Tapping Mode Imaging Research Articles

Large-range high-speed dynamic-mode atomic force microscope imaging: adaptive tapping towards minimal force

In this paper, a software-hardware integrated approach is proposed for high-speed, large-range tapping mode imaging of atomic force microscope (AFM). High speed AFM imaging is needed in various applications, particularly in interrogating dynamic processes at nanoscale such as polymer crystallization process. Achieving high speed in tapping-mode AFM imaging is challenging as the probe-sample interaction during the imaging process is highly nonlinear, making the tapping motion highly sensitive to the probe sample spacing, and thereby, difficult to maintain at high speed. Increasing the speed via hardware bandwidth enlargement, however, leads to a substantially reduction of the imaging area. Contrarily, the imaging speed can be increased without loss of the scan size through control (algorithm)-based approach. For example, the recently-developed adaptive multiloop mode (AMLM) technique has demonstrated its efficacy in increasing the tapping-mode imaging speed without loss of scan size. Further improvement, however, has been limited by the hardware bandwidth and the online signal processing speed and computation complexity involved. Thus, in this paper, the AMLM technique is further enhanced to optimize the probe tapping regulation, and integrated with a field programmable gate array platform to further increase the imaging speed without loss of quality and scan range. Experimental implementation of the proposed approach demonstrates that high-quality imaging can be achieved at a high-speed scanning rate of 100 Hz and higher, and over a large imaging area of over 20 μm.

Topography and mechanical measurements of primary Schwann cells and neuronal cells with atomic force microscope for understanding and controlling nerve growth

Peripheral nerve injuries require a piece of substantial information for a satisfactory treatment. The prior peripheral nerve injury knowledge, can improve nerve repair, and its growth at molecular and cellular level. In this study, we employed an atomic force microscope (AFM) to investigate the topography and mechanical properties of the primary Schwann cells and neuronal cells. Tapping mode images and contact points force-volume maps provide the cells topography. Two different probes were used to acquire the micro and nanomechanical properties of the primary Schwann cells, NG-108–15 neuronal cells, and growth cones. Moreover, the sharp probe was only used to investigate neurites nanomechanics. A significant difference in the elastic moduli found between primary Schwann cells, and neuronal cells, with both probes, with consistent results. The elastic moduli of the growth cones were found higher, than the neuronal cells and primary Schwann cells, with both probes. Furthermore, the modulus variations were also found between neurites. These results have significant implications for a better understanding of the peripheral nerve system (PNS) in terms of defining the optimal pattern surface and nerve guidance conduits.

(Invited) Investigation of Corrosion Inhibitor Adsorption on Mild Steel By Electrochemical Atomic Force Microscopy

Mild steel has wide application in oil and gas industry due to its excellent mechanical properties and low costs. However, it is susceptible to corrosion attack in a typical water-containing service environment. Organic corrosion inhibitors can adsorb on steel surfaces to form protective layers even when added in a small amount. As conceptualized in the literature, the decrease in corrosion rates due to addition of inhibitor should be directly related to their surface coverage. However, studies have so far not provided any direct evidence of such a link, primarily due to limitations of conventional electrochemical analysis that have been used to investigate them. In the present work, a specially designed holder for electrochemical atomic force microscopy (EC-AFM) has been used to study the influence of a tetra-decyl-dimethyl-benzyl-ammonium inhibitor model compound on mitigation of corrosion. AFM results shows that when the bulk solution inhibitor concentration was at 0.5 CMC, partial coverage of the surface by the inhibitor film and patchy corrosion (which could lead to localized attack) of the UNS G1018 steel surface were observed by contact mode AFM imaging. When inhibitor bulk concentration was above the surface saturation value (1 CMC), full surface protection was indicated by the almost unchanged AFM topography images from 10 minutes to 4 hours exposure to the inhibited solution. The LPR results obtained with EC-AFM showed that the corrosion rates of this steel decreased as the bulk solution concentration of inhibitor in separate tests was increased from 0, 0.5 CMC to 1 CMC, while further increase of concentrations (from 1 CMC to 2 CMC) didn’t lead to any apparent reduction of general corrosion rate. Although the LPR corrosion rates at 1 CMC and 2 CMC were extremely low (0.05 - 0.1 mm/year), the values didn’t reduce to 0 under these full coverage conditions. Additional AFM tapping mode imaging results on mica indicated the formation of a porous film (a layer with ‘holes’) which is used to explain the types of adsorption layers possible and their probable influence on corrosion.

Soft sample deformation, damage and induced electromechanical property changes in contact- and tapping-mode atomic force microscopy

Probe-induced soft sample damage in atomic force microscopy (AFM), as well as the resulting alteration of local mechanical and electrical properties of the material are explored, specifically comparing contact-mode and intermittent-contact-mode imaging methods. In our experiments, performed on conductive polymer films, induced changes are present in contact-mode imaging while they are negligible or absent in tapping-mode imaging. To understand this result, a viscoelastic parameter extraction is performed, which suggests that permanent sample deformation can readily occur for tip-sample interactions with a duration on the timescale of contact-mode interactions. Using the extracted viscoelastic parameters, a dynamic AFM simulation is conducted, which suggests that the material responds more elastically with reduced or absent sample damage in tapping-mode AFM, due to the higher rate of mechanical deformation and shorter timescales.

The Attachment of Carbon Nanotubes to Atomic Force Microscopy Tips Using the Pick-Up Method

In the last 30 years research has shown that the resolution and reproducibility of data acquired using the atomic force microscope (AFM) can be improved through the development of new imaging modes or by modifying the AFM tip. One method that has been explored since the 1990s is to attach carbon nanotubes (CNT) to AFM tips. CNTs possess a small diameter, high aspect ratio, high strength and demonstrate a high degree of wear resistance. While early indications suggested the widespread use of these types of probes would be routine this has not been the case. A number of methods for CNT attachment have been proposed and explored including chemical vapor deposition (CVD), dielectrophoresis and manual attachment inside a scanning electron microscope (SEM). One of the earliest techniques developed is known as the pick-up method and involves adhering CNTs to AFM tips by simply scanning the AFM tip, in tapping mode, across a CNT-covered surface until a CNT attaches to the AFM tip. In this work we will further investigate how, for example, high force tapping mode imaging can improve the stability and success rate of the pick-up method. We will also discuss methods to determine CNT attachment to AFM probes including changes in AFM image resolution, amplitude versus distance curves and SEM imaging. We demonstrate that the pick-up method can be applied to a range of AFM probes, including contact mode probes with relatively soft spring constants (0.28 N/m). Finally, we demonstrate that the pick-up method can be used to attach CNTs to two AFM tips simultaneously. This is significant as it demonstrates the techniques potential for attaching CNTs to multiple AFM tips which could have applications in AFM-based data storage, devices such as the Snomipede, or making CNT-AFM tips more commercially viable.

AFM Microcantilever With a Collocated AlN Sensor-Actuator Pair: Enabling Efficient Q-Control for Dynamic Imaging

This manuscript presents a novel microcantilever with an embedded piezoelectric sensor-actuator pair for dynamic atomic force microscopy (AFM). The transducer pair is constructed from a two-layered AlN stack. Stacking the piezoelectric transducers in this manner leads to a minimal feedthrough from actuation to sense electrode, granting a high dynamic range frequency response for dynamic mode AFM. The cantilever’s design allows for dual mode operation at 1st and 2nd resonance modes. High resolution tapping mode imaging results are reported, while the cantilever is operated at these modes. A feedback control loop is used to modify quality factor of the 1st mode of the cantilever, using a positive position feedback (PPF) controller. A faster response time is achieved by reducing the ${Q}$ -factor, enabling the cantilever to track the topography at a higher scan rate. Results of bimodal AFM imaging are reported, using amplitude changes of the 1st mode for surface topography, while material properties are encoded in phase changes of the 2nd mode. [2020-0096]

Real-time and in situ observation of structural evolution of giant block copolymer thin film under solvent vapor annealing by atomic force microscopy.

An instrumentation technique for real-time, in situ and real space observation of microphase separation was proposed for ultra-high molecular weight block copolymer thin films (1010 kg mol−1, domain spacing of 180 nm) under high solvent vapor swelling conditions. This is made possible by a combination of a homebuilt chamber which is capable of supplying sufficient amount of vapor, and force–distance curve measurements which gives real-time swollen film thickness and allow active feedback for controlling the degree of swelling. We succeeded in monitoring the domain coarsening of perpendicular lamellar structures in polystyrene-block-poly(methyl methacrylate) thin films for eight hours via tapping mode imaging. During the annealing process, the thickness reached a maximum of 8.5 times that of the original film. The series of temporal real space topographic images obtained via this method allowed us to study, for the first time, the growth exponent of the correlation length under solvent vapor annealing.

Lights Out! Nano-Scale Topography Imaging of Sample Surface in Opaque Liquid Environments with Coated Active Cantilever Probes.

Atomic force microscopy is a powerful topography imaging method used widely in nanoscale metrology and manipulation. A conventional Atomic Force Microscope (AFM) utilizes an optical lever system typically composed of a laser source, lenses and a four quadrant photodetector to amplify and measure the deflection of the cantilever probe. This optical method for deflection sensing limits the capability of AFM to obtaining images in transparent environments only. In addition, tapping mode imaging in liquid environments with transparent sample chamber can be difficult for laser-probe alignment due to multiple different refraction indices of materials. Spurious structure resonance can be excited from piezo actuator excitation. Photothermal actuation resolves the resonance confusion but makes optical setup more complicated. In this paper, we present the design and fabrication method of coated active scanning probes with piezoresistive deflection sensing, thermomechanical actuation and thin photoresist polymer surface coating. The newly developed probes are capable of conducting topography imaging in opaque liquids without the need of an optical system. The selected coating can withstand harsh chemical environments with high acidity (e.g., 35% sulfuric acid). The probes are operated in various opaque liquid environments with a custom designed AFM system to demonstrate the imaging performance. The development of coated active probes opens up possibilities for observing samples in their native environments.

Probing phospholipid microbubbles by atomic force microscopy to quantify bubble mechanics and nanostructural shell properties

Microbubbles (MBs), which are used as ultrasonic contrast agents, have distinct acoustic signatures which enable them to significantly enhance visualisation of the vasculature. Research is progressing to develop MBs which act as drug/gene delivery vehicles for site-specific therapeutics. In order to manufacture effective theranostic vehicles, it is imperative to understand the mechanical and nanostructural properties of these agents; this will enrich the understanding of how the structural, biophysical and chemical properties of these bubbles impact their functionality. We produced microfluidic phospholipid-based MBs due to their favourable properties, such as biocompatibility and echogenicity, as well as the ability to modify the shell for targeting applications. We have drawn upon atomic force microscopy to conduct force spectroscopy and tapping-mode imaging investigations. We have, for the first time to our knowledge, been able to accurately quantify the thickness and lipid configuration of phospholipid-shelled MBs - showing a trilayer as opposed to the conventional monolayer structure. Furthermore, we have measured MB stiffness and employed different mechanical theories to quantify the Young’s modulus. We show that the Reissner theory is inappropriate for mechanical characterisation of phospholipid MBs, however, the Hertz model does offer biologically relevant comparisons. Analysis using the Alexander-de Gennes polymer brush theory has allowed us to provide new information regarding how the thickness of the polyethylene glycol brushes, end-grafted to our phospholipid microbubbles, changes with diameter.

Measurement of Radial Elasticity and Original Height of DNA Duplex Using Tapping-Mode Atomic Force Microscopy.

Atomic force microscopy (AFM) can characterize nanomaterial elasticity. However, some one-dimensional nanomaterials, such as DNA, are too small to locate with an AFM tip because of thermal drift and the nonlinearity of piezoelectric actuators. In this study, we propose a novel approach to address the shortcomings of AFM and obtain the radial Young’s modulus of a DNA duplex. The elastic properties are evaluated by combining physical calculations and measured experimental results. The initial elasticity of the DNA is first assumed; based on tapping-mode scanning images and tip–sample interaction force simulations, the calculated elastic modulus is extracted. By minimizing the error between the assumed and experimental values, the extracted elasticity is assigned as the actual modulus for the material. Furthermore, tapping-mode image scanning avoids the necessity of locating the probe exactly on the target sample. In addition to elasticity measurements, the deformation caused by the tapping force from the AFM tip is compensated and the original height of the DNA is calculated. The results show that the radial compressive Young’s modulus of DNA is 125–150 MPa under a tapping force of 0.5–1.3 nN; its original height is 1.9 nm. This approach can be applied to the measurement of other nanomaterials.

Nanoscale Chemical Imaging of Individual, Chemotherapeutic Cytarabine-loaded Liposomal Nanocarriers.

Dosage of chemotherapeutic drugs is a tradeoff between efficacy and side-effects. Liposomes are nanocarriers that increase therapy efficacy and minimize side-effects by delivering otherwise difficult to administer therapeutics with improved efficiency and selectivity. Still, variabilities in liposome preparation require assessing drug encapsulation efficiency at the single liposome level, an information that, for non-fluorescent therapeutic cargos, is inaccessible due to the minute drug load per liposome. Photothermal induced resonance (PTIR) provides nanoscale compositional specificity, up to now, by leveraging an atomic force microscope (AFM) tip contacting the sample to transduce the sample's photothermal expansion. However, on soft samples (e.g. liposomes) PTIR effectiveness is reduced due to the likelihood of tip-induced sample damage and inefficient AFM transduction. Here, individual liposomes loaded with the chemotherapeutic drug cytarabine are deposited intact from suspension via nES-GEMMA (nano-electrospray gas-phase electrophoretic mobility molecular analysis) collection and characterized at the nanoscale with the chemically-sensitive PTIR method. A new tapping-mode PTIR imaging paradigm based on heterodyne detection is shown to be better adapted to measure soft samples, yielding cytarabine distribution in individual liposomes and enabling classification of empty and drug-loaded liposomes. The measurements highlight PTIR capability to detect ≈ 103 cytarabine molecules (≈ 1.7 zmol) label-free and non-destructively.

Characterization of CRM Binders with Wax Additives Using an Atomic Force Microscopy (AFM) and an Optical Microscopy

Abstract This paper investigates the change in micromorphology of two binders and their respective warm mix asphalt binders before and after aging using atomic force microscopy (AFM) and optical microscopy. The control (PG 64-22) and crumb rubber modified (CRM) binders were blended with two different types of wax additives (L, a wax-based composition including crystal controller, and S, a long chain aliphatic hydrocarbon) and then artificially short-term and long-term aged using the rolling thin film oven and pressure aging vessel procedures. L is a wax based composition including crystal controller and S is a long chain aliphatic hydrocarbon. The AFM tapping-mode imaging technique was used to analyze the microstructure and the Superpave asphalt binder test was used to measure the rheology properties. In general, the results of this study indicated that (1) a correlation was observed between the changes in micromorphology (the distribution of dispersed domains and matrix) and rheology properties regardless of the aging, binder, and wax types; (2) the addition of crumb rubber into the base binder decreased the dimension of the catana phase (“bee-like”) structure, which increased the quantity of these structures; (3) the opposite trend was found between the aged control binder and the aged CRM binder regarding the size of the bee-like structure; (4) the long-term aging seemed to have a significant effect on the microstructures of the warm control and CRM binders; and (5) the inclusion of S and L was observed to have a similar trend for both binder types in terms of increased size of the bee-like structure and no bee-like structure, respectively.

Viscoelastic mapping of spruce-polyurethane bond line area using AM-FM atomic force microscopy

Amplitude modulation-frequency modulation atomic force microscopy (AM-FM AFM) with the capability of mapping simultaneously the topography and the nanomechanical properties of materials provides elastic information including Young's modulus, and loss tangent as viscoelastic information. The AM-FM mode operating principle involves the excitation and detection of two cantilever resonances simultaneously. Amplitude modulation (AM), the first resonance, is used for tapping mode imaging, while a higher resonance mode is operated in frequency modulation (FM). At resonance, the cantilever frequency and phase respond sensitively to changes in sample properties to provide a direct measurement of substrate mechanical properties and compositional information that only requires tuning of the cantilever resonance to provide quantitative information. In this study for the first time, by using an AM-FM AFM, the viscoelastic properties of the spruce wood and polyurethane adhesive (PUR) bond line area as a part of cross laminated timber (CLT) system were investigated. It was found that PUR adhesive rarely penetrates into the wood, and in contrast to viscous properties (tan δ), the elastic properties increase from the wood part towards the adhesive part in the bond line. AM-FM AFM technique proved to be able to effectively map viscoelastic properties of bond line region.

High-speed dynamic-mode atomic force microscopy imaging of polymers: an adaptive multiloop-mode approach.

Adaptive multiloop-mode (AMLM) imaging to substantially increase (over an order of magnitude) the speed of tapping-mode (TM) imaging is tested and evaluated through imaging three largely different heterogeneous polymer samples in experiments. It has been demonstrated that AMLM imaging, through the combination of a suite of advanced control techniques, is promising to achieve high-speed dynamic-mode atomic force microscopy imaging. The performance, usability, and robustness of the AMLM in various imaging applications, however, is yet to be assessed. In this work, three benchmark polymer samples, including a PS–LDPE sample, an SBS sample, and a Celgard sample, differing in feature size and stiffness of two orders of magnitude, are imaged using the AMLM technique at high-speeds of 25 Hz and 20 Hz, respectively. The comparison of the images obtained to those obtained by using TM imaging at scan rates of 1 Hz and 2 Hz showed that the quality of the 25 Hz and 20 Hz AMLM imaging is at the same level of that of the 1 Hz TM imaging, while the tip–sample interaction force is substantially smaller than that of the 2 Hz TM imaging.

Crystal Initiation Structures in Developing Enamel: Possible Implications for Caries Dissolution of Enamel Crystals.

Investigations of developing enamel crystals using Atomic and Chemical Force Microscopy (AFM, CFM) have revealed a subunit structure. Subunits were seen in height images as collinear swellings about 30 nM in diameter on crystal surfaces. In friction mode they were visible as positive regions. These were similar in size (30–50 nM) to collinear spherical structures, presumably mineral matrix complexes, seen in developing enamel using a freeze fracturing/freeze etching procedure. More detailed AFM studies on mature enamel suggested that the 30–50 nM structures were composed of smaller units, ~10–15 nM in diameter. These were clustered in hexagonal or perhaps a spiral arrangement. It was suggested that these could be the imprints of initiation sites for mineral precipitation. The investigation aimed at examining original freeze etched images at high resolution to see if the smaller subunits observed using AFM in mature enamel were also present in developing enamel i.e., before loss of the organic matrix. The method used was freeze etching. Briefly samples of developing rat enamel were rapidly frozen, fractured under vacuum, and ice sublimed from the fractured surface. The fractured surface was shadowed with platinum or gold and the metal replica subjected to high resolution TEM. For AFM studies high-resolution tapping mode imaging of human mature enamel sections was performed in air under ambient conditions at a point midway between the cusp and the cervical margin. Both AFM and freeze etch studies showed structures 30–50 nM in diameter. AFM indicated that these may be clusters of somewhat smaller structures ~10–15 nM maybe hexagonally or spirally arranged. High resolution freeze etching images of very early enamel showed ~30–50 nM spherical structures in a disordered arrangement. No smaller units at 10–15 nM were clearly seen. However, when linear arrangements of 30–50 nM units were visible the picture was more complex but also smaller units including ~10–15 nM units could be observed.Conclusions: Structures ~10–15 nM in diameter were detected in developing enamel. While the appearance was complex, these were most evident when the 30–5 nM structures were in linear arrays. Formation of linear arrays of subunits may be associated with the development of mineral initiation sites and attendant processing of matrix proteins.

Evaluating Asphalt Binders Prepared with Different Processes to Meet the Same Performance Grade: Use of Atomic Force Microscope

This paper summarizes the results of a laboratory testing program conducted to evaluate the microstructure, adhesion, and other mechanical properties of asphalt binders meeting the same performance grade (PG) but produced by using different processes and modifiers. To that end, atomic force microscope (AFM) tapping mode imaging and force spectroscopy experiments were conducted on different straight-run and modified asphalt binders meeting the same PG. In addition, bitumen bond strength and semicircular beam (SCB) tests were conducted on the different binders evaluated and on the mixes prepared by using those binders, respectively, for comparison. The AFM images indicated that the microstructure of the modified binders was different from those of the straight-run binders. The AFM force spectroscopy test results showed that binders with the same PG could have significantly different adhesion properties. The results of the SCB tests indicated that the fatigue performance was affected by the adhesion properties of the binders evaluated. The AFM bonding energy had a very good correlation with the flexibility index parameter obtained from SCB test results. The results of this paper suggest that the adhesion properties of asphalt binders should be included in their evaluation process and specifications.

Sub-nanometer Resolution Imaging with Amplitude-modulation Atomic Force Microscopy in Liquid

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown that when operated in amplitude-modulation (tapping) mode, atomic or molecular-level resolution images can be achieved over a wide range of soft and hard samples in liquid. In these situations, small oscillation amplitudes (SAM-AFM) enhance the resolution by exploiting the solvated liquid at the surface of the sample. Although the technique has been successfully applied across fields as diverse as materials science, biology and biophysics and surface chemistry, obtaining high-resolution images in liquid can still remain challenging for novice users. This is partly due to the large number of variables to control and optimize such as the choice of cantilever, the sample preparation, and the correct manipulation of the imaging parameters. Here, we present a protocol for achieving high-resolution images of hard and soft samples in fluid using SAM-AFM on a commercial instrument. Our goal is to provide a step-by-step practical guide to achieving high-resolution images, including the cleaning and preparation of the apparatus and the sample, the choice of cantilever and optimization of the imaging parameters. For each step, we explain the scientific rationale behind our choices to facilitate the adaptation of the methodology to every user's specific system.

Sub-nanometer Resolution Imaging with Amplitude-modulation Atomic Force Microscopy in Liquid.

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown that when operated in amplitude-modulation (tapping) mode, atomic or molecular-level resolution images can be achieved over a wide range of soft and hard samples in liquid. In these situations, small oscillation amplitudes (SAM-AFM) enhance the resolution by exploiting the solvated liquid at the surface of the sample. Although the technique has been successfully applied across fields as diverse as materials science, biology and biophysics and surface chemistry, obtaining high-resolution images in liquid can still remain challenging for novice users. This is partly due to the large number of variables to control and optimize such as the choice of cantilever, the sample preparation, and the correct manipulation of the imaging parameters. Here, we present a protocol for achieving high-resolution images of hard and soft samples in fluid using SAM-AFM on a commercial instrument. Our goal is to provide a step-by-step practical guide to achieving high-resolution images, including the cleaning and preparation of the apparatus and the sample, the choice of cantilever and optimization of the imaging parameters. For each step, we explain the scientific rationale behind our choices to facilitate the adaptation of the methodology to every user's specific system.

Probing Single Individual Proteins Unfold and Refold with 1-μs Resolution: Improved AFM-Based Single Molecule Force Spectroscopy

Protein folding is understood as a set of transitions between states. Yet, an over-simplified view of the folding process emerges if briefly populated states remain undetected due to ensemble averaging and/or limited instrumental precision. Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is widely used to mechanically measure the folding and unfolding of proteins. However, the temporal resolution of a standard cantilever is 50-1,000 μs, masking rapid transitions and short-lived intermediates. Recently, SMFS with 0.7-μs temporal resolution was achieved using ultrashort (L=9 µm) cantilevers on a custom-built, high-speed AFM. By modifying such cantilevers with a focused ion beam, we optimized them for SMFS rather than tapping-mode imaging. To enhance usability and throughput, we detected these cantilevers on a commercial AFM retrofitted with a detection laser featuring a 3-μm spot size. The improved capabilities of the modified cantilevers were demonstrated in two biophysical experiments. First, we unfolded a polyprotein, a popular assay, where these cantilevers maintained a 1-μs response time while eliminating cantilever ringing (Q≈0.5). In particular, these cantilever had improved short-term precision by avoiding periods of 30-90 pN (peak-to-peak) force modulation exhibited by unmodified ultrashort cantilevers undergoing underdamped motion at ∼500 kHz. In the second assay, we unfolded bacteriorhodopsin (bR), a model membrane protein. The resulting force-extension curves show unprecedented detail, increasing the number of intermediates resolved while unfolding a pair of transmembrane helices from 2 to 14. Equilibrium measurements revealed the cooperative folding of a 3-amino-acid structural element, resolved those states in <15-μs, and deduced the transition's underlying energy landscape. These bR results sharpen the picture of membrane protein folding and, more broadly, the instrumental enhancements demonstrate a new experimental regime: studying the equilibrium folding and unfolding of individual proteins with 1-µs resolution.

Label-Free Intramolecular Chemical Microscopy of a Protein-RNA Complex

Obtaining structural information from single biomolecules using imaging methods is challenging due to resolution limitations and difficulties in labelling specific sites of biomolecules. Atomic force microscopy based imaging of interaction forces between a specially designed probe molecule and a biomolecular complex of interest can, in principle, allow determining the locations of various regions within the complex, if the probe is designed to exhibit affinity to these regions. Here we demonstrate this concept with protein-RNA complex by targeting specific segments of the RNA with a DNA probe having sequence complementarity with these segments. To map the interaction forces between the probe and the sample, we used T-shaped cantilevers that allow mapping interaction forces during the tapping mode imaging process [1]. We worked with stem loop binding proteins in complex with RNA [2] and we targeted the flanking RNA regions adjacent to the stem loop. We show that locations and sequence identities of the RNA segments can be imaged with this approach. Repeated measurements of the same protein-RNA complex showed the consistency of the images. Our results show that it is possible obtain chemically-specific structural information from single biomolecules in physiologically relevant conditions without using labels.1. Kim, D., Sahin, O. Nature Nanotechnology 10, 264-269 (2015).2. Tan, D., Mazluff, W. F., Dominski, Z., Tong, L. Science 339, 318-321 (2013).