The authors have no acknowledgements.

<ol>
	<li>Barth, J. V., Costantini, G., Kern, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+atomic+and+molecular+nanostructures+at+surfaces.">Engineering atomic and molecular nanostructures at surfaces.</a> Nature. 437, 671-679 (2005).</li><li>Otero, R., Rosei, F., Besenbacher, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+tunneling+microscopy+manipulation+of+complex+organic+molecules+on+solid+surfaces.">Scanning tunneling microscopy manipulation of complex organic molecules on solid surfaces.</a> Annu. Rev. Phys. Chem. 57, 497-525 (2006).</li><li>Urgel, J. I., Ecija, D., Auw&#228;rter, W., Barth, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Manipulation+of+Gadolinium+Coordinated+Supramolecules+by+Low-Temperature+Scanning+Tunneling+Microscopy.">Controlled Manipulation of Gadolinium Coordinated Supramolecules by Low-Temperature Scanning Tunneling Microscopy.</a> Nano Lett. 14, 1369-1373 (2014).</li><li>Fournier, N., Wagner, C., Weiss, C., Temirov, R., Tautz, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-controlled+lifting+of+molecular+wires.">Force-controlled lifting of molecular wires.</a> Phys. Rev. B. 84, 035435 (2011).</li><li>Wagner, C., Fournier, N., Tautz, F. S., Temirov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+the+Binding+Energies+of+the+Organic-Metal+Perylene-Tetracarboxylic-Dianhydride/Au(111)+Bonds+by+Molecular+Manipulation+Using+an+Atomic+Force+Microscope.">Measurement of the Binding Energies of the Organic-Metal Perylene-Tetracarboxylic-Dianhydride/Au(111) Bonds by Molecular Manipulation Using an Atomic Force Microscope.</a> Phys. Rev. Lett. 109 (7), 076102 (2012).</li><li>Wagner, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-additivity+of+molecule-surface+van+der+Waals+potentials+from+force+measurements.">Non-additivity of molecule-surface van der Waals potentials from force measurements.</a> Nat. Commun. 5, 5568 (2014).</li><li>Green, M. F. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+a+hydrogen-bonded+molecular+monolayer+with+a+hand-controlled+scanning+probe+microscope.">Patterning a hydrogen-bonded molecular monolayer with a hand-controlled scanning probe microscope.</a> Beilstein Journal of Nanotechnology. 5, 1926-1932 (2014).</li><li>Leinen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virtual+reality+visual+feedback+for+hand-controlled+scanning+probe+microscopy+manipulation+of+single+molecules.">Virtual reality visual feedback for hand-controlled scanning probe microscopy manipulation of single molecules.</a> Beilstein J. Nanotechnol. 6, 2148-2153 (2015).</li><li>Wagner, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+Quantum+Dot+Microscopy.">Scanning Quantum Dot Microscopy.</a> Phys. Rev. Lett. 115 (2), 026101 (2015).</li><li>Mura, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+theoretical+analysis+of+H-bonded+supramolecular+assemblies+of+PTCDA+molecules.">Experimental and theoretical analysis of H-bonded supramolecular assemblies of PTCDA molecules.</a> Phys. Rev. B. 81 (19), 195412 (2010).</li><li>Besocke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+easily+operable+scanning+tunneling+microscope.">An easily operable scanning tunneling microscope.</a> Surf. Sci. Lett. (1-2), 145-153 (1987).</li><li>Giessibl, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+atomic+force+microscopy.">Advances in atomic force microscopy.</a> Rev. Mod. Phys. 75 (3), 949-983 (2003).</li><li>Albrecht, T. R., Gru&#776;tter, P., Horne, D., Rugar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+modulation+detection+using+high-Q+cantilevers+for+enhanced+force+microscope+sensitivity.">Frequency modulation detection using high-Q cantilevers for enhanced force microscope sensitivity.</a> J. Appl. Phys. 69 (2), 668-673 (1991).</li><li>Temirov, R., Lassise, A., Anders, F. B., Tautz, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kondo+effect+by+controlled+cleavage+of+a+single-molecule+contact.">Kondo effect by controlled cleavage of a single-molecule contact.</a> Nanotechnology. 19 (6), 065401 (2008).</li><li>Gl&#246;ckler, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+ordered+structures+and+submolecular+scanning+tunnelling+microscopy+contrast+of+PTCDA+and+DM-PBDCI+monolayers+on+Ag(111)+and+Ag(110).">Highly ordered structures and submolecular scanning tunnelling microscopy contrast of PTCDA and DM-PBDCI monolayers on Ag(111) and Ag(110).</a> Surf. Sci. 405 (1), 1-20 (1998).</li><li>Simon, G. H., Heyde, M., Rust, H. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recipes+for+cantilever+parameter+determination+in+dynamic+force+spectroscopy:+spring+constant+and+amplitude.">Recipes for cantilever parameter determination in dynamic force spectroscopy: spring constant and amplitude.</a> Nanotechnology. 18 (25), 255503 (2007).</li><li>Rohlfing, M., Temirov, R., Tautz, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+structure+and+scanning+tunneling+data+of+a+prototype+organic-inorganic+interface+PTCDA+on+Ag+(111).">Adsorption structure and scanning tunneling data of a prototype organic-inorganic interface PTCDA on Ag (111).</a> Phys. Rev. B. 76 (11), 115421 (2007).</li><li>Guthold, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Manipulation+of+Molecular+Samples+with+the+nanoManipulator.">Controlled Manipulation of Molecular Samples with the nanoManipulator.</a> IEEE/ASME Trans. Mechatronics. 5 (2), 189-198 (2000).</li></ol>

The authors have nothing to disclose and no competing financial interests.

Like other SPM-based methods, the molecular manipulation experiments described in this paper also depend to some extent on the properties of the SPM tip. The tip apex structure (which cannot be fully controlled) determines the strength of the tip-molecule bond. Hence the strength of the tip-molecule contact may vary considerably and thus sometimes may be too low. Hence within the protocol we refer to some basic tests of tip quality and tip treatment procedures. However, a more severe tip treatment might be required in some cases to achieve satisfactory manipulation results.

Another critical aspect of contacting manipulation with a NC-AFM/STM is the oscillation of the qPlus sensor during manipulation. With increasing amplitude the controlled manipulation becomes more difficult since the tuning fork moves the tip vertically up and down by a total of 2A0. In the limit of large amplitudes the tip is always retracted from the surface along a vertical trajectory which crucially affects the manipulation. Therefore, if the noise characteristics of the used NC-AFM/STM does not permit working with amplitudes well below 1 &#197; one should consider trying manipulation in STM mode, i.e., without exciting the tuning fork. While no information about the junction stiffness can be obtained in this case, the conductivity alone might be sufficient to monitor manipulation.

The use of HMD for the visualization of tip trajectory data has advantages but also limitations. A clear advantage is the gain in precision and intuition (in real time!) if one works in a true 3D virtual scene environment. In that respect we find the virtual reality approach to be much more effective than &#34;offline&#34; inspection of the trajectory data on a standard display. On the other hand wearing HMD complicates the operation with lab-equipment so that the important measurement data has to be projected into the VR (see Figure 6) to be viewed on-line. This limitation may in future be overcome with the augmented virtual reality interfaces in which the 3D virtual reality scene is directly superimposed over the real image of the laboratory environment.

Although there exist other approaches which couple a virtual reality environment and MCS to control an AFM those setups are specified for micro- and sub-micrometer scale manipulation under ambient conditions18. With respect to the manipulation of individual molecules with atomic precision our combination of MCS, HMD, and LT-SPM is unique. Hand-controlled manipulation gives a unique intuitive access to the problem of molecular manipulation. With the level of control offered, one can perform spectroscopic studies of single molecules in different configurations, understanding more about the fundamental physics in such metal-molecule-metal junctions. Using the method described in this paper will allow one to &#34;learn&#34; the complex potential energy surfaces which determine the probability for success of a specific manipulation attempt. Having acquired sufficient intuition one could delegate that learning to a computer, finally automating the manipulation process. Another consequential future development of the method is its combination with an atomistic real-time simulation of the manipulation process which would allow an even more direct feedback for the experimenter.

The low-temperature non-contact atomic force/scanning tunneling microscope (LT NC-AFM/STM, in the following simply termed SPM) is the tool of choice for atomically precise manipulation of individual atoms or molecules1-3. SPM-based manipulation is typically limited to two dimensions and consists of a series of abrupt and often stochastic manipulation events (jumps). This essentially limits the control over the process. Contacting the molecule in question by a single chemical bond at a well-defined atomic position leads to an approach that can overcome these limitations4-9. Throughout its manipulation the contacted molecule is connected to the SPM tip so that moving the molecule in all three dimensions by appropriate displacements of the tip becomes possible. This creates the possibility for various complex manipulation procedures performed in 3D space. However the contacting manipulation may be hindered by interactions of the manipulated molecule with the surface or/and other molecules in its surroundings, which may create forces that are large enough to rupture the tip-molecule contact. Therefore a particular 3D trajectory of the SPM tip may or may not result in a successful manipulation event. A question thus arises how to define protocols that lead to successful completion of manipulation in the circumstances when the tip-molecule bond has a limited strength, while the interactions of the manipulated molecule with its environment are not a-priori well characterized.
Here this question is approached in the most intuitive manner imaginable. The experimenter is allowed to control the displacements of the SPM tip simply by moving their hand7. This is achieved by coupling the SPM to a commercial motion capture system, some of the specifications of which are provided below. The advantage of &#34;hand controlled manipulation&#34; (HCM) is in the experimenter&#39;s ability to try out different manipulation trajectories quickly and learn from their failure or success.
The HCM setup has been used to conduct a proof-of-principle experiment in which a word (&#34;J&#220;LICH&#34;) was stenciled in a closed layer of perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) molecules on Ag(111), removing 48 molecules, one by one, with HCM7. Lifting a molecule from the surface cleaves its intermolecular hydrogen bonds which bind the molecules in the monolayer10. Typically the total strength of the present intermolecular bonds exceeds the strength of the single chemical bond between the outermost atom of the tip and a carboxylic oxygen atom of PTCDA by which the molecule is contacted (see Figure 1). That may lead to the rupture of the tip-molecule contact and the following failure of the manipulation attempt. The experimenter&#39;s task is thus to determine a tip trajectory that breaks the resisting intermolecular bonds sequentially rather than simultaneously, so that the total force applied to the tip-molecule contact never exceeds its strength.
Although the desired trajectory may in principle be simulated, due to the size and complexity of the system involved the necessary simulations would take a prohibitively large amount of time. In contrast to that, using HCM it was possible to remove the first molecule after 40 minutes. Towards the end of the experiment the extraction took already much less time which confirms the effectiveness of the learning procedure. Additionally, the accuracy and versatility of the HCM method was evidenced in the act of reverse manipulation when a molecule extracted from the neighboring location was used to close the void left after the erroneous removal of another molecule from the monolayer.
Motion capture approach, while being fast and intuitive, is limited to the generation of tip-trajectory data. For further systematic development of new molecular manipulation protocols it is equally important to be able to view the tip trajectory data in real time as well as to analyze previously generated data. Therefore, the functionality of the HCM setup is enhanced substantially by adding virtual reality goggles which allow the experimentalist to see the data plotted in the 3D virtual scene where the tip trajectory is augmented by the current (I) and frequency shift (&#916;f) values measured by the SPM in real-time8 (see below). In addition to that, the virtual reality scene shows a model of the manipulated molecule that serves as a visual scale reference. Thus the HCM setup complimented by the virtual reality interface is suitable for systematic mapping of the manipulation trajectory space and successive refinement of the promising manipulation protocols. Besides that the system also facilitates the knowledge transfer between different experiments. The following paragraphs give a description of the setup and some of its specifications that are relevant for manipulation experiments.
The experiments are performed in ultra-high vacuum (UHV) at a base pressure of 1 x 10-10 mbar with a commercial SPM consisting of a preparation chamber and an analysis chamber. The preparation chamber is equipped with: Ar+ source used for sample sputtering, sample transfer via manipulator (allows heating and cooling of a sample), low-energy electron diffraction (LEED), a customized Knudsen cell (K-cell) containing PTCDA powder purified by sublimation. The analysis chamber is equipped with: LN2 bath cryostat with a volume of 12 L and a holding time of 46 hr, LHe bath cryostat (5 L, 72 hr), Besocke11 beetle-type SPM equipped with a tuning fork sensor12 (TFS) consisting of a quartz tuning fork with an electrically connected PtIr tip (for STM operation), which is cut and sharpened by a focused ion beam (FIB) (Figure 2).
<img alt="Figure 1" src="/files/ftp_upload/54506/54506fig2.jpg" /> 
Figure 2. Tuning fork sensor. (a) Image of a commercial tuning fork sensor with attached PtIr tip. (b) SEM image of the PtIr tip apex cut with FIB. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54506/54506fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The AFM is operated in the frequency-modulated (FM) mode13 where the TFS is excited at the resonance (f0 &#8776; 31,080 Hz) with a dither piezo. The piezoelectric signal of the oscillating tuning fork is amplified and used by a phase locked loop (PLL), which keeps the amplitude of the TFS&#39;s oscillation constant and tracks changes of its resonance frequency, &#916;f = f - f0, that originates from the gradient of the force acting on the tip. As shown in Figure 3 the SPM tip position is controlled by voltages (ux, uy, uz) applied to a set of x-, y-, z-piezos (piezo constants at 5 K: x=15, y=16, z=6 &#197;/V). The ux, uy, uz-voltages (&#177;10 V at 20 bit resolution) are generated at the SPM electronics outputs. They are further amplified by a high voltage (HV) amplifier that has a maximum output voltage of &#177;200 V.
<img alt="Figure 1" src="/files/ftp_upload/54506/54506fig3.jpg" /> 
Figure 3. Schematics of the HCM setup. The position of (tracked object) TO that has multiple (infrared) IR sources installed on its surface is tracked by two infrared cameras of the motion capture system (MCS). TipControl software obtains the TO coordinates (x,y,z) from MCS and passes it to the remote voltage source (RVS) which generates a set of voltages (vx,vy,vz) that are summed with the voltages (ux,uy,uz) produced by the SPM electronics for control of the SPM tip position. The added voltage passes through a high-voltage (HV) amplifier and is further applied to the piezo-positioning system of the SPM tip. The setup allows manual control of the tip positioning when the SPM feedback (FB) loop is open. The (x,y,z) position of the tip as well as I(x,y,z) and &#916;f(x,y,z) are passed to the VRinterface software that plots it in the 3D virtual scene seen by the operator wearing the head-mounted display (HMD). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54506/54506fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The tunneling current that flows between the SPM tip and the surface is measured by a transimpedance amplifier with a variable gain that ranges from 1 x 103 to 1 x 109 V/A (bandwidth at gain 1 x 109 V/A is 1 kHz). The output of the amplifier is fed into the STM feedback (FB) loop to regulate the tip height above the surface in constant current scanning mode. The stability of the junction (with the TFS oscillation turned off) is 1-3 pm. The piezoelectric oscillation signal of the TFS is amplified in two stages: (1) preamplifier fixed to the LN2&#160;shield (gain 1 x 108 V/A, bandwidth 20 kHz), and (2) external voltage amplifier with variable gain from 1 x 101 to 5 x 104 and a bandwidth of 1 MHz.
For HCM experiments, the SPM setup is extended with: motion capture system (MCS), remotely controllable multichannel voltage source (RVS), summing amplifier and virtual reality head mounted display (HMD). All of the listed devices except the summing amplifier were acquired commercially.
MSC is an infrared (IR) marker-tracking system that allows millimeter resolution of spatial displacements at a rate of 100 Hz. The system consists of two IR cameras, a trackable object (TO) and the control software. The MCS software obtains the x-, y-, z-coordinates of the TO in 3D space by analyzing its images obtained by the two cameras. MCS provides a programming library that allows use of the coordinates of TO in a separate software program.
The coordinates of TO (xTO, yTO, zTO) are passed to a custom-developed software program &#34;TipControl&#34;. Figure 4 shows a screenshot of the graphical user interface. The software is activated by the &#34;start&#34; button in the window. After activation (&#964;=0) the software sets all vx-, vy-, vz-voltages on RVS (voltage range &#177;10 V at 16 bit resolution, 50 msec latency per voltage step) according to the following expression <img alt="Equation 1" src="/files/ftp_upload/54506/54506eq1.jpg" />etc., where cx, cy, cz are the factors that convert 5 cm of the displacement of TO into 1 &#197; displacement of the SPM tip. The factors px(t), py(t), pz(t) have values defined by the status of the x-, y-, z-checkboxes in the software window. If the box is checked then the corresponding p(t) is set to 1. All p(t) are set to 0 at the moment when the &#34;pause&#34; button is pressed in the software window. That allows the operator to temporarily &#34;freeze&#34; the position of the tip. Pressing the &#34;reset all&#34; button in the software window sets vx-, vy-, vz-voltages to zero which returns the tip to its initial position defined by the SPM software. The text field &#34;manual command to RVS&#34; in the software window can be used to set any of the vx-, vy-, vz-voltages to any value in the allowed range of &#177;10 V. The vx-, vy-, vz-voltages generated by RVS are added to the ux-, uy-, uz-output voltage signals of SPM electronics via a summing amplifier (gain 1, bandwidth 50 kHz, output range &#177;10 V).
<img alt="Figure 1" src="/files/ftp_upload/54506/54506fig4.jpg" /> 
Figure 4. Screenshot of the interface window. Two indicators exhibit the status of connection with MCS and RVS systems. Checkboxes are used to activate hand-control along selected spatial axes. The button &#8220;Start&#8221; initiates data flow between MCS, TipControl and RVS according to the scheme shown in Figure 3. Button &#8220;Pause&#8221; stops the data flow. Button &#8220;Reset All&#8221; sets all RVS voltages to zero. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54506/54506fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For visualization of the experimental data (tip trajectory, I, &#916;f) a head mounted display (HMD) is used. The HMD provides a stereoscopic view (split HD display &#8212; one half for each eye, 1,920 x 1,080 pixels at 75 Hz). A dedicated IR camera tracks the position and orientation of HMD in 3D space using IR-LEDs fixed on the surface of HMD. The HMD tracking system allows the operator to change the view inside the 3D virtual reality scene by a turn of their head or simply moving their body.
The custom-written software &#34;VRinterface&#34; collects the data both from SPM and MCS, renders it in the 3D scene using OpenGL and displays it in the HMD with the help of the HMDs software development kit (SDK). VRinterface retrieves the actual x-, y-, z-coordinates of the tip directly from the tip software (few millisecond latency) while I and &#916;f signals are read directly from the outputs of the SPM electronics (latency &#8776; 250 msec). Figure 5 shows a screenshot of the 3D virtual scene as seen by the operator wearing HMD during HCM. Inside the 3D virtual scene the tip apex is rendered as a white sphere. The coloring of the recorded tip trajectories reflects values of either log(I(x,y,z)) or &#916;f(x,y,z). Switching between log(I(x,y,z)) or &#916;f(x,y,z) color modes is done by the press of a button. Another button initiates the recording (and displaying) of experimental tip trajectory data. When pressed again the button stops the recording. The virtual scene also shows a static PTCDA molecule which is used as a visual aid during manipulation. The operator aligns its orientation manually to fit the orientation of the real molecule on the surface by using buttons on a keyboard.
Caution: Because the head tracking of HMD relies on IR-LEDs, it may interfere with the MCS since it also uses IR light to track the position of TO. Therefore TO has to have a unique shape recognized by the MCS. This helps MCS to discriminate between the signals that come from TO and those coming from IR-LEDs of HMD.
<img alt="Figure 1" src="/files/ftp_upload/54506/54506fig5.jpg" /> 
Figure 5. Screenshot of the 3D virtual scene displayed to the operator in HMD during HCM. A set of white spheres forms a model Ag(111) surface. Orientation of the model surface may not necessarily coincide with the orientation of the sample. A model of the PTCDA molecule is placed above the model surface. C, O, H atoms of PTCDA are shown in black, red and white respectively. For the purpose of convenience azimuthal orientation of the model molecule can be adjusted to fit the orientation of the real molecule chosen for manipulation. The tip position is marked by a single white sphere representing the outermost tip apex atom. The real-time I(x,y,z) and &#916;f(x,y,z) data are displayed as bar indicators placed next to the tip. Previously recorded as well as the currently executed manipulations are displayed as 3D trajectories whose color represents either log(I(x,y,z)) or &#916;f(x,y,z) values measured at corresponding positions of the trajectory. The figure shows trajectories that are colored with log(I(x,y,z)) signal. The color contrast can be switched between log(I(x,y,z)) and &#916;f(x,y,z) modes by press of a button. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54506/54506fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>LN&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>caution: cryogenic liquid</td></tr><tr><td>LHe</td><td></td><td></td><td>caution: cryogenic liquid</td></tr><tr><td>PTCDA</td><td></td><td></td><td>caution: irritating substance</td></tr><tr><td>Knudsen cell (K-cell)</td><td></td><td></td><td>custom</td></tr><tr><td>ErLEED</td><td>Specs</td><td></td><td>used with power supply ErLEED 1,000 A</td></tr><tr><td>combient LT NC-AFM/STM</td><td>Createc</td><td></td><td></td></tr><tr><td>qPlus sensor</td><td>Createc</td><td></td><td>TFS</td></tr><tr><td>preamplifier</td><td>Createc</td><td></td><td>amplifier for tuning fork signal fixed to LN&lt;sub&gt;2&lt;/sub&gt; shield (stage 1)</td></tr><tr><td>Low-Noise Voltage Preamplifier</td><td>Standford Research System</td><td>SR560</td><td>external amplifier for tuning fork signal (stage 2)</td></tr><tr><td>Variable Gain Low Noise Current Amplifier</td><td>Femto</td><td>DLPCA-200</td><td>amplifier for tunneling current</td></tr><tr><td>Bonita</td><td>Vicon</td><td>B10, SN: MXBN-0B10-3658</td><td>MCS IR camera</td></tr><tr><td>Apex Interaction Device</td><td>Vicon</td><td>SN: AP0062</td><td>MCS trackable object (TO)</td></tr><tr><td>MX Calibration Wand</td><td>Vicon</td><td></td><td>MCS calibration object</td></tr><tr><td>Tracker</td><td>Vicon</td><td></td><td>MCS software</td></tr><tr><td>BS series voltage supply</td><td>stahl-electronics</td><td>BS 1-4</td><td>RVS</td></tr><tr><td>summing amplifier&amp;nbsp;</td><td></td><td></td><td>custom, gain 1, based on operational amplifier TL072</td></tr><tr><td>Oculus Rrift Development Kit 2</td><td>Oculus VR</td><td></td><td>HMD</td></tr><tr><td>TipControl</td><td></td><td></td><td>custom-written software</td></tr><tr><td>VRinterface</td><td></td><td></td><td>custom-written software</td></tr></tbody></table>

hand controlled manipulation of single molecules via a scanning probe microscope with a 3d virtual reality interface

Considering organic molecules as the functional building blocks of future nanoscale technology, the question of how to arrange and assemble such building blocks in a bottom-up approach is still open. The scanning probe microscope (SPM) could be a tool of choice; however, SPM-based manipulation was until recently limited to two dimensions (2D). Binding the SPM tip to a molecule at a well-defined position opens an opportunity of controlled manipulation in 3D space. Unfortunately, 3D manipulation is largely incompatible with the typical 2D-paradigm of viewing and generating SPM data on a computer. For intuitive and efficient manipulation we therefore couple a low-temperature non-contact atomic force/scanning tunneling microscope (LT NC-AFM/STM) to a motion capture system and fully immersive virtual reality goggles. This setup permits &#34;hand controlled manipulation&#34; (HCM), in which the SPM tip is moved according to the motion of the experimenter&#39;s hand, while the tip trajectories as well as the response of the SPM junction are visualized in 3D. HCM paves the way to the development of complex manipulation protocols, potentially leading to a better fundamental understanding of nanoscale interactions acting between molecules on surfaces. Here we describe the setup and the steps needed to achieve successful hand-controlled molecular manipulation within the virtual reality environment.

Note: This part shows work published in7,8.
Applying HCM to the problem of lifting PTCDA/Ag(111) out of a layer, we were able to write a pattern by sequentially removing individual molecules (Figure 9). In total 48 molecules were removed, 40 of which could be redeposited to the clean Ag(111), showing that the molecules stay intact during the manipulation process. This allows using HCM to correct &#34;writing errors&#34; by taking a molecule from a different location and filling an accidentally created vacancy (insets of Figure 9)7.
Successful trajectories that allowed to remove a molecule from the layer are displayed in Figure 10. They bunch in a relative narrow solid angle, the direction of which suggests that the molecule can be removed from within the layer in a &#34;peeling&#34; motion. This peeling facilitates a gradual cleaving of the intermolecular H-bonds and keeps the total forces acting on the tip-molecule bond under a critical threshold7.
The VR feedback allows to execute several reproducible manipulations, each time following the same previously recorded trajectory. Reproducibility is indicated by strong similarities in the I(x,y,z) and &#916;f(x,y,z) data in Figure 11. Because the shape of the trajectory depends also on the exact shape of the tip apex we perform an experiment in which one and the same molecule is manipulated along similar trajectories maintaining the same tip apex structure. An unchanged tip shape is verified by the reproducibility of the contact point between lifting attempts. The molecule is not completely pulled out of the layer but each time returned to its vacancy after the recording is stopped (as long as the bond did not break by itself), to lower the probability of triggering a modification of the apex as a consequence of high forces during complete removal. Figure 11 shows the 3D trajectories of such an experiment, where the molecule was repeatedly pulled along two trajectories8.
<img alt="Figure 1" src="/files/ftp_upload/54506/54506fig1.jpg" /> 
Figure 1. Illustration of the manipulation process. (a) Extraction of a single PTCDA molecule from an island by contacting manipulation (four tip apex atoms are shown). (b) Example I(z) and &#916;f(z) curves recorded during the extraction of a molecule by HCM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54506/54506fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/54506/54506fig6.jpg" /> 
Figure 6. STM image of a compact PTCDA island on Ag(111). The image size is 600 x 600 &#197;2 and the tunneling conditions are I = 0.1 nA and Vb = -0.35&#160;V. A void seen in the film is the result of extraction of three molecules. Extracted molecules were re-deposited back onto the surface (bottom left corner of the image). The inset shows a 50 x 30 &#197;2 STM image that reveals the typical contrast of PTCDA that stems from its lowest unoccupied molecular orbital (LUMO). The reactive carboxylic oxygen atoms of PTCDA that are used for contacting the molecule are marked by two red crosses. The molecular orientation can be derived according to Ref. 17. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54506/54506fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/54506/54506fig7.jpg" /> 
Figure 7. Frequency response spectrum. Shown is the frequency response of the tuning fork sensor (TFS) measured in UHV and at 5 K. Corresponding Q-factor value is about 70,000. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54506/54506fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/54506/54506fig8.jpg" /> 
Figure 8. I(z) curves recorded during the test of the tip-molecule contact. The contact is established by approaching the tip towards one of the carboxylic oxygen atoms of PTCDA. The approach distance &#916;z from the stabilization point I = 0.1 nA, Vb = -0.35&#160;Vis about 4 &#197;. Both, approach (black) and retraction (red) curves were recorded with the bias Vb = -5 mV. Sharp jump observed at z= 0 &#197; in the approach curve occurs due to the oxygen atom of PTCDA snapping to the tip and forming the tip-molecule contact. After the contact was formed the tip was retracted vertically back to its initial position. As the tip was retracted its contact to the molecule ruptured at z = 0.3 &#197;. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54506/54506fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/54506/54506fig9.jpg" /> 
Figure 9. Example for the nano-structuring of a molecular layer by HCM. The STM image (stabilization point: I = 0.1 A, Vb = -0.35 V) shows&#160;47 vacancies created by consecutive removal of individual PTCDA molecules by HCM without using the 3D virtual reality visual feedback. The insets show a sequence of manipulations made to correct a &#34;writing error&#34;, filling a vacancy by a molecule removed from the island edge. (Adapted from Ref. 7) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54506/54506fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/54506/54506fig10.jpg" /> 
Figure 10. A perspective view showing 34 manipulation trajectories that all led to the successful removal of PTCDA from the monolayer. All of the trajectories were recorded while creating the pattern shown in Figure 9. The inset shows a projection of a 7 &#197; radius sphere around the contacting point and indicates where successful (red) and unsuccessful (black) trajectories penetrate that sphere. All successful trajectories concentrate in a relatively narrow solid angle &#937; (adapted from Ref. 7). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54506/54506fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/54506/54506fig11.jpg" /> 
Figure 11. Manipulation trajectory refinement and reproducibility. Projection of 3D tip trajectories recorded using HCM with 3D virtual reality visual feedback on a molecule within a PTCDA/Ag(111) island. The inset in (a) shows three manipulation attempts performed by the experimenter in search of the (successful) kinked trajectory shown in (a) and (b). The grey curve is the trajectory obtained from averaging the trajectories shown in Figure 10. Top and side views of seven manipulation attempts following the averaged trajectory (no success) and seven attempts along a newly found kinked trajectory (all successful). The color coding shows (a) log(I(x,y,z)) and (b) &#916;f(x,y,z). (Adapted from Ref. 8) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54506/54506fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Hand Controlled Manipulation of Single Molecules via a Scanning Probe Microscope with a 3D Virtual Reality Interface at JoVE.com

Hand Controlled Manipulation of Single Molecules via a Scanning Probe Microscope with a 3D Virtual Reality Interface

We demonstrate the precise manipulation of individual organic molecules on a metal surface with the tip of a scanning probe microscope driven in 3D by the experimenter's hand using a motion capture system and fully immersive virtual reality goggles.

Considering organic molecules as the functional building blocks of future nanoscale technology, the question of how to arrange and assemble such building ...

hand-controlled-manipulation-single-molecules-via-scanning-probe

Nanyang Technological University

Research

JoVE Journal

Engineering

9.0K Views.  Forschungszentrum Jülich. We demonstrate the precise manipulation of individual organic molecules on a metal surface with the tip of a scanning probe microscope driven in 3D by the experimenter's hand using a motion capture system and fully immersive virtual reality goggles.

Video: Hand Controlled Manipulation of Single Molecules via a Scanning Probe Microscope with a 3D Virtual Reality Interface

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

Novel 3D/VR Interactive Environment for MD Simulations, Visualization and Analysis

The increasing development of computing (hardware and software) in the last decades has impacted scientific research in many fields including materials science, biology, chemistry and physics among many others. A new computational system for the accurate and fast simulation and 3D/VR visualization of nanostructures is presented here, using the open-source molecular dynamics (MD) computer program LAMMPS. This alternative computational method uses modern graphics processors, NVIDIA CUDA technology and specialized scientific codes to overcome processing speed barriers common to traditional computing methods. In conjunction with a virtual reality system used to model materials, this enhancement allows the addition of accelerated MD simulation capability. The motivation is to provide a novel research environment which simultaneously allows visualization, simulation, modeling and analysis. The research goal is to investigate the structure and properties of inorganic nanostructures (e.g., silica glass nanosprings) under different conditions using this innovative computational system. The work presented outlines a description of the 3D/VR Visualization System and basic components, an overview of important considerations such as the physical environment, details on the setup and use of the novel system, a general procedure for the accelerated MD enhancement, technical information, and relevant remarks. The impact of this work is the creation of a unique computational system combining nanoscale materials simulation, visualization and interactivity in a virtual environment, which is both a research and teaching instrument at UC Merced.

A new computational system featuring GPU-accelerated molecular dynamics simulation and 3D/VR visualization, analysis and manipulation of nanostructures has been implemented, representing a novel approach to advance materials research and promote innovative investigation and alternative methods to learn about material structures with dimensions invisible to the human eye.

The increasing development of computing (hardware and software) in the last decades has impacted scientific research in many fields including materials ...

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 ...

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in these structures are held together by physical interactions mediated by a network of extracellular polymeric substances. Bacterial biofilms impact many human activities and the understanding of their properties is crucial for a better control of their development &#8212; maintenance or eradication &#8212; depending on their adverse or beneficial outcome. This paper describes a novel methodology aiming to measure in situ the local physical properties of the biofilm that had been, until now, examined only from a macroscopic and homogeneous material perspective. The experiment described here&#160;involves introducing magnetic particles into a growing biofilm to seed local probes that can be remotely actuated without disturbing the structural properties of the biofilm. Dedicated magnetic tweezers were developed to exert a defined force on each particle embedded in the biofilm. The setup is mounted on the stage of a microscope to enable the&#160;recording of time-lapse images of the particle-pulling period. The particle trajectories are then extracted from the pulling sequence and the local viscoelastic parameters are derived from each particle displacement curve, thereby providing the 3D-spatial distribution of the parameters. Gaining insights into the biofilm mechanical profile is essential from an engineer&#39;s point of view for biofilm control purposes but also from a fundamental perspective to clarify the relationship between the architectural properties and the specific biology of these structures.

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

This paper shows an original methodology based on the remote actuation of magnetic particles seeded in a bacterial biofilm and the development of dedicated magnetic tweezers to measure in situ the local mechanical properties of the complex living material built by micro-organisms at interfaces.

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in ...

Bioengineering

Scanning SQUID Study of Vortex Manipulation by Local Contact

Local, deterministic manipulation of individual vortices in type 2 superconductors is challenging. The ability to control the position of individual vortices is necessary in order to study how vortices interact with each other, with the lattice, and with other magnetic objects. Here, we present a protocol for vortex manipulation in thin superconducting films by local contact, without applying current or magnetic field. Vortices are imaged using a scanning superconducting quantum interference device (SQUID), and vertical stress is applied to the sample by pushing the tip of a silicon chip into the sample, using a piezoelectric element. Vortices are moved by tapping the sample or sweeping it with the silicon tip. Our method allows for effective manipulation of individual vortices, without damaging the film or affecting its topography. We demonstrate how vortices were relocated to distances of up to 0.8 mm. The vortices remained stable at their new location up to five days. With this method, we can control vortices and move them to form complex configurations. This technique for vortex manipulation could also be implemented in applications such as vortex based logic devices.

We present a protocol for manipulation of individual vortices in thin superconducting films, using local mechanical contact. The method does not include applying current, magnetic field or additional fabrication steps.

Local, deterministic manipulation of individual vortices in type 2 superconductors is challenging. The ability to control the position of individual ...

Visualizing Solution Structure at Solid-Liquid Interfaces using Three-Dimensional Fast Force Mapping

Amongst the challenges for a variety of research fields are the visualization of solid-liquid interfaces and understanding how they are affected by the solution conditions such as ion concentrations, pH, ligands, and trace additives, as well as the underlying crystallography and chemistry. In this context, three-dimensional fast force mapping (3D FFM) has emerged as a promising tool for investigating solution structure at interfaces. This capability is based on atomic force microscopy (AFM) and allows the direct visualization of interfacial regions in three spatial dimensions with sub-nanometer resolution. Here we provide a detailed description of the experimental protocol for acquiring 3D FFM data. The main considerations for optimizing the operating parameters depending on the sample and application are discussed. Moreover, the basic methods for data processing and analysis are discussed, including the transformation of the measured instrument observables into tip-sample force maps that can be linked to the local solution structure. Finally, we shed light on some of the outstanding questions related to 3D FFM data interpretation and how this technique can become a central tool in the repertoire of surface science.

Here, we present a protocol for using three-dimensional fast force mapping - an atomic force microscopy technique - for visualizing solution structure at solid-liquid interfaces with the subnanometer resolution by mapping the tip-sample interactions within the interfacial region.

Amongst the challenges for a variety of research fields are the visualization of solid-liquid interfaces and understanding how they are affected by the ...

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. ...

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

Developmental Biology

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.

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

Improved Imaging of DNA Star Motifs by PORT High-Speed Atomic Force Microscopy

High-Speed Atomic Force Microscopy Imaging of DNA Three-Point-Star Motif Self Assembly Using Photothermal Off-Resonance Tapping

High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due to its ability to image under physiological conditions in liquid environments. The photothermal off-resonance tapping (PORT) mode uses a drive laser to oscillate the cantilever in a controlled manner. This direct cantilever actuation is effective in the MHz range. Combined with operating the feedback loop on the time domain force curve rather than the resonant amplitude, PORT enables high-speed imaging at up to ten frames per second with direct control over tip-sample forces. PORT has been shown to enable imaging of delicate assembly dynamics and precise monitoring of patterns formed by biomolecules. Thus far, the technique has been used for a variety of dynamic in vitro studies, including the DNA 3-point-star motif assembly patterns shown in this work. Through a series of experiments, this protocol systematically identifies the optimal imaging parameter settings and ultimate limits of the HS-PORT AFM imaging system and how they affect biomolecular assembly processes. Additionally, it investigates potential undesired thermal effects induced by the drive laser on the sample and surrounding liquid, particularly when the scanning is limited to small areas. These findings provide valuable insights that will drive the advancement of PORT mode's application in studying complex biological systems.

Here, we show the imaging protocol for observing biomolecular interactions with photothermal off-resonance tapping (PORT), where we optimized imaging parameters, identified system limits, and investigated potential improvements in imaging three-point-star DNA motif assembly.

High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due ...

Hand Controlled Manipulation of Single Molecules via a Scanning Probe Microscope with a 3D Virtual Reality Interface

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