The authors Ivo W. Rangelow and Thomas Sattel would like to acknowledge the German Federal Ministry of Education and Research (BMBF) and the German Federal Ministry of Economics Affairs and Climate Action (BMWK) for supporting parts of the presented methods by funding the projects FKZ:13N16580 &#34;Active Probes with diamond tip for quantum metrology and nanofabrication&#34; within the research line KMU-innovativ: Photonics and Quantum Technologies and KK5007912DF1 &#34;Conjungate Nano-Positioner-Scanner for fast and large metrological tasks in Atomic Force Microscopy&#34; within the funding line Central Innovation Program for small and medium sized industries (ZIM). Part of the work reported here was funded by the European Union Seventh Framework Programme FP7/2007-2013 under Grant Agreement No. 318804 &#34;Single Nanometre Manufacturing: Beyond CMOS.&#34; The authors Ivo W. Rangelow and Eberhard Manske gratefully acknowledge the support by the Deutsche Forschungsgemeinschaft (DFG) in the framework of Research Training Group &#34;Tip- and laser-based 3D-Nanofabrication in extended macroscopic working areas&#34; (GRK 2182) at the Technische Universit&#228;t Ilmenau, Germany.

1. Sample preparation for large-scale inspection
<ol>
	<li>Prepare the sample with a suitable size for the AFM (see Table of Materials). 
	NOTE: Wafer-shape samples with an in-plane diameter&#160;from 75 mm to 300 mm and an expected out-of-plane height variation below 200 &#181;m can fit on the AFM sample stage. In this study, an extreme ultraviolet (EUV) mask on a 4 inch wafer is used (see Table of Materials).</li>
	<li>Clean the sample to remove contaminants and keep the samples inside a clean room or low-dust environment, such as a vacuum chamber or a nitrogen-purged cabinet.
	<ol>
		<li>Remove large dust particles by blowing the sample with a compressed nitrogen spray gun, or rinse with deionized water if the sample does not react with water. To avoid damaging the sample, use a small flow rate below 0.1 m3/min.</li>
		<li>Optionally, apply plasma cleaning to remove organic contaminants. Place the sample into the plasma treatment machine. Close the chamber and pump down the pressure to 600 mTorr. Expose the sample to plasma for 30 s for cleaning. 
		NOTE: Step 1.3.2 is optional for contamination removal. In this study, fabrication and characterization were performed inside a clean room, so this step was not needed.</li>
	</ol>
	</li>
	<li>Identify suitable imaging strategies, including areas of interest, scanning range, spatial/pixel resolution, and scanning line speed.
	<ol>
		<li>Determine whether the sample needs an overall panorama or automated imaging of multiple smaller selected areas. 
		NOTE: For nanofabricated samples with desired structures, it is often easier to determine the key areas that are more prone to defects for inspection purposes. For other samples, taking a low spatial resolution image for a quick overview, and then zooming into the smaller area of interest for high spatial resolution imaging can be easier.</li>
		<li>Estimate the size of the features based on prior knowledge of the sample to determine the desired spatial resolution to resolve these features.</li>
		<li>Use the ratio of the imaging range and spatial resolution to determine pixel resolution.</li>
		<li>Select an initial scan line speed based on prior experience with the sample, or use the AFM system software later, based on the sample material, probe dynamics, and desired spatial resolution.</li>
	</ol>
	</li>
</ol>
2. AFM instrument calibration and experiment setup
<ol>
	<li>Select the appropriate active AFM cantilever array. 
	NOTE: The AFM active cantilever stiffness, first resonance frequency, and unused probe tip sharpness for each cantilever in the probe array are characterized from production. The data can be retrieved from the manufacturer and loaded into the software automatically before imaging. Selecting the appropriate cantilever recommended by the software based on the sample material or application is helpful to ensure successful imaging. Due to manufacturing variability, the properties of each cantilever can be different but on a similar level.</li>
	<li>Turn on the AFM by switching on the main power of the controller and wait for the system to initialize. Turn on the host computer and open the AFM system software.</li>
	<li>Perform active cantilever probe installation.
	<ol>
		<li>Raise the probe scanner up by clicking on the Active Cantilever Installation. Wait for the probe holder to be raised up from the sample stage and stop automatically.</li>
		<li>Mount and secure the AFM cantilever probe array on the probe holder. 
		NOTE: The AFM cantilever arrays are attached to a nano-SD card shape holder and can be handled directly by hand for probe installation. With the fundamental difference in sensing and actuation principles, there is no need to perform any laser alignment.</li>
		<li>Click on the Probe Auto Settings and load the probe information provided by the manufacturer (see Table of Materials). Ensure to match the serial number on the probe and in the software.</li>
		<li>Conduct a probe frequency sweep to verify the resonance of each cantilever for imaging. Click on Cantilever Tuning and click on Sweep in the pop-up window. Specify the Start Frequency and End Frequency if the range is known. If not, the software will automatically update the values using the default settings. 
		NOTE: This step is, in principle, optional for new probes as they have been calibrated after production. However, for probes that have been used previously, it is recommended to perform this sweep just in case any properties have changed during the previous operation (e.g., attachment of contaminant particles that shifts the cantilever resonance). Due to thermomechanical actuation, the heating power is proportional to the square of the driving voltage. For tapping non-contact mode operation, a second harmonic of the input voltage sine wave component is generated due to the square relation in Equation 2. This second harmonic is typically matched with the cantilever resonance during excitation, as the direct current (DC) offset signal does not affect its amplitude. Therefore, the DC components control the average cantilever deflection, and the alternating current (AC) component of the drive signal is automatically set to half of the cantilever resonance frequency for imaging.</li>
	</ol>
	</li>
	<li>Load and secure the sample in place. Ensure the bottom surface in contact with the sample is parallel to the top surface with the features to be imaged. Fine-tune the sample stage tilt using the micrometer knobs to ensure the sample is flat. Add spacers if the tilt is too large for the fine adjustment positioners to compensate for.</li>
	<li>Adjust the in-plane XY position of the sample using the micrometer on the AFM stage. Use an optical microscope image to locate the area of interest and position the relative position of the cantilever array onto the first area of interest to be imaged.</li>
	<li>Establish a global coordinate by clicking on the XYZ Zero button. 
	NOTE: For the creation of a panoramic image, this step can be conducted roughly using the optical microscope view. When imaging selected areas of a nanofabricated sample, it may be necessary to precisely align the XYZ coordinate of the fabrication equipment with the AFM coordinate. This step can be performed more precisely by performing the AFM imaging and clicking on the XYZ Zero button again.</li>
	<li>Close and seal the acoustic shield. 
	&#8203;NOTE: The acoustic shield helps reduce vibration propagation through the air. Moreover, the sealed cover can also reduce the chance of dust particles landing on the sample, since large-scale imaging can take a long time to complete. This protective cover can be optional in a clean room environment without vibration sources.</li>
</ol>
3. Topography imaging and parameter tuning
<ol>
	<li>Select the tab Imaging Parameter Setup (active cantilever eigenmode, scanning speed, set-point, etc.) and input the desired imaging parameters.
	<ol>
		<li>For a single panoramic image, enter the coordinate of the top left corner of the image and the scan size. For imaging multiple selected regions automatically, repeat this process for all the regions to be imaged. Add extra padding around the imaging area for post-processing image stitching. 
		NOTE: For the present study, the top left corner of the EUV mask was set with pre-defined features, and the system was configured to capture four images with 130 &#181;m by 130 &#181;m simultaneously using each cantilever.</li>
		<li>Enter the desired in-plane pixel resolution (typically thousands of pixels per scan line) and use the default recommended line scan speed from the software for imaging. If needed, manually adjust the line scan speed for each region to be imaged. 
		NOTE: In this study, a pixel resolution of 26,000 pixels per line, corresponding to 5 nm per pixel, was used to obtain high-resolution images.</li>
		<li>For non-contact mode, use the default drive amplitude, frequency, and setpoint in the software obtained from the cantilever characteristics, or manually input the setpoint for each cantilever on the array. Leave the PID controller settings as default.</li>
	</ol>
	</li>
	<li>Engage the active cantilever probe array with the sample.
	<ol>
		<li>Click on Initialize Cantilever to pre-bend the cantilever before imaging.</li>
		<li>Click on Start Non-Contact Drive to excite the cantilever resonance.</li>
		<li>Click on Engage to let the system automatically bring the sample and the probe into contact. Imaging will start automatically after this step is completed.</li>
	</ol>
	</li>
	<li>Adjust the PID controller parameters for each cantilever based on the scanned trace/image. Adjust the PID parameters to improve the matching between the trace and retrace lines, which helps to capture the topography changes. The imaging speed can also be adjusted correspondingly to improve the topography tracking performance.</li>
	<li>Save the current data on the screen by clicking on the Save button. The data will also be automatically saved during the scan at the end of each frame.</li>
	<li>Stop imaging by clicking on the Stop button. The system will stop imaging and automatically retract the cantilever array from the sample. The cantilever drive will also be stopped so that it will be safe to uninstall the probe.</li>
	<li>Remove the probe and the sample carefully and turn off the system.</li>
</ol>
4. Post-processing and image analysis
<ol>
	<li>Open the AFM image analysis software supplied by the vendor.
	<ol>
		<li>Click on Auto Process to apply the default post-processing sequence, including sample tilt correction, pixel level outlier removal, and scan line matching.</li>
		<li>Apply further corrections on image defects manually from observation of the images. With advanced AFM instruments, such defects are rare, but images can still be improved with these steps. 
		NOTE: For large amounts of images, it is possible to automate the process using Macro or Python scripts through batch processing with the same operation. This was not required for the present study.</li>
	</ol>
	</li>
	<li>Use the software to form a panoramic image by clicking on the Panorama Image button and selecting multiple images to be stitched. 
	NOTE: Image stitching is performed automatically by directly using the overlapping space of the adjacent images. Alternatively, image merging tries to maximize the correlation of the overlapping pixels on the edges. Various options are available in these commands and can be optimized to improve the overall merging performance.</li>
	<li>Save the data for further analysis based on the specific sample.</li>
</ol>

Parallel Active Cantilever Arrays in AFMS to Enable High-Throughput Inspections

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The authors have no conflicts of interest.

As demonstrated in the representative results, an active cantilever array can be used to capture multiple images of a static sample in parallel. This scalable setup can significantly improve the imaging throughput of large-area samples, making it suitable for inspecting nanofabricated devices on semiconductor wafers. The technique is also not limited to man-made structures; as long as the topography variation within a group of active cantilevers is not too large for the cantilever array to handle, high-throughput imaging...

Atomic force microscopes (AFMs) can capture 3D topography images with nanoscale spatial resolution. Researchers have extended the capability of AFMs to create sample property maps in mechanical, electrical, magnetic, optical, and thermal domains. In the meantime, improving imaging throughput has also been the focus of research to adapt AFMs to new experimental needs. There are primarily two application domains for high-throughput AFM imaging: the first category is high-speed imaging of a small area to capture dynamic changes in the sample due to biological or chemical reactions1,2; the second category is for high-spatial resolution, large-scale imaging of static samples during an inspection, which is discussed in detail in this work. With transistor size shrinking down to the nanoscale, the semiconductor industry urgently needs high-throughput AFMs to inspect wafer-scale nanofabricated devices with nanoscale spatial resolution3.
The characterization of nanofabricated devices on a wafer can be challenging due to the vast scale difference between wafer and transistor features. Large defects can be spotted with optical microscopes automatically4. In addition, scanning electron microscopes (SEMs) are widely used for inspection down to tens of nanometers in 2D5. For 3D information and higher resolution, the AFM is a more suitable tool if its throughput can be improved.
With limited imaging throughput, one approach is to image selected wafer areas where nanofabrication defects are more likely to happen6. This would require prior knowledge of the design and fabrication process. Alternatively, combining other modalities, such as an optical microscope or SEM with an AFM for overview and zoom, is possible7,8. A wide-range, high-precision positioning system is needed to properly align the coordinate system between the fabrication and characterization tools. Moreover, an automated AFM system to image various selected areas is necessary to realize this functionality.
As an alternative, researchers have investigated different ways to increase AFM scanning speed. Since enabling high-throughput AFMs is a systematic precision instrumentation challenge, researchers have investigated various methods, including using smaller AFM probes, redesigning high-bandwidth nano-positioners9,10,11,12 and driving electronics13, optimizing modes of operation, imaging control algorithms14,15,16,17, etc. With these efforts, the effective relative tip and sample speed can be increased to a maximum of around tens of millimeters per second for commercially available single-probe AFM systems.
To further improve the imaging throughput, adding multiple probes to operate in parallel is a natural solution. However, the optical beam deflection (OBD) system utilized for cantilever deflection sensing is relatively bulky, which makes the addition of multiple probes relatively challenging. Individual cantilever deflection control can also be difficult to realize.
To overcome this limitation, embedded sensing, and actuation principles without bulky external components, are preferred. As detailed in previously published reports18,19, deflection sensing with piezoresistive, piezoelectric, and optomechanical principles can be considered embedded sensing, with the former two being more mature and easier to implement. For embedded actuation, thermomechanical with electrical heating or piezoelectric principles can both be utilized. Although piezoelectric principles can operate in a wider temperature range down to cryogenic environments, they can only support tapping mode AFM operations, since static deflection cannot be measured due to the charge leakage and static actuation suffering from hysteresis and creep. In prior work, active cantilever probe arrays using a piezoresistive sensor and the piezoelectric sensor have been developed for large-range imaging20,21, but have not been further scaled up for large-scale imaging or commercialized. In this work, the combination of piezoresistive sensing and thermomechanical actuation are selected as embedded transducers with static deflection control capability.
In this work, a novel &#34;Quattro&#34;22 parallel active cantilever array is used as the probe23 for simultaneous imaging using active cantilevers. To measure the cantilever deflection, piezoresistive sensors in a Wheatstone bridge configuration19 are nanofabricated at the base of each micro-cantilever to measure the internal stress, which is linearly proportional to the cantilever tip deflection. This compact embedded sensor can also achieve sub-nanometer resolution as the conventional OBD sensor. The governing equation of the Wheatstone bridge voltage output Uout in response to the applied force F or cantilever deflection z is shown in Equation 119 for a cantilever with length L, width W, and thickness H, piezoresistive sensor coefficient PR, and effective elastic modulus of the cantilever E bridge supply voltage Ub.
<img alt="Equation 1" src="/files/ftp_upload/65210/65210eq01.jpg" style="margin: auto;" />&#160; &#160; (1)
As dynamic tapping/non-contact mode operation is preferred for noninvasive imaging to avoid disturbing the sample, a thermomechanical actuator made of serpentine shape aluminum wires is used to heat up the bimorph cantilever made with aluminum/magnesium alloy24, silicon, and silicon oxide materials. At the microscopic scale, the time constant of thermal processes is much smaller, and the cantilever resonance at tens to hundreds of kilohertz can be excited by driving the heater with an electrical signal. The cantilever free end deflection zh controlled by the heater temperature&#160;&#916;T relative ambience is shown in Equation 219for cantilever length L with a constant K, depending on the bimorph material thermo coefficient of expansion and geometric thickness and area. It should be noted that the&#160;&#916;T&#160;is proportional to the heater power P, which is equal to the square of the applied voltage V divided by its resistance R.
<img alt="Equation 2" src="/files/ftp_upload/65210/65210eq02.jpg" style="margin: auto;" />&#160; &#160; (2)
As an added benefit, static deflection can also be controlled in addition to resonance excitation. This can be a particularly helpful capability to regulate the probe-sample interaction of each cantilever individually. Moreover, multiple cantilevers on the same base chip can be excited individually with the embedded thermomechanical actuator, which is impossible in conventional resonance excitation with piezo-generated acoustic waves.
Combining piezoresistive sensing and thermomechanical actuation, the active cantilever probe has enabled a wide range of applications, including collocated AF microscopy in SE microscopy, imaging in opaque liquid, and scanning probe lithography, with more details available in review25. For high-throughput inspection purposes, the active cantilever array is created with a representative AFM implementation example involving four parallel cantilevers, as shown in Figure 1. In the future, an industrial-scale system will be developed using eight parallel active cantilevers and tens of positioners28. To illustrate the scale using an example, with an in-plane spatial resolution of 100 nm, imaging an area of 100 mm by 100 mm would result in over 106 scan lines and 1012 pixels. With a scanning speed of 50 mm/s per cantilever, this would require a total of over 555.6 h of scanning (23+ days) for a single cantilever, which is too long to be practically useful. Using the active cantilever array technology with tens of positioners, the required imaging time can be reduced by around two orders of magnitude to 5-10 h (less than half a day) without making any compromise to the resolution, which is a reasonable time scale for industrial inspection purpose.
To capture large-area, high-resolution images, the nano-positioning system is also upgraded. For imaging wafer-scale large samples, scanning the probe instead of the sample is preferred, in order to reduce the size of the objects being moved. With the separation distance between active cantilevers at 125 &#181;m, the scanner covers an area slightly larger than this range so that images from each cantilever can be stitched together during post-processing. Upon completion of a scan, the coarse positioner automatically repositions the probe to a new adjacent area to continue the imaging process. While the embedded thermomechanical actuatorregulates the deflection of each cantilever, the averaged deflection of all parallel cantilevers is regulated with another proportional-integral-derivative (PID) controller to assist the cantilevers during topography tracking. The scanner controller also ensures that the bending of each cantilever does not exceed a maximum threshold value, which may cause other probes to lose contact with the surface if the topography variation is too large.
The level of topography variation that can be tracked for cantilevers on the same base chip should it be limited, since the static deflection control range of the cantilever is on the order of tens of microns. For semiconductor wafers, the sample topography variations are typically on the sub-micrometer scale, so they should not be much of a big problem. However, with the addition of more cantilevers, the sample plane tilt with respect to the line of cantilevers can become a problem. In practice, eight parallel cantilevers with spacings close to 1 mm would still allow 1&#176; of tilt angle, while adding more cantilevers can make the tilting control more difficult to realize. Therefore, using multiple groups of eight-cantilever probes placed on separated probe scanners is an ongoing effort to fully realize the potential of the parallel active cantilever probe principle.
After data collection, a post-processing operation is necessary to retrieve the desired information. The process generally involves removing scanning artifacts, stitching adjacent images to form an overall panorama, and optionally identifying the structure defects by comparing them to the desired geometry using suitable algorithms26. It is worth noting that the amount of data accumulated can be enormous for a large range of images, and data-driven learning algorithms are also being developed for more efficient processing27.
This article illustrates the general process of acquiring high-resolution AFM images using the parallel active cantilever array integrated into a custom AFM system. Detailed implementation of the system is available in22,28,29,30, and it is being commercialized with the model number listed in Table of Materials. All four cantilevers were operated in tapping mode excited by the embedded thermal-mechanical actuator. Representative results on calibration samples, nanofabrication masks, and highly oriented pyrolytic graphite (HOPG) samples (see Table of Materials) are provided to illustrate the effectiveness of this new AFM tool for large-area inspection.

<table><tbody><tr><td>Active-Cantilever&amp;nbsp;</td><td>nano analytik GmbH</td><td>AC-10-2012</td><td>AFM Probe</td></tr><tr><td>E-Beam</td><td>EBX-30, INC</td><td>012323-15</td><td>Mask patterning instrument</td></tr><tr><td>Highly Oriented Pyrolytic Graphite &amp;ndash; HOPG</td><td>TED PELLA, INC</td><td>626-10</td><td>AFM calibration sample</td></tr><tr><td>Mask Sample</td><td>Nanda Technologies GmbH</td><td>Test substrate</td><td>EUV Mask Sample substrate</td></tr><tr><td>NANO-COMPAS-PRO&amp;nbsp;</td><td>nano analytik GmbH</td><td>23-2016</td><td>AFM Software</td></tr><tr><td>nanoMetronom 20</td><td>nano analytik GmbH</td><td>1-343-2020</td><td>AFM Instrument</td></tr></tbody></table>

active probe atomic force microscopy with quattro-parallel cantilever arrays for high-throughput large-scale sample inspection

An Atomic Force Microscope&#160;(AFM) is&#160;a powerful and versatile tool for nanoscale surface studies to capture 3D topography images of samples. However, due to their limited imaging throughput, AFMs have not been widely adopted for large-scale inspection purposes. Researchers have developed high-speed AFM systems to record dynamic process videos in chemical and biological reactions at tens of frames per second, at the cost of a small imaging area of up to several square micrometers. In contrast, inspecting large-scale nanofabricated structures, such as semiconductor wafers, requires nanoscale spatial resolution imaging of a static sample over hundreds of square centimeters with high productivity. Conventional AFMs use a single passive cantilever probe with an optical beam deflection system, which can only collect one pixel at a time during AFM imaging, resulting in low imaging throughput. This work utilizes an array of active cantilevers with embedded piezoresistive sensors and thermomechanical actuators, which allows simultaneous multi-cantilever operation in parallel operation for increased imaging throughput. When combined with large-range nano-positioners and proper control algorithms, each cantilever can be individually controlled to capture multiple AFM images. With data-driven post-processing algorithms, the images can be stitched together, and defect detection can be performed by comparing them to the desired geometry. This paper introduces principles of the custom AFM using the active cantilever arrays, followed by a discussion on practical experiment considerations for inspection applications. Selected example images of silicon calibration grating, highly-oriented pyrolytic graphite, and extreme ultraviolet lithography masks are captured using an array of four active cantilevers (&#34;Quattro&#34;) with a 125 &#181;m tip separation distance. With more engineering integration, this high-throughput, large-scale imaging tool can provide 3D metrological data for extreme ultraviolet (EUV) masks, chemical mechanical planarization (CMP) inspection, failure analysis, displays, thin-film step measurements, roughness measurement dies, and laser-engraved dry gas seal grooves.

To demonstrate the effectiveness of AFM large-range imaging using parallel active cantilevers for topography imaging, the stitched images of a calibration grating, taken by four cantilevers operated in parallel, are shown in Figure 2. The silicon wafer calibration structure has 45 &#181;m long features with a height of 14 nm. Each cantilever covers an area of 125 &#181;m by 125 &#181;m, which gives a stitched panoramic image of 500 &#181;m by 125 &#181;m. The scanning speed was set to 10 lin...

Watch this Scientific Journal Video about Author Spotlight: Introduction to Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays at JoVE.com

Author Spotlight: Introduction to Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays

Large-scale sample inspection with nanoscale resolution has a wide range of applications, especially for nanofabricated semiconductor wafers. Atomic force microscopes can be a great tool for this purpose, but are limited by their imaging speed. This work utilizes parallel active cantilever arrays in AFMs to enable high-throughput and large-scale inspections.

Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays for High-Throughput Large-Scale Sample Inspection

An Atomic Force Microscope&#160;(AFM) is&#160;a powerful and versatile tool for nanoscale surface studies to capture 3D topography images of samples. ...

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1.4K Views.  Massachusetts Institute of Technology. Scansondemicroscopie op basis van nieuwe micro-elektromechanische systeemsondes worden actieve cantilevers genoemd. Draagarmen met geïntegreerde bediening en detectie bieden verschillende voordelen ten opzichte van passieve draagarmen, die afhankelijk zijn van piëzo-elektrische excitatie en optische straalafbuigingsmeting. De meest recente ontwikkeling op dit gebied is de realisatie van arrays van actieve cantilevers voor high-throughput parallelle SPM-beeldvorming.De reeksen van act...

Video: Auteur in de schijnwerpers: Inleiding tot actieve sonde atomaire krachtmicroscopie met quattro-parallelle cantilever-arrays

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

Chemistry

Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or &#8220;tapping mode&#8221; AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.
PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.
The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.

A method for investigating the structure of a protein photoreceptor using atomic force microscopy (AFM) is described in this paper. PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) reveals intact protein dimers on a mica surface.

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Biochemistry

Functionalization of Single-walled Carbon Nanotubes with Thermo-reversible Block Copolymers and Characterization by Small-angle Neutron Scattering

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous solution. In a carbon nanotube/PEO105-PPO70-PEO105 (poloxamer 407) aqueous solution, the amphiphilic poloxamer 407 adsorbs onto the carbon nanotube surfaces and self-assembles into continuous layers, driven by intermolecular interactions between constituent molecules. The addition of 5-methylsalicylic acid changes the self-assembled structure from spherical-micellar to a cylindrical morphology. The fabricated poloxamer 407/carbon nanotube hybrid particles exhibit thermo-responsive structural features so that the density and thickness of poloxamer 407 layers are also reversibly controllable by varying temperature. The detailed structural properties of the poloxamer 407/carbon nanotube particles in suspension can be characterized by small-angle neutron scattering experiments and model fit analyses. The distinct curve shapes of the scattering intensities depending on temperature control or addition of aromatic additives are well described by a modified core-shell cylinder model consisting of a carbon nanotube core cylinder, a hydrophobic shell, and a hydrated polymer layer. This method can provide a simple but efficient way for the fabrication and in-situ characterization of carbon nanotube-based nano particles with a structure-tunable encapsulation.

A method for the functionalization of carbon nanotubes with structure-tunable polymeric encapsulation layers and structural characterization using small-angle neutron scattering is presented.

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous ...

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer&#8217;s and Parkinson&#8217;s diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

Co-localizing Kelvin Probe Force Microscopy with Other Microscopies and Spectroscopies: Selected Applications in Corrosion Characterization of Alloys

Kelvin probe force microscopy (KPFM), sometimes referred to as surface potential microscopy, is the nanoscale version of the venerable scanning Kelvin probe, both of which measure the Volta potential difference (VPD) between an oscillating probe tip and a sample surface by applying a nulling voltage equal in magnitude but opposite in sign to the tip-sample potential difference. By scanning a conductive KPFM probe over a sample surface, nanoscale variations in surface topography and potential can be mapped, identifying likely anodic and cathodic regions, as well as quantifying the inherent material driving force for galvanic corrosion.
Subsequent co-localization of KPFM Volta potential maps with advanced scanning electron microscopy (SEM) techniques, including back scattered electron (BSE) images, energy dispersive spectroscopy (EDS) elemental composition maps, and electron backscattered diffraction (EBSD) inverse pole figures can provide further insight into structure-property-performance relationships. Here, the results of several studies co-localizing KPFM with SEM on a wide variety of alloys of technological interest are presented, demonstrating the utility of combining these techniques at the nanoscale to elucidate corrosion initiation and propagation.
Important points to consider and potential pitfalls to avoid in such investigations are also highlighted: in particular, probe calibration and the potential confounding effects on the measured VPDs of the testing environment and sample surface, including ambient humidity (i.e., adsorbed water), surface reactions/oxidation, and polishing debris or other contaminants. Additionally, an example is provided of co-localizing a third technique, scanning confocal Raman microscopy, to demonstrate the general applicability and utility of the co-localization method to provide further structural insight beyond that afforded by electron microscopy-based techniques.

Kelvin probe force microscopy (KPFM) measures surface topography and differences in surface potential, while scanning electron microscopy (SEM) and associated spectroscopies can elucidate surface morphology, composition, crystallinity, and crystallographic orientation. Accordingly, the co-localization of SEM with KPFM can provide insight into the effects of nanoscale composition and surface structure on corrosion.

Kelvin probe force microscopy (KPFM), sometimes referred to as surface potential microscopy, is the nanoscale version of the venerable scanning Kelvin ...

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

Exploring Multiphase Polymeric Systems by Nanoscale Infrared Spectroscopy

Advances in Nanoscale Infrared Spectroscopy to Explore Multiphase Polymeric Systems

Multiphase polymeric systems encompass local domains with dimensions that can vary from a few tens of nanometers to several micrometers. Their composition is commonly assessed using infrared spectroscopy, which provides an average fingerprint of the various materials contained in the volume probed. However, this approach does not offer any details on the arrangement of the phases in the material. Interfacial regions between two polymeric phases, often in the nanoscale range, are also challenging to access. Photothermal nanoscale infrared spectroscopy monitors the local response of materials excited by infrared light with the sensitive probe of an atomic force microscope (AFM). While the technique is suitable for interrogating small features, such as individual proteins on pristine gold surfaces, the characterization of three-dimensional multicomponent materials is more elusive. This is due to a relatively large volume of material undergoing photothermal expansion, defined by the laser focalization onto the sample and by the thermal properties of the polymeric constituents, compared to the nanoscale region probed by the AFM tip. Using a polystyrene (PS) bead and a polyvinyl alcohol (PVA) film, we evaluate the spatial footprint of photothermal nanoscale infrared spectroscopy for surface analysis as a function of the position of PS in the PVA film. The effect of the feature position on the nanoscale infrared images is investigated, and spectra are acquired. Some perspectives on the future advances in the field of photothermal nanoscale infrared spectroscopy are provided, considering the characterization of complex systems with embedded polymeric structures.

Author Spotlight: Advances in Nanoscale Infrared Spectroscopy to Explore Multiphase Polymeric Systems  

This protocol describes the application of atomic force microscopy and nanoscale infrared spectroscopy to evaluate the performance of photothermal nanoscale infrared spectroscopy in the characterization of three-dimensional multi-polymeric samples.

Multiphase polymeric systems encompass local domains with dimensions that can vary from a few tens of nanometers to several micrometers. Their composition ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays for High-Throughput Large-Scale Sample Inspection

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