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High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due to its ability to image under physiological conditions in liquid environments. The photothermal off-resonance tapping (PORT) mode uses a drive laser to oscillate the cantilever in a controlled manner. This direct cantilever actuation is effective in the MHz range. Combined with operating the feedback loop on the time domain force curve rather than the resonant amplitude, PORT enables high-speed imaging at up to ten frames per second with direct control over tip-sample forces. PORT has been shown to enable imaging of delicate assembly dynamics and precise monitoring of patterns formed by biomolecules. Thus far, the technique has been used for a variety of dynamic in vitro studies, including the DNA 3-point-star motif assembly patterns shown in this work. Through a series of experiments, this protocol systematically identifies the optimal imaging parameter settings and ultimate limits of the HS-PORT AFM imaging system and how they affect biomolecular assembly processes. Additionally, it investigates potential undesired thermal effects induced by the drive laser on the sample and surrounding liquid, particularly when the scanning is limited to small areas. These findings provide valuable insights that will drive the advancement of PORT mode's application in studying complex biological systems.
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Fenómenos Mecánicos , Nanotecnología , Microscopía de Fuerza Atómica/métodos , Imagen Molecular , ADNRESUMEN
Super-resolution techniques expand the abilities of researchers who have the knowledge and resources to either build or purchase a system. This excludes the part of the research community without these capabilities. Here we introduce the openSIM add-on to upgrade existing optical microscopes to Structured Illumination super-resolution Microscopes (SIM). The openSIM is an open-hardware system, designed and documented to be easily duplicated by other laboratories, making super-resolution modality accessible to facilitate innovative research. The add-on approach gives a performance improvement for pre-existing lab equipment without the need to build a completely new system.
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Dynamic atomic force microscopy (AFM) modes that operate at frequencies far away from the resonance frequency of the cantilever (off-resonance tapping (ORT) modes) can provide high-resolution imaging of a wide range of sample types, including biological samples, soft polymers, and hard materials. These modes offer precise and stable control of vertical force, as well as reduced lateral force. Simultaneously, they enable mechanical property mapping of the sample. However, ORT modes have an intrinsic drawback: a low scan speed due to the limited ORT rate, generally in the low-kilohertz range. Here, we analyze how the conventional ORT control method limits the topography tracking quality and hence the imaging speed. The closed-loop controller in conventional ORT restricts the sampling rate to the ORT rate and introduces a large closed-loop delay. We present an alternative ORT control method in which the closed-loop controller samples and tracks the vertical force changes during a defined time window of the tip-sample interaction. Through this, we use multiple samples in the proximity of the maximum force for the feedback loop, rather than only one sample at the maximum force instant. This method leads to improved topography tracking at a given ORT rate and therefore enables higher scan rates while refining the mechanical property mapping.
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Invading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to microbial structures. How intracellular bacterial pathogens adapt to these stresses is not fully understood. Here, we report the discovery of a virulence mechanism by which changes to the mechanical stiffness of the mycobacterial cell surface confer refraction to killing during infection. Long-term time-lapse atomic force microscopy was used to reveal a process of "mechanical morphotype switching" in mycobacteria exposed to host intracellular stress. A "soft" mechanical morphotype switch enhances tolerance to intracellular macrophage stress, including cathelicidin. Both pharmacologic treatment, with bedaquiline, and a genetic mutant lacking uvrA modified the basal mechanical state of mycobacteria into a soft mechanical morphotype, enhancing survival in macrophages. Our study proposes microbial cell mechanical adaptation as a critical axis for surviving host-mediated stressors.
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Mycobacterium , Macrófagos/metabolismo , Fagocitos , Membrana CelularRESUMEN
High-speed atomic force microscopy (HS-AFM) is a technique capable of revealing the dynamics of biomolecules and living organisms at the nanoscale with a remarkable temporal resolution. The phase delay in the feedback loop dictates the achievable speed of HS-AFM instruments that rely on fast nanopositioners operated predominantly in conjunction with piezoelectric actuators (PEAs). The high capacitance and high operating voltage of PEAs make them difficult to drive. The limited bandwidth of associated high-voltage piezo-amplifiers is one of the bottlenecks to higher scan speeds. In this study, we report a high-voltage, wideband voltage amplifier comprised of a separate amplification and novel voltage-follower power stage, requiring no global feedback. The reported amplifier can deliver a current over ±2 amps, offers a small-signal bandwidth of 1 MHz, and exhibits an exceptionally low phase lag, making it particularly well suited for the needs of next-generation HS-AFMs. We demonstrate its capabilities by reporting its achievable bandwidth under various PEA loads and showcasing its merit for HS-AFM by imaging tubulin protofilament dynamics at sub-second frame rates.
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Time-lapse light microscopy combined with in vitro neuronal cultures has provided a significant contribution to the field of Developmental Neuroscience. The establishment of the neuronal polarity, i.e., formation of axons and dendrites, key structures responsible for inter-neuronal signaling, was described in 1988 by Dotti, Sullivan and Banker in a milestone paper that continues to be cited 30 years later. In the following decades, numerous fluorescently labeled tags and dyes were developed for live cell imaging, providing tremendous advancements in terms of resolution, acquisition speed and the ability to track specific cell structures. However, long-term recordings with fluorescence-based approaches remain challenging because of light-induced phototoxicity and/or interference of tags with cell physiology (e.g., perturbed cytoskeletal dynamics) resulting in compromised cell viability leading to cell death. Therefore, a label-free approach remains the most desirable method in long-term imaging of living neurons. In this paper we will focus on label-free high-resolution methods that can be successfully used over a prolonged period. We propose novel tools such as scanning ion conductance microscopy (SICM) or digital holography microscopy (DHM) that could provide new insights into live cell dynamics during neuronal development and regeneration after injury.
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Microscopía , Neuronas , Neuronas/fisiología , Microscopía/métodos , Supervivencia Celular , Células CultivadasRESUMEN
Malignant transformation and tumour progression are associated with cancer-cell softening. Yet how the biomechanics of cancer cells affects T-cell-mediated cytotoxicity and thus the outcomes of adoptive T-cell immunotherapies is unknown. Here we show that T-cell-mediated cancer-cell killing is hampered for cortically soft cancer cells, which have plasma membranes enriched in cholesterol, and that cancer-cell stiffening via cholesterol depletion augments T-cell cytotoxicity and enhances the efficacy of adoptive T-cell therapy against solid tumours in mice. We also show that the enhanced cytotoxicity against stiffened cancer cells is mediated by augmented T-cell forces arising from an increased accumulation of filamentous actin at the immunological synapse, and that cancer-cell stiffening has negligible influence on: T-cell-receptor signalling, production of cytolytic proteins such as granzyme B, secretion of interferon gamma and tumour necrosis factor alpha, and Fas-receptor-Fas-ligand interactions. Our findings reveal a mechanical immune checkpoint that could be targeted therapeutically to improve the effectiveness of cancer immunotherapies.
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Inmunoterapia Adoptiva , Neoplasias , Animales , Inmunoterapia , Interferón gamma , Ratones , Neoplasias/terapia , Linfocitos TRESUMEN
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.
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Microscopía de Sonda de Barrido , Orgánulos , Microscopía de Sonda de Barrido/métodos , Microscopía de Fuerza Atómica , Membrana CelularRESUMEN
Discovering mechanisms governing organelle assembly is a fundamental pursuit in biology. The centriole is an evolutionarily conserved organelle with a signature 9-fold symmetrical chiral arrangement of microtubules imparted onto the cilium it templates. The first structure in nascent centrioles is a cartwheel, which comprises stacked 9-fold symmetrical SAS-6 ring polymers emerging orthogonal to a surface surrounding each resident centriole. The mechanisms through which SAS-6 polymerization ensures centriole organelle architecture remain elusive. We deploy photothermally-actuated off-resonance tapping high-speed atomic force microscopy to decipher surface SAS-6 self-assembly mechanisms. We show that the surface shifts the reaction equilibrium by ~104 compared to solution. Moreover, coarse-grained molecular dynamics and atomic force microscopy reveal that the surface converts the inherent helical propensity of SAS-6 polymers into 9-fold rings with residual asymmetry, which may guide ring stacking and impart chiral features to centrioles and cilia. Overall, our work reveals fundamental design principles governing centriole assembly.
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Proteínas de Ciclo Celular/química , Centriolos/química , Chlamydomonas reinhardtii/química , Cinética , Microscopía de Fuerza Atómica , Modelos Químicos , Simulación de Dinámica Molecular , Biogénesis de Organelos , Conformación Proteica , Multimerización de ProteínaRESUMEN
High-resolution live-cell imaging is necessary to study complex biological phenomena. Modern fluorescence microscopy methods are increasingly combined with complementary, label-free techniques to put the fluorescence information into the cellular context. The most common high-resolution imaging approaches used in combination with fluorescence imaging are electron microscopy and atomic-force microscopy (AFM), originally developed for solid-state material characterization. AFM routinely resolves atomic steps, however on soft biological samples, the forces between the tip and the sample deform the fragile membrane, thereby distorting the otherwise high axial resolution of the technique. Here we present scanning ion-conductance microscopy (SICM) as an alternative approach for topographical imaging of soft biological samples, preserving high axial resolution on cells. SICM is complemented with live-cell compatible super-resolution optical fluctuation imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. Finally, we employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffraction-resolution.
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Imagenología Tridimensional , Microscopía Fluorescente , Análisis de la Célula Individual , Animales , Células COS , Chlorocebus aethiops , Citoesqueleto/metabolismo , Iones , Imagen Óptica , Tubulina (Proteína)/metabolismoRESUMEN
Centrioles are evolutionarily conserved multi-protein organelles essential for forming cilia and centrosomes. Centriole biogenesis begins with self-assembly of SAS-6 proteins into 9-fold symmetrical ring polymers, which then stack into a cartwheel that scaffolds organelle formation. The importance of this architecture has been difficult to decipher notably because of the lack of precise tools to modulate the underlying assembly reaction. Here, we developed monobodies against Chlamydomonas reinhardtii SAS-6, characterizing three in detail with X-ray crystallography, atomic force microscopy and cryo-electron microscopy. This revealed distinct monobody-target interaction modes, as well as specific consequences on ring assembly and stacking. Of particular interest, monobody MBCRS6-15 induces a conformational change in CrSAS-6, resulting in the formation of a helix instead of a ring. Furthermore, we show that this alteration impairs centriole biogenesis in human cells. Overall, our findings identify monobodies as powerful molecular levers to alter the architecture of multi-protein complexes and tune centriole assembly.
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Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Proteínas Algáceas/química , Proteínas Algáceas/metabolismo , Proteínas Portadoras/química , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/química , Centriolos/ultraestructura , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/ultraestructura , Microscopía por Crioelectrón , Cristalografía por Rayos X , Microscopía de Fuerza Atómica , Modelos Moleculares , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
Advanced in vitro models called "organ-on-a-chip" can mimic the specific cellular environment found in various tissues. Many of these models include a thin, sometimes flexible, membrane aimed at mimicking the extracellular matrix (ECM) scaffold of in vivo barriers. These membranes are often made of polydimethylsiloxane (PDMS), a silicone rubber that poorly mimics the chemical and physical properties of the basal membrane. However, the ECM and its mechanical properties play a key role in the homeostasis of a tissue. Here, we report about biological membranes with a composition and mechanical properties similar to those found in vivo. Two types of collagen-elastin (CE) membranes were produced: vitrified and nonvitrified (called "hydrogel membrane"). Their mechanical properties were characterized using the bulge test method. The results were compared using atomic force microscopy (AFM), a standard technique used to evaluate the Young's modulus of soft materials at the nanoscale. Our results show that CE membranes with stiffnesses ranging from several hundred of kPa down to 1 kPa can be produced by tuning the CE ratio, the production mode (vitrified or not), and/or certain parameters such as temperature. The Young's modulus can easily be determined using the bulge test. This method is a robust and reproducible to determine membrane stiffness, even for soft membranes, which are more difficult to assess by AFM. Assessment of the impact of substrate stiffness on the spread of human fibroblasts on these surfaces showed that cell spread is lower on softer surfaces than on stiffer surfaces.
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Matriz Extracelular , Dispositivos Laboratorio en un Chip , Membrana Celular , Módulo de Elasticidad , Humanos , Microscopía de Fuerza AtómicaRESUMEN
Mycobacteria have unique cell envelopes, surface properties, and growth dynamics, which all play a part in the ability of these important pathogens to infect, evade host immunity, disseminate, and resist antibiotic challenges. Recent atomic force microscopy (AFM) studies have brought new insights into the nanometer-scale ultrastructural, adhesive, and mechanical properties of mycobacteria. The molecular forces with which mycobacterial adhesins bind to host factors, like heparin and fibronectin, and the hydrophobic properties of the mycomembrane have been unraveled by AFM force spectroscopy studies. Real-time correlative AFM and fluorescence imaging have delineated a complex interplay between surface ultrastructure, tensile stresses within the cell envelope, and cellular processes leading to division. The unique capabilities of AFM, which include subdiffraction-limit topographic imaging and piconewton force sensitivity, have great potential to resolve important questions that remain unanswered on the molecular interactions, surface properties, and growth dynamics of this important class of pathogens.
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Membrana Celular/ultraestructura , Mycobacterium/ultraestructura , Adhesinas Bacterianas/metabolismo , Antibacterianos/farmacología , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Interacciones Hidrofóbicas e Hidrofílicas , Lípidos de la Membrana/química , Lípidos de la Membrana/fisiología , Microscopía de Fuerza Atómica , Mycobacterium/química , Mycobacterium/crecimiento & desarrollo , Mycobacterium/fisiología , Propiedades de SuperficieRESUMEN
In this work, we report on the integration of an atomic force microscope (AFM) into a helium ion microscope (HIM). The HIM is a powerful instrument, capable of imaging and machining of nanoscale structures with sub-nanometer resolution, while the AFM is a well-established versatile tool for multiparametric nanoscale characterization. Combining the two techniques opens the way for unprecedented in situ correlative analysis at the nanoscale. Nanomachining and analysis can be performed without contamination of the sample and environmental changes between processing steps. The practicality of the resulting tool lies in the complementarity of the two techniques. The AFM offers not only true 3D topography maps, something the HIM can only provide in an indirect way, but also allows for nanomechanical property mapping, as well as for electrical and magnetic characterization of the sample after focused ion beam materials modification with the HIM. The experimental setup is described and evaluated through a series of correlative experiments, demonstrating the feasibility of the integration.
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Bacterial resistance to conventional antibiotics is of major concern. Antimicrobial peptides (AMPs) are considered excellent alternatives. Among them, D-cateslytin (D-Ctl, derivative of a host defense peptide) has shown high efficiency against a broad spectrum of bacteria. The first target of AMPs is the outer membrane of the bacterium. However, the role of bacterial cell-wall structures on D-Ctl's mechanism of action has not yet been understood. In this study, we investigated the activity of D-Ctl on two isogenic strains of E. coli: one is devoid of any parietal structures; the other constitutively overexpresses only type 1 fimbriae. We studied the damage caused by D-Ctl at several initial concentrations of bacteria and D-Ctl, and exposure times to D-Ctl were examined using a combination of epifluorescence microscopy, atomic force microscopy (AFM), and Fourier transform infrared spectroscopy in attenuated total reflectance mode (ATR-FTIR). The analysis of nanomechanical and spectrochemical properties related to the antibacterial mechanism showed a concentration dependent activity. Whereas the membrane permeabilization was evidenced for all concentrations of D-Ctl and both mutants, no pore formation was observed. The bacterial stiffness is modified dramatically concomitantly to major membrane damage and changes in the spectral fingerprints of the bacteria. In the case of the occurrence of type 1 fimbriae only, an intracellular activity was additionally detected. Our results evidenced that D-Ctl activity is highly impacted by the cell-wall external structures and surface properties of the bacteria.
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Antibacterianos/farmacología , Pared Celular/efectos de los fármacos , Cromogranina A/farmacología , Escherichia coli/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Pared Celular/metabolismo , Escherichia coli/metabolismo , Fimbrias Bacterianas/clasificación , Fimbrias Bacterianas/metabolismo , Pruebas de Sensibilidad MicrobianaRESUMEN
The bacterial cell envelope is essential for viability, the environmental gatekeeper and first line of defense against external stresses. For most bacteria, the envelope biosynthesis is also the site of action of some of the most important groups of antibiotics. It is a complex, often multicomponent structure, able to withstand the internally generated turgor pressure. Thus, elucidating the architecture and dynamics of the cell envelope is important, to unravel not only the complexities of cell morphology and maintenance of integrity but also how interventions such as antibiotics lead to death. To address these questions requires the capacity to visualize the cell envelope in situ via high-spatial resolution approaches. In recent years, atomic force microscopy (AFM) has brought novel molecular insights into the assembly, dynamics, and functions of bacterial cell envelopes. The ultrafine resolution and physical sensitivity of the technique have revealed a wealth of ultrastructural features that are invisible to traditional optical microscopy techniques or imperceptible in their true physiological state by electron microscopy. Here, we discuss recent progress in our use of AFM imaging for understanding the architecture and dynamics of the bacterial envelope. We survey recent studies that demonstrate the power of the technique to observe isolated membranes and live cells at (sub)nanometer resolution and under physiological conditions and to track in vitro structural dynamics in response to growth or to drugs.
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Bacterias/ultraestructura , Membrana Celular/ultraestructura , Pared Celular/ultraestructura , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Membrana Celular/efectos de los fármacos , Pared Celular/efectos de los fármacos , Proteínas de la Membrana , Microscopía de Fuerza Atómica/métodosRESUMEN
Mechanisms to control cell division are essential for cell proliferation and survival 1. Bacterial cell growth and division require the coordinated activity of peptidoglycan synthases and hydrolytic enzymes 2-4 to maintain mechanical integrity of the cell wall 5. Recent studies suggest that cell separation is governed by mechanical forces 6,7. How mechanical forces interact with molecular mechanisms to control bacterial cell division in space and time is poorly understood. Here, we use a combination of atomic force microscope (AFM) imaging, nanomechanical mapping, and nanomanipulation to show that enzymatic activity and mechanical forces serve overlapping and essential roles in mycobacterial cell division. We find that mechanical stress gradually accumulates in the cell wall concentrated at the future division site, culminating in rapid (millisecond) cleavage of nascent sibling cells. Inhibiting cell wall hydrolysis delays cleavage; conversely, locally increasing cell wall stress causes instantaneous and premature cleavage. Cells deficient in peptidoglycan hydrolytic activity fail to locally decrease their cell wall strength and undergo natural cleavage, instead forming chains of non-growing cells. Cleavage of these cells can be mechanically induced by local application of stress with AFM. These findings establish a direct link between actively controlled molecular mechanisms and passively controlled mechanical forces in bacterial cell division.
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Mycobacteria grow by inserting new cell wall material in discrete zones at the cell poles. This pattern implies that polar growth zones must be assembled de novo at each division, but the mechanisms that control the initiation of new pole growth are unknown. Here, we combine time-lapse optical and atomic force microscopy to measure single-cell pole growth in mycobacteria with nanometer-scale precision. We show that single-cell growth is biphasic due to a lag phase of variable duration before the new pole transitions from slow to fast growth. This transition and cell division are independent events. The difference between the lag and interdivision times determines the degree of single-cell growth asymmetry, which is high in fast-growing species and low in slow-growing species. We propose a biphasic growth model that is distinct from previous unipolar and bipolar models and resembles "new end take off" (NETO) dynamics of polar growth in fission yeast.
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Modelos Biológicos , Mycobacterium/citología , Mycobacterium/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , División Celular , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía de Fuerza Atómica , Mycobacterium/genética , Análisis Espacio-Temporal , Imagen de Lapso de TiempoRESUMEN
Solid-state nanopores provide a highly sensitive tool for single-molecule sensing and probing nanofluidic effects in solutions. Glass nanopipettes are a cheap and robust type of solid-state nanopore produced from pulling glass capillaries with opening orifice diameters down to below tens of nanometers. Sub-50 nm nanocapillaries allow an unprecedented resolution for translocating single molecules or for scanning ion conductance microscopy imaging. Due to the small opening orifice diameters, such nanocapillaries are difficult to fill with solutions, compromising their advantages of low cost, availability, and experimental simplicity. We present a simple and cheap method to reliably fill nanocapillaries down to sub-10 nm diameters by microwave radiation heating. Using a large statistic of filled nanocapillaries, we determine the filling efficiency and physical principle of the filling process using sub-50 nm quartz nanocapillaries. Finally, we have used multiple nanocapillaries filled by our method for high-resolution scanning ion conductance microscopy imaging.
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Employing polymer cantilevers has shown to outperform using their silicon or silicon nitride analogues concerning the imaging speed of atomic force microscopy (AFM) in tapping mode (intermittent contact mode with amplitude modulation) by up to one order of magnitude. However, tips of the cantilever made out of a polymer material do not meet the requirements for tip sharpness and durability. Combining the high imaging bandwidth of polymer cantilevers with making sharp and wear-resistant tips is essential for a future adoption of polymer cantilevers in routine AFM use. In this work, we have developed a batch fabrication process to integrate silicon nitride tips with an average tip radius of 9 ± 2 nm into high-speed SU8 cantilevers. Key aspects of the process are the mechanical anchoring of a moulded silicon nitride tip and a two-step release process. The fabrication recipe can be adjusted to any photo-processable polymer cantilever.