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 ...

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11.7K 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

Concurrent Quantitative Conductivity and Mechanical Properties Measurements of Organic Photovoltaic Materials using AFM

Organic photovoltaic (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale inhomogeneity of OPV materials affects performance of photovoltaic devices. Thus, understanding of spatial variations in composition as well as electrical properties of OPV materials is of paramount importance for moving PV technology forward.1,2 In this paper, we describe a protocol for quantitative measurements of electrical and mechanical properties of OPV materials with sub-100 nm resolution. Currently, materials properties measurements performed using commercially available AFM-based techniques (PeakForce, conductive AFM) generally provide only qualitative information. The values for resistance as well as Young's modulus measured using our method on the prototypical ITO/PEDOT:PSS/P3HT:PC61BM system correspond well with literature data. The P3HT:PC61BM blend separates onto PC61BM-rich and P3HT-rich domains. Mechanical properties of PC61BM-rich and P3HT-rich domains are different, which allows for domain attribution on the surface of the film. Importantly, combining mechanical and electrical data allows for correlation of the domain structure on the surface of the film with electrical properties variation measured through the thickness of the film.

Organic photovoltaic (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale inhomogeneity of OPV materials affects performance of photovoltaic devices. In this paper, we describe a protocol for quantitative measurements of electrical and mechanical properties of OPV materials with sub-100 nm resolution.

Organic photovoltaic  (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale  inhomogeneity of OPV materials affects performance ...

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 ...

Dynamic Pore-scale Reservoir-condition Imaging of Reaction in Carbonates Using Synchrotron Fast Tomography

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve geologic seals and allow CO2 to escape. However, the dissolution processes at reservoir conditions are poorly understood. Thus, time-resolved experiments are needed to observe and predict the nature and rate of dissolution at the pore scale. Synchrotron fast tomography is a method of taking high-resolution time-resolved images of complex pore structures much more quickly than traditional &#181;-CT. The Diamond Lightsource Pink Beam was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 hours. The images were segmented and the porosity and permeability were measured using image analysis and network extraction. Porosity increased uniformly along the length of the sample; however, the rate of increase of both porosity and permeability slowed at later times.

Synchrotron fast tomography was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 h.

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve ...

Evanescent Field Based Photoacoustics: Optical Property Evaluation at Surfaces

Here, we present a protocol to estimate material and surface optical properties using the photoacoustic effect combined with total internal reflection. Optical property evaluation of thin films and the surfaces of bulk materials is an important step in understanding new optical material systems and their applications. The method presented can estimate thickness, refractive index, and use absorptive properties of materials for detection. This metrology system uses evanescent field-based photoacoustics (EFPA), a field of research based upon the interaction of an evanescent field with the photoacoustic effect. This interaction and its resulting family of techniques allow the technique to probe optical properties within a few hundred nanometers of the sample surface. This optical near field allows for the highly accurate estimation of material properties on the same scale as the field itself such as refractive index and film thickness. With the use of EFPA and its sub techniques such as total internal reflection photoacoustic spectroscopy (TIRPAS) and optical tunneling photoacoustic spectroscopy (OTPAS), it is possible to evaluate a material at the nanoscale in a consolidated instrument without the need for many instruments and experiments that may be cost prohibitive.

Here we present a protocol to estimate material and surface optical properties using the photoacoustic effect combined with total internal reflection. This technique evanescent field-based photoacoustics can be used to create a photoacoustic metrology system to estimate materials&#39; thicknesses, bulk and thin film refractive indices, and explore their optical properties.

Here, we present a protocol to estimate material and surface optical properties using the photoacoustic effect combined with total internal reflection. ...

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 ...

Measurement of Quantum Interference in a Silicon Ring Resonator Photon Source

Silicon photonic chips have the potential to realize complex integrated quantum information processing circuits, including photon sources, qubit manipulation, and integrated single-photon detectors. Here, we present the key aspects of preparing and testing a silicon photonic quantum chip with an integrated photon source and two-photon interferometer. The most important aspect of an integrated quantum circuit is minimizing loss so that all of the generated photons are detected with the highest possible fidelity. Here, we describe how to perform low-loss edge coupling by using an ultra-high numerical aperture fiber to closely match the mode of the silicon waveguides. By using an optimized fusion splicing recipe, the UHNA fiber is seamlessly interfaced with a standard single-mode fiber. This low-loss coupling allows the measurement of high-fidelity photon production in an integrated silicon ring resonator and the subsequent two-photon interference of the produced photons in a closely integrated Mach-Zehnder interferometer. This paper describes the essential procedures for the preparation and characterization of high-performance and scalable silicon quantum photonic circuits.

Silicon photonic chips have the potential to realize complex integrated quantum systems. Presented here is a method for preparing and testing a silicon photonic chip for quantum measurements.

Silicon photonic chips have the potential to realize complex integrated quantum information processing circuits, including photon sources, qubit ...

Characterization of Ultra-fine Grained and Nanocrystalline Materials Using Transmission Kikuchi Diffraction

One of the challenges in microstructure analysis nowadays resides in the reliable and accurate characterization of ultra-fine grained (UFG) and nanocrystalline materials. The traditional techniques associated with scanning electron microscopy (SEM), such as electron backscatter diffraction (EBSD), do not possess the required spatial resolution due to the large interaction volume between the electrons from the beam and the atoms of the material. Transmission electron microscopy (TEM) has the required spatial resolution. However, due to a lack of automation in the analysis system, the rate of data acquisition is slow which limits the area of the specimen that can be characterized. This paper presents a new characterization technique, Transmission Kikuchi Diffraction (TKD), which enables the analysis of the microstructure of UFG and nanocrystalline materials using an SEM equipped with a standard EBSD system. The spatial resolution of this technique can reach 2 nm. This technique can be applied to a large range of materials that would be difficult to analyze using traditional EBSD. After presenting the experimental set up and describing the different steps necessary to realize a TKD analysis, examples of its use on metal alloys and minerals are shown to illustrate the resolution of the technique and its flexibility in term of material to be characterized.

This paper provides a detailed method to characterize the microstructure of ultra-fine grained and nanocrystalline materials using a scanning electron microscope equipped with a standard electron backscatter diffraction system. Metal alloys and minerals presenting refined microstructures are analyzed using this technique, showing the diversity of its possible applications.

One of the challenges in microstructure analysis nowadays resides in the reliable and accurate characterization of ultra-fine grained (UFG) and ...

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 ...