The NSF-MRI program (CHE: 1229103) is acknowledged for funding the purchase of new control electronics, software, liquid cells, and other equipment needed to assemble a dual AFM/STM. We acknowledge the shared facilities at The University of Chicago NSF-MRSEC program (DMR-0820054) for assistance with AFM instrumentation, training, and imaging, and&#160;for instrument time made available by the Materials Research Facilities Network (DMR-0820054). We particularly thank Dr. Qiti Guo, Dr. Justin Jureller, and Prof. Ka Yee Lee for welcoming our students before the funding of the NSF-MRI proposal that brought the necessary instrumentation to our campus. We acknowledge funding from a Title III STEM grant (ID: P031C110157) awarded to Northeastern Illinois University that provided summer research stipends for students and faculty as well as support for consumable supplies. Finally, we acknowledge Bruker-Nano, Inc. for continued instrumental support and for permission to reproduce a plot showing the mechanism of PeakForce QNM and to use the word ScanAsyst to describe the automated software.

1. Computer and Microscope Set Up
<ol>
	<li>Open the valve of the N2 cylinder and adjust the knobs to ensure the air table is floating and level.</li>
	<li>Turn on the computer, controller, and fiber optic light in this order.</li>
	<li>Set the scanner to AFM/LFM mode and center the camera over the AFM head.</li>
	<li>Open software. Select experiment category &#8220;Nanomechanical Properties&#8221;, &#8220;Quantitative Nanomechanical Mapping&#8221; under experiment group, and &#8220;PeakForce QNM in Fluid&#8221; under experiment. Click Load Experiment and then Setup.</li>
	<li>Focus the camera using the Z objective to see the surface of the stage. Using the coarse up/down piezos move the stage approximately 1 mm below the surface of the AFM head aperture.</li>
</ol>
2. Preparation of Mica Surface
<ol>
	<li>Press an adhesive sticker onto the face of a clean sample support disk (15 mm diameter, 0.8 mm thick). Push a mica V-4 grade disk (25 x 25 x 26 mm) onto the adhesive until firmly attached.</li>
	<li>Rub a piece of clear tape onto the mica ensuring it is fully adhered with no air bubbles. Pull the tape off the mica from one side to evenly cleave it. Repeat if necessary to insure the mica is flat.</li>
	<li>Grip the support disk, clean mica side up, with a pair of circular tweezers (disk grippers), and place onto the scanner. The mica should be below the level of the AFM head aperture.</li>
</ol>
3. Fluid Cell Assembly and Imaging of Mica Surface
<ol>
	<li>Rinse the fluid cell, O-ring, syringe adapter, hose adapter, and the ejection hose in 100% ethanol. Rinse the fluid cell in DI water. Let all parts air dry completely.</li>
	<li>Push the hose adapter into the port on the clean fluid cell. Gently press the O-ring into the center indentation of the fluid cell using flat headed tweezers.</li>
	<li>Press the spring lightly on the probe retainer clip and turn it away from the groove for the tip.</li>
	<li>Grasp the probe on the long end away from the tip with flat headed tweezers. Place the probe into the groove with the tip facing the center of the cell. Push the spring in and adjust the retainer clip over the probe to secure it (Figure 1B).</li>
	<li>Check the clamp on the AFM to ensure it is fully retracted (Figure 1C).</li>
	<li>Place the fluid cell, O-ring and probe facing downwards, on top of the mica sample. Allow the fluid cell to rest onto the studs of the AFM. Lower the clamp to stabilize the fluid cell on the stage. Press the down switch on the microscope until a visual inspection shows the O-ring firmly compressed between the support disk and the fluid cell.</li>
	<li>While watching the monitor, focus the coarse adjustment knob to visualize the top of the fluid cell on the screen.</li>
	<li>Move the microscope until there is a clear image of the tip using the X/Y adjustment knobs and the Z objective. The tip will appear like a metallic, golden triangle. Move the tip closer to the sample surface using the down piezo lever on the microscope. Focus on the reflection of the tip. Keep track of the actual cantilever in relationship to its reflection. At this stage, they should be closely aligned, but not completely overlapping.</li>
	<li>Attach the syringe adapter to a sterile 1 ml syringe. Attach one end of the ejection hose to the syringe adapter and place the other end of the hose in the container of buffer solution. Fully extend the plunger of the syringe.</li>
	<li>Attach the hose to the hose adapter on the fluid cell. Gently press the plunger until fluid fully fills the center chamber. Check the cell to insure there are no air bubbles. Check to see there is no fluid spilling out from the fluid cell onto the microscope. Place the syringe on a raised solid support.</li>
	<li>Use the camera feed and the X/Y translation knobs to locate the laser on the screen (a diffuse red dot). Using the laser movement control knobs, move the laser onto the tip.</li>
	<li>Finely adjust the laser and mirror controls until the sum signal displayed on the microscope is maximized (value = 4 - 6).</li>
	<li>Adjust the angle of the quadrant diode using the knobs on the left rear side and the top left side of the scan head to lower the vertical and horizontal deflections displayed on the microscope as close to zero as possible (&#177; 0.1).</li>
	<li>In the software, select acquisition options. At a minimum, select height (trace and retrace), peak force error, and deformation. The other options are user dependent and can be tailored based on the specific information desired by the experimentalist.</li>
	<li>Select &#8220;line&#8221; for RT Plane Fit in each acquisition window. Select &#8220;none&#8221; for the OT Plane Fit in the height windows. Set the X and Y offset and scan angle to zero, if not already done.</li>
	<li>Set the scan rate to 1 Hz, the sample per line to 256, the ScanAsyst auto control to individual, and the ScanAsyst auto Z limit to off. Change the Z limit to 500 nm. Set scan size to 1 &#181;m.</li>
	<li>Click engage. At this point, the tip approaches the surface. While the tip is approaching, observe Z piezo voltage change in the lower left hand corner of the screen. If the tip goes outside of range, withdraw the tip and re-approach. If this is unsuccessful, the tip may need to be changed in order to have a successful approach. Once the tip is engaged, the lines will start to appear on each of the acquisition windows that will compose an image.</li>
	<li>Take images of mica at 1 x 1 &#181;m and zoom in slowly to ensure that the mica surface is clean and flat. Click continuous capture to save images as they appear. Zoom in and move around the surface as desired.</li>
</ol>
4. Protein Deposition and Preparation of Sample for Imaging
<ol>
	<li>Obtain purified protein sample38 and keep on ice. If stored in a freezer, thaw sample on ice for 5 min.</li>
	<li>Dilute the protein to 0.0010 mg/ml using refrigerated imaging buffer solution. The imaging buffer used here is 15 mM Tris-HCl (pH = 8.0), 150 mM NaCl, and 15 mM MgCl2.</li>
	<li>Fill a 4 chamber (100 x 15 mm) Petri dish with refrigerated imaging buffer solution. Fill each chamber with at least 5 ml of solution. Gently mix the diluted protein solution with the tip attached to micropipettor.</li>
	<li>Apply 200 &#181;l of the diluted protein solution to the surface of mica already prepared. After 30 sec, pick up the protein/mica surface with the circular tweezers (disk grippers). Hold the sample parallel to the lab bench.</li>
	<li>While holding the sample, immediately immerse the sample in the buffer solution contained in the first quadrant of the Petri dish (step 4.2) while ensuring that the sample is parallel to the basin of the Petri dish. After 1 sec, remove the sample.</li>
	<li>Continuing to hold the sample parallel to the basin of the Petri dish, repeat step 4.5 for the remaining three quadrants.</li>
	<li>Place the disk on a dry dust free cloth to soak up excess moisture and transfer the disk onto the stage of the microscope. Do not touch the actual surface with the cloth. Do not allow the sample to dry.</li>
	<li>Repeat steps 3.6 - 3.16.</li>
</ol>
5. Imaging Proteins on Mica
<ol>
	<li>Click on check parameters box on the left hand side of the screen. Spring size and tip radius should be set based on the tip being used.</li>
	<li>Click engage. Similarly to the mica imaging process (step 3.17), observe the z-piezo window as the tip approaches.</li>
	<li>Allow the scanner to image the same area for at least two consecutive passes. Click continuous capture to save each image collected.</li>
	<li>Move the scanner to a new region and/or change the image size as desired.</li>
	<li>After the automated software has finished adjusting the scanning parameters for a particular image size, turn the software off.</li>
	<li>Manually adjust scan rate, z limit, the number of lines, and voltage in order to fine tune the focus of the image. If the image becomes too far out of focus, turn software back on to return the original starting position. To increase the resolution of an image while zooming in on a protein, decrease the scan rate and increase the number of lines proportionally.</li>
	<li>After the experiment, clean the liquid-cell with 100% ethanol and rinse with DI H2O. Allow the liquid cell to fully dry.</li>
	<li>Open the data software to process raw data images. First, flatten the image by clicking on the flatten icon. Select Zeroth order and click execute. Depending on the image, a plane fit may be necessary to obtain an appropriately flat image. In this case, select the plane fit icon and draw a plane of a flat area of the image using the cursor. Click execute. Repeat as necessary</li>
	<li>Select the cross-section tab to measure the dimensions of the protein. Draw a line with the cursor move it over the molecule or area to be analyzed. Repeat for multiple areas and the software will generate an overlay. Export the picture or the data used to generate picture in a format compatible to other software and drawing programs.</li>
</ol>

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R., Zhang, J., Brunzelle, J. S., Vierstra, R. D., Forest, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+structure+of+Deinococcus+bacteriophytochrome+yields+new+insights+into+phytochrome+architecture+and+evolution.">High resolution structure of Deinococcus bacteriophytochrome yields new insights into phytochrome architecture and evolution.</a> J. Biol. Chem. 282, 12298-12309 (2007).</li><li>Wagner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+analysis+of+Deinococcus+radiodurans+bacteriophytochrome+reveals+key+amino+acids+necessary+for+the+photochromicity+and+proton+exchange+cycle+of+phytochromes.">Mutational analysis of Deinococcus radiodurans bacteriophytochrome reveals key amino acids necessary for the photochromicity and proton exchange cycle of phytochromes.</a> J. Biol. Chem. 283, 12212-12226 (2008).</li></ol>

There are no competing financial interests or conflicts of interest.

AFM is a scanning probe microscopy method fully capable of imaging proteins and other biological macromolecules in physiologically relevant conditions. In comparison to X-ray crystallography and NMR, one limitation of AFM is its inability to achieve the same resolution, particularly lateral resolution. When using AFM to analyze a molecule on any surface, the impact of the surface and the probe on the image of the molecule must also be considered when data are analyzed. Deconvoluting of the probe&#8217;s impact on the acq...

Atomic force microscopy (AFM) has become a very important tool for investigating the structural and mechanical properties of surfaces, thin films, and single molecules since its invention in 1986 (Figure 1).1-3 Using a liquid-cell, the method has become particularly useful in studies of biological macromolecules and even living cells in a physiologically relevant environment.4-10 Tapping-mode AFM has traditionally been used for imaging soft materials or loosely bound molecules to the surface, since contact-mode AFM is typically unsuitable due to the damage caused by the lateral forces exerted on the sample by the cantilever.11 Tapping-mode AFM substantially reduces these forces by having the tip intermittently touch the surface rather than being in constant contact. In this mode, the cantilever is oscillated at or near its resonant frequency normal to the surface. Similarly to contact-mode AFM, topography is analyzed by plotting the movement of the z-piezo as a function of xy (distance).
The cantilever dynamics can be quite unstable at or near resonance; therefore, they are very challenging to automate outside of a &#8220;steady-state&#8221; situation. Specifically, these dynamics depend on both the sample properties and scanning environment. For a soft molecule adsorbed to a hard(er) surface, a well-tuned feedback loop for the molecule may lead to feedback oscillation for the surface. Operation in fluid further complicates the tuning of the cantilever. Changes in temperature or fluid levels require constant readjustment of set point, gains, and other imaging parameters. These adjustments tend to be very time-consuming and challenging for users.
Peak Force Quantitative Nanomechanical Property Mapping (PF-QNM), like tapping mode AFM, avoids lateral interactions by intermittently contacting the sample (Figure 2).12-15 However, PF-QNM operates in non-resonant mode and frequencies much lower than tapping-mode AFM. This eliminates the tuning challenges of tapping-mode AFM, particularly those exacerbated by the presence of fluid. With PF-QNM, images are collected by taking a force response curve at every point of contact. With the addition of ScanAsyst software,15 adjustment of the scanning parameters can be automated and a high-resolution image obtained in a matter of minutes by even inexperienced users. Once the user becomes more familiar with the AFM, any or all of the automated parameters may be disabled at any time which permits the experimentalist to fine tune the image quality manually. Since its inception, PF-QNM has been applied to map bacteriorhodopsin, a membrane protein, and other native proteins at the submolecular level.16-18 For bacteriorhodopsin, there is a direct correlation between protein flexibility and X-ray crystallographic structures.12 PF-QNM has been utilized to investigate living cells with high resolution.19,20 Furthermore, PF-QNM data has elucidated important connections between structure and mechanics within the erythrocyte membrane that are critical for cell integrity and function.21
We have employed scanning probe microscopy (SPM) methods,22 including AFM,23 to study the structure of red-light photoreceptors called bacteriophytochromes (BphPs).24,25 They consist of a light sensing module covalently linked to a signaling-effector module such as histidine kinase (HK).26 The light-sensing module typically contains a bilin chromophore which undergoes structural transformation upon absorption of a photon, with a series of structural changes reaching the signaling-effector module and leading to a global transformation of the entire protein.24,27-29 Based on this transformation, there are two distinct light absorbing states of BphPs, a red and far-red light absorbing state, denoted as Pr and Pfr. Pr is thermally stable, dark-adapted state for most BphPs.28 The molecular basis of Pr/Pfr photoconversion is not entirely understood due to limited structural knowledge of these proteins. With the exception of one structure from D. radiodurans,30 all published X-ray crystallographic structures of these proteins are in the dark-adapted state and lack effector domain. The intact BphPs are too large to be effectively studied by Nuclear Magnetic Resonance (NMR) and are notoriously difficult to crystallize in their intact form (particularly in the light-adapted state) for X-ray crystallography. BphPs have recently been engineered as infrared fluorescent protein markers (IFP&#8217;s).31 Structural characterization of these proteins can further aid in effective IFP design.32-36
The focus of this article is to present a procedure for imaging of BphPs using liquid-cell AFM via PF-QNM. The method is demonstrated by studies of the light-adapted state of the bacteriophytochrome RpBphP3 (P3) from the photosynthetic bacterium R. palustris. The AFM procedure presented here is convenient and straightforward approach for imaging of proteins as well as other biological macromolecules. With this method, structural details of individual molecules can be collected in a short period of time, similar to an upper-level science course laboratory session. Through measuring cross-sections and completing further dimensional analyses, experimental data can be compared to useful computational models.37-42

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog #</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td>O4997-100</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Acros Organics</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Acros Organics</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Multimode 8 AFM</td>
			<td>Bruker-Nano</td>
			<td>492-008-011</td>
			<td>equipped with Nanoscope V controller and J scanner</td>
		</tr>
		<tr>
			<td>Probe</td>
			<td>Bruker-Nano</td>
			<td>SNL-10, ScanAsyst-Fluid+</td>
			<td></td>
		</tr>
		<tr>
			<td>Tapping Mode Fluid Cell</td>
			<td>Bruker-Nano</td>
			<td>MTFML</td>
			<td></td>
		</tr>
		<tr>
			<td>Mica V-4 Grade</td>
			<td>SPI supplies</td>
			<td>1150503</td>
			<td>25 x 25 x .26 mm</td>
		</tr>
		<tr>
			<td>Sample support disk</td>
			<td>nanoSurf</td>
			<td>BT02236</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Plasta-Medic, Inc.</td>
			<td></td>
			<td>100 mm x 15 mm&#160;</td>
		</tr>
		<tr>
			<td>micropipettors</td>
			<td>Denville Scientific</td>
			<td>XL 3000i</td>
			<td></td>
		</tr>
		<tr>
			<td>RpBphP3</td>
			<td></td>
			<td></td>
			<td>Prepared according to cited references</td>
		</tr>
		<tr>
			<td>Nanoscope software</td>
			<td>Bruker-Nano</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber Optic Light</td>
			<td>Digital Instruments Inc.</td>
			<td>F0-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Pelco AFM Disc Gripper</td>
			<td>Ted Pella Inc</td>
			<td>1668</td>
			<td>12 mm</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>McKesson</td>
			<td>102-ST1C</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Denville Scientific</td>
			<td>C2171</td>
			<td></td>
		</tr>
		<tr>
			<td>The Pymol Molecular Graphic System v.1.5.0.1</td>
			<td>Schrodinger, LLC</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Representative AFM images of a photoreceptor protein, P3, in its light-adapted state are presented in Figures 3 and 4. A freshly-cleaved mica substrate (Figure 3A) is a suitable, flat surface for protein adsorption. Collecting an image of clean mica as a negative control is important for several reasons. First, it insures the liquid cell is clean and no residual materials from previous experiments will contaminate the surface. Second, it tests the quality of the probe. I...

Watch this Scientific Journal Video about Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping at JoVE.com

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

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 ...

atomic-force-microscopy-red-light-photoreceptors-using-peakforce

Research

JoVE Journal

Engineering

11.6K Views.  Northeastern Illinois University. 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. 

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

Preparation and Friction Force Microscopy Measurements of Immiscible, Opposing Polymer Brushes

Solvated polymer brushes are well known to lubricate high-pressure contacts, because they can sustain a positive normal load while maintaining low friction at the interface. Nevertheless, these systems can be sensitive to wear due to interdigitation of the opposing brushes. In a recent publication, we have shown via molecular dynamics simulations and atomic force microscopy experiments, that using an immiscible polymer brush system terminating the substrate and the slider surfaces, respectively, can eliminate such interdigitation. As a consequence, wear in the contacts is reduced. Moreover, the friction force is two orders of magnitude lower compared to traditional miscible polymer brush systems. This newly proposed system therefore holds great potential for application in industry. Here, the methodology to construct an immiscible polymer brush system of two different brushes each solvated by their own preferred solvent is presented. The procedure how to graft poly(N-isopropylacrylamide) (PNIPAM) from a flat surface and poly(methyl methacrylate) (PMMA) from an atomic force microscopy (AFM) colloidal probe is described. PNIPAM is solvated in water and PMMA in acetophenone. Via friction force AFM measurements, it is shown that the friction for this system is indeed reduced by two orders of magnitude compared to the miscible system of PMMA on PMMA solvated in acetophenone.

The methodology to perform friction force microscopy experiments for contacting brushes is presented: Two polymer brushes that are grafted from (a) substrates and (b) colloidal probes are slid to show that, by using two contacting immiscible brush systems, friction in sliding contacts is reduced compared to miscible brush systems.

Solvated polymer brushes are well known to lubricate high-pressure contacts, because they can sustain a positive normal load while maintaining low ...

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

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.

We present a method for achieving sub-nanometer resolution images with amplitude-modulation (tapping mode) atomic force microscopy in liquid. The method is demonstrated on commercial atomic force microscopes. We explain the rationale behind our choices of parameters and suggest strategies for resolution optimization.

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown ...

An Efficient Method for Selective Desalination of Radioactive Iodine Anions by Using Gold Nanoparticles-Embedded Membrane Filter

Here, we demonstrate a detail protocol for the preparation of nanomaterials-embedded composite membranes and its application to the efficient and ion-selective removal of radioactive iodines. By using citrate-stabilized gold nanoparticles (mean diameter: 13 nm) and cellulose acetate membranes, gold nanoparticle-embedded cellulose acetate membranes (Au-CAM) have easily been fabricated. The nano-adsorbents on Au-CAM were highly stable in the presence of high concentration of inorganic salts and organic molecules. The iodide ions in aqueous solutions could rapidly be captured by this engineered membrane. Through a filtration process using an Au-CAM containing filter unit, excellent removal efficiency (&gt;99%) as well as ion-selective desalination result was achieved in a short time. Moreover, Au-CAM provided good reusability without significant decrease of its performances. These results suggested that the present technology using the engineered hybrid membrane will be a promising process for the large-scale decontamination of radioactive iodine from liquid wastes.

An efficient method for the rapid and ion-selective desalination of radioactive iodine in several aqueous solutions is described by using gold nanoparticles-immobilized cellulose acetate membrane filters.

Here, we demonstrate a detail protocol for the preparation of nanomaterials-embedded composite membranes and its application to the efficient and ...

Surface Mapping of Earth-like Exoplanets using Single Point Light Curves

Spatially resolving exoplanet features from single-point observations is essential for evaluating the potential habitability of exoplanets. The ultimate goal of this protocol is to determine whether these planetary worlds harbor geological features and/or climate systems. We present a method of extracting information from multi-wavelength single-point light curves and retrieving surface maps. It uses singular value decomposition (SVD) to separate sources that contribute to light curve variations and infer the existence of partially cloudy climate systems. Through analysis of the time series obtained from SVD, physical attributions of principal components (PCs) could be inferred without assumptions of any spectral properties. Combining with viewing geometry, it is feasible to reconstruct surface maps if one of the PCs are found to contain surface information. Degeneracy originated from convolution of the pixel geometry and spectrum information determines the quality of reconstructed surface maps, which requires the introduction of regularization. For the purpose of demonstrating the protocol, multi-wavelength light curves of Earth, which serves as a proxy exoplanet, are analyzed. Comparison between the results and the ground truth is presented to show the performance and limitation of the protocol. This work provides a benchmark for future generalization of exoplanet applications.

The protocol extracts information from light curves of exoplanets and constructs their surface maps. It uses light curves of Earth, which serves as a proxy exoplanet, to demonstrate the approach.

Spatially resolving exoplanet features from single-point observations is essential for evaluating the potential habitability of exoplanets. The ultimate ...

Optimizing Magnetic Force Microscopy Resolution and Sensitivity to Visualize Nanoscale Magnetic Domains

Magnetic force microscopy (MFM) enables mapping local magnetic fields across a sample surface with nanoscale resolution. To perform MFM, an atomic force microscopy (AFM) probe whose tip has been magnetized vertically (i.e., perpendicular to the probe cantilever) is oscillated at a fixed height above the sample surface. The resultant shifts in the oscillation phase or frequency, which are proportional to the magnitude and sign of the vertical magnetic force gradient at each pixel location, are then tracked and mapped. Although the spatial resolution and sensitivity of the technique increases with decreasing lift height above the surface, this seemingly straightforward path to improved MFM images is complicated by considerations such as minimizing topographical artifacts due to shorter range van der Waals forces, increasing the oscillation amplitude to further improve sensitivity, and the presence of surface contaminants (in particular water due to humidity under ambient conditions). In addition, due to the orientation of the probe's magnetic dipole moment, MFM is intrinsically more sensitive to samples with an out-of-plane magnetization vector. Here, high-resolution topographical and magnetic phase images of single and bicomponent nanomagnet artificial spin-ice (ASI) arrays obtained in an inert (argon) atmosphere glovebox with &lt;0.1 ppm O2 and H2O are reported. Optimization of lift height and drive amplitude for high resolution and sensitivity while simultaneously avoiding the introduction of topographical artifacts is discussed, and detection of the stray magnetic fields emanating from either end of the nanoscale bar magnets (~250 nm long and &lt;100 nm wide) aligned in the plane of the ASI sample surface is shown. Likewise, using the example of a Ni-Mn-Ga magnetic shape memory alloy (MSMA), MFM is demonstrated in an inert atmosphere with magnetic phase sensitivity capable of resolving a series of adjacent magnetic domains each ~200 nm wide.

Magnetic force microscopy (MFM) employs a vertically magnetized atomic force microscopy probe to measure sample topography and local magnetic field strength with nanoscale resolution. Optimizing MFM spatial resolution and sensitivity requires balancing decreasing lift height against increasing drive (oscillation) amplitude, and benefits from operating in an inert atmosphere glovebox.

Magnetic force microscopy (MFM) enables mapping local magnetic fields across a sample surface with nanoscale resolution. To perform MFM, an atomic force ...

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 ...

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

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.

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

Bio-Hybrid AFM Probe with Mosquito Labrum for Studying Biting Behaviors

Development of a Bio-Hybrid Mosquito Stinger-Based Atomic Force Microscopy Probe

Mosquitoes, notorious as the deadliest animals to humans due to their capacity to transmit diseases, pose a persistent challenge to public health. The primary prevention strategy currently in use involves chemical repellents, which often prove ineffective as mosquitoes rapidly develop resistance. Consequently, the invention of new preventive methods is crucial. Such development hinges on a thorough understanding of mosquito biting behaviors, necessitating an experimental setup that accurately replicates actual biting scenarios with controllable testing parameters and quantitative measurements. To bridge this gap, a bio-hybrid atomic force microscopy (AFM) probe was engineered, featuring a biological stinger - specifically, a mosquito labrum - as its tip. This bio-hybrid probe, compatible with standard AFM systems, enables a near-authentic simulation of mosquito penetration behaviors. This method marks a step forward in the quantitative study of biting mechanisms, potentially leading to the creation of effective barriers against vector-borne diseases (VBDs) and opening new avenues in the fight against mosquito-transmitted illnesses.

Author Spotlight: Development of Bio-Hybrid AFM Cantilevers for Quantitative Analysis of Mosquito Biting Mechanisms

Quantitative and controlled investigations into insect biting behaviors are crucial for devising effective strategies to combat vector-borne diseases. In this context, a method for fabricating a bio-hybrid atomic force microscopy (AFM) probe is introduced.

Mosquitoes, notorious as the deadliest animals to humans due to their capacity to transmit diseases, pose a persistent challenge to public health. The ...