sides. At the same time, these methods will enable opportunities and paradigms for scientific discovery and nanostructure fabrication.

Reactive oxygen FIB spin milling enables correlative workflow for 3D super-resolution light microscopy and serial FIB/SEM of cultured cells.

Correlative light and electron microscopy (CLEM) is a powerful tool for defining the ultrastructural context of molecularly-labeled biological specimens, particularly when superresolution fluorescence microscopy (SRM) is used for CLEM. Current CLEM, however, is limited by the stark differences in sample preparation requirements between the two modalities. For CLEM using SRM, the small region of interest (ROI) of either or both modalities also leads to low success rate and imaging throughput. To overcome these limitations, here we present a CLEM workflow based on a novel focused ion beam/scanning electron microscope (FIB/SEM) compatible with common SRM for imaging biological specimen with ultrahigh 3D resolution and improved imaging throughput. By using a reactive oxygen source in a plasma FIB (PFIB) and a rotating sample stage, the novel FIB/SEM was able to achieve several hundreds of micrometer large area 3D analysis of resin embedded cells through a process named oxygen serial spin mill (OSSM). Compared with current FIB mechanisms, OSSM offers gentle erosion, highly consistent slice thickness, reduced charging during SEM imaging, and improved SEM contrast without increasing the dose of post-staining and fixation. These characteristics of OSSM-SEM allowed us to pair it with interferometric photoactivated localization microscopy (iPALM), a recent SRM technique that affords 10-20 nm isotropic spatial resolution on hydrated samples, for 3D CLEM imaging. We demonstrate a CLEM workflow generalizable to using other SRM strategies using mitochondria in human osteosarcoma (U2OS) cells as a model system, where immunostained TOM20, a marker for the mitochondrial outer membrane, was used for iPALM. Owing to the large scan area of OSSM-SEM, it is now possible to select as many FOVs as needed for iPALM and conveniently re-locate them in EM, this improving the imaging throughput. The significantly reduced dose of post-fixation also helped to better preserve the sample ultrastructures as evidenced by the excellent 3D registration between OSSM-SEM and iPALM images and by the accurate localization of TOM20 (by iPALM) to the peripheries of mitochondria (by OSSM-SEM). These advantages make OSSM-SEM an ideal modality for CLEM applications. As OSSM-SEM is still in development, we also discuss some of the remaining issues and the implications to biological imaging with SEM alone or with CLEM.

Nanoscale Magnetization and Current Imaging Using Time-Resolved Scanning-Probe Magnetothermal Microscopy.

Magnetic microscopy that combines nanoscale spatial resolution with picosecond scale temporal resolution uniquely enables direct observation of the spatiotemporal magnetic phenomena that are relevant to future high-speed, high-density magnetic storage and logic technologies. Magnetic microscopes that combine these metrics has been limited to facility-level instruments. To address this gap in lab-accessible spatiotemporal imaging, we develop a time-resolved near-field magnetic microscope based on magnetothermal interactions. We demonstrate both magnetization and current density imaging modalities, each with spatial resolution that far surpasses the optical diffraction limit. In addition, we study the near-field and time-resolved characteristics of our signal and find that our instrument possesses a spatial resolution on the scale of 100 nm and a temporal resolution below 100 ps. Our results demonstrate an accessible and comparatively low-cost approach to nanoscale spatiotemporal magnetic microscopy in a table-top form to aid the science and technology of dynamic magnetic devices with complex spin textures.

Electrophysiology using coaxial atom probe array: live imaging reveals hidden circuits of a hippocampal neural network.

Since the 1960s, it is held that when a neuron fires, a nerve spike passes only through the selective branches, the calculated choice is a key to learning by rewiring. It is argued by chemically estimating the membrane's ion channel density that different axonal branches get active to pass the spike-branches blink at firing at different time domains. Here, using a new time-lapse dielectric imaging, we visualize the classic branch selection process; thenceforth, hidden circuits operating at different time domains become visible. The fractal grid of coaxial probes captures wireless snapshots of material's vibration at various depths below the membrane by setting a suitable frequency. Thus far, branch selection observed emitted energy or particle but never the emitters, what they do. As each dielectric material transmits and reflects signals of different frequencies, we image live how filaments search for many branch-made circuits, choose a unique pathway 103 times faster than a single nerve spike. It reveals that neural branches and circuit visible in a microscope are not absolute, there coexist many circuits each operating in different dime domains, operating at a time.NEW & NOTEWORTHY Using dielectric resonance scanner, we show electromagnetic field connections between physically separated neurons. Electromagnetic field creates field lines that pass through gap junctions, connect Axon initial segment with the dendrites through Soma, and connect axonal or dendritic branches even if there is no synaptic junction. Consequently, many distinct loops connecting various branches form coexisting circuits. Our discovery suggests that physically appearing neural circuit is a fractional view of many simultaneously operating circuits in different time domains in a neural network.

Enabling autonomous scanning probe microscopy imaging of single molecules with deep learning.

Scanning probe microscopies allow investigating surfaces at the nanoscale, in real space and with unparalleled signal-to-noise ratio. However, these microscopies are not used as much as it would be expected considering their potential. The main limitations preventing a broader use are the need of experienced users, the difficulty in data analysis and the time-consuming nature of experiments that require continuous user supervision. In this work, we addressed the latter and developed an algorithm that controlled the operation of an Atomic Force Microscope (AFM) that, without the need of user intervention, allowed acquiring multiple high-resolution images of different molecules. We used DNA on mica as a model sample to test our control algorithm, which made use of two deep learning techniques that so far have not been used for real time SPM automation. One was an object detector, YOLOv3, which provided the location of molecules in the captured images. The second was a Siamese network that could identify the same molecule in different images. This allowed both performing a series of images on selected molecules while incrementing the resolution, as well as keeping track of molecules already imaged at high resolution, avoiding loops where the same molecule would be imaged an unlimited number of times. Overall, our implementation of deep learning techniques brings SPM a step closer to full autonomous operation.

Towards a Fully Automated Scanning Probe Microscope for Biomedical Applications.

The increase in capabilities of Scanning Probe Microscopy (SPM) has resulted in a parallel increase in complexity that limits the use of this technique outside of specialised research laboratories. SPM automation could substantially expand its application domain, improve reproducibility and increase throughput. Here, we present a bottom-up design in which the combination of positioning stages, orientation, and detection of the probe produces an SPM design compatible with full automation. The resulting probe microscope achieves sub-femtonewton force sensitivity whilst preserving low mechanical drift (2.0±0.2 nm/min in-plane and 1.0±0.1 nm/min vertically). The additional integration of total internal reflection microscopy, and the straightforward operations in liquid, make this instrument configuration particularly attractive to future biomedical applications.

Scanning Probe Microscopies: Imaging and Biomechanics in Reproductive Medicine Research.

Basic and translational research in reproductive medicine can provide new insights with the application of scanning probe microscopies, such as atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). These microscopies, which provide images with spatial resolution well beyond the optical resolution limit, enable users to achieve detailed descriptions of cell topography, inner cellular structure organization, and arrangements of single or cluster membrane proteins. A peculiar characteristic of AFM operating in force spectroscopy mode is its inherent ability to measure the interaction forces between single proteins or cells, and to quantify the mechanical properties (i.e., elasticity, viscoelasticity, and viscosity) of cells and tissues. The knowledge of the cell ultrastructure, the macromolecule organization, the protein dynamics, the investigation of biological interaction forces, and the quantification of biomechanical features can be essential clues for identifying the molecular mechanisms that govern responses in living cells. This review highlights the main findings achieved by the use of AFM and SNOM in assisted reproductive research, such as the description of gamete morphology; the quantification of mechanical properties of gametes; the role of forces in embryo development; the significance of investigating single-molecule interaction forces; the characterization of disorders of the reproductive system; and the visualization of molecular organization. New perspectives of analysis opened up by applying these techniques and the translational impacts on reproductive medicine are discussed.

Optical and Electron Microscopy for Analysis of Nanomaterials.

Not only is fabrication important for research in materials science, but also materials characterization and analysis. Special microscopes capable of ultra-high magnification are more essential for observing and analyzing nanoparticles than for macro-size particles. Recently, electron microscopy (EM) and scanning probe microscopy (SPM) are commonly used for observing and analyzing nanoparticles. In this chapter, the basic principles of various techniques in optical and electron microscopy are described and classified. In particular, techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are explained.

A de-waxing methodology for scanning probe microscopy.

A de-waxing protocol that successfully removes paraffin from tissue microarray (TMA) cores of fixed tissue obtained from oral cancer is described. The success of the protocol is demonstrated by the comparison of Fourier transform infrared (FTIR) results obtained on paraffin-embedded and de-waxed tissue and the absence of any significant correlations between infrared scanning near-field optical microscopy (SNOM) images of de-waxed tissue obtained at the three main paraffin IR peaks. The success of the protocol in removing paraffin from tissue is also demonstrated by images obtained with scanning electron microscopy (SEM) and by energy dispersive spectra (EDS) of a de-waxed CaF2 disc which shows no significant contribution from carbon. The FTIR spectra of the de-waxed TMA core overlaps that obtained from OE19 oesophageal cancer cells which had never been exposed to paraffin.

Mapping the surface potential, charge density and adhesion of cellulose nanocrystals using advanced scanning probe microscopy.

Cellulose nanocrystals (CNC) are the focus of significant attention in the broad area of sustainable technologies for possessing many desirable properties such as a large surface area, high strength and stiffness, outstanding colloidal stability, excellent biocompatibility and biodegradability, low weight and abundance in nature. Yet, a fundamental understanding of the micro- and nanoscale electrical charge distribution on nanocellulose still remains elusive. Here we present direct quantification and mapping of surface charges on CNCs at ambient condition using advanced surface probe microscopy techniques such as Kelvin probe force microscopy (KPFM), electrostatic force microscopy (EFM) and force-distance (F-D) curve measurements. We show by EFM measurements that the surface charge in the solid-state (as contrasted with liquid dispersions) present at ambient condition on CNCs provided by Innotech Alberta is intrinsically negative and the charge density is estimated to be 13 µC/cm2. These charges also result in CNCs having two times the adhesive force exhibited by SiO2 substrates in adhesion mapping studies. The origin of negative surface charge is likely due to the formation of CNCs through sulfuric acid hydrolysis where sulfate half esters groups remained on the surface (Johnston et al., 2018).

Volcano-Shaped Scanning Probe Microscopy Probe for Combined Force-Electrogram Recordings from Excitable Cells.

Atomic force microscopy based approaches have led to remarkable advances in the field of mechanobiology. However, linking the mechanical cues to biological responses requires complementary techniques capable of recording these physiological characteristics. In this study, we present an instrument for combined optical, force, and electrical measurements based on a novel type of scanning probe microscopy cantilever composed of a protruding volcano-shaped nanopatterned microelectrode (nanovolcano probe) at the tip of a suspended microcantilever. This probe enables simultaneous force and electrical recordings from single cells. Successful impedance measurements on mechanically stimulated neonatal rat cardiomyocytes in situ were achieved using these nanovolcano probes. Furthermore, proof of concept experiments demonstrated that extracellular field potentials (electrogram) together with contraction displacement curves could simultaneously be recorded. These features render the nanovolcano probe especially suited for mechanobiological studies aiming at linking mechanical stimuli to electrophysiological responses of single cells.