The authors acknowledge financial support from a variety of sources including the UK Engineering and Physical Sciences Research Council (grant refs. EP/K011685/1, EP/K024485/1) and a graduate studentship for JH; the Leverhulme Trust (RPG-2014-292); the Wellcome Trust Institutional Strategic Support Fund (105610/Z/14/Z); the British Council (216196834); and the University of Manchester for a University of Manchester Research Institute (UMRI pump priming fund) and a Presidential Doctoral Scholarship to SW. Technical assistance by Dr. Andreas Lieb (Nanosurf AG) is also gratefully acknowledged.

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-Area+Scanning+Probe+Nanolithography+Facilitated+by+Automated+Alignment+of+Probe+Arrays.">Large-Area Scanning Probe Nanolithography Facilitated by Automated Alignment of Probe Arrays.</a> RSC Adv. 5, 61402-61409 (2015).</li><li>Noh, H., 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="Multipoint+Force+Feedback"+Leveling+of+Massively+Parallel+Tip+Arrays+in+Scanning+Probe+Lithography.">"Multipoint Force Feedback" Leveling of Massively Parallel Tip Arrays in Scanning Probe Lithography.</a> Small. 11, 4526-4531 (2015).</li><li>Dalby, M. J., 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=The+Control+of+Human+Mesenchymal+Cell+Differentiation+Using+Nanoscale+Symmetry+and+Disorder.">The Control of Human Mesenchymal Cell Differentiation Using Nanoscale Symmetry and Disorder.</a> Nat. Mater. 6, 997-1003 (2007).</li><li>Connell, C. D., Higgins, M. J., Moulton, S. E., Wallace, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-Bioelectronics+via+Dip-Pen+Nanolithography.">Nano-Bioelectronics via Dip-Pen Nanolithography.</a> J. Mater. Chem. C. 3, 6431-6444 (2015).</li><li>Giam, L. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irreversible,+Direct+Bonding+of+Nanoporous+Polymer+Membranes+to+PDMS+or+Glass+Microdevices.">Irreversible, Direct Bonding of Nanoporous Polymer Membranes to PDMS or Glass Microdevices.</a> Lab Chip. 10, 548-552 (2010).</li><li>Liao, X., Braunschweig, A. B., Zheng, Z., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-+and+Time-Dependent+Feature+Size+and+Shape+Control+in+Molecular+Printing+via+Polymer-Pen+Lithography.">Force- and Time-Dependent Feature Size and Shape Control in Molecular Printing via Polymer-Pen Lithography.</a> Small. 6, 1082-1086 (2010).</li><li>Sheehan, P. E., Whitman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol+Diffusion+and+The+Role+of+Humidity+in+"Dip+Pen+Nanolithography".">Thiol Diffusion and The Role of Humidity in "Dip Pen Nanolithography".</a> Phys. Rev. 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R., Anthony, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Cell Culture: Essential Methods. , 153-172 (2010).</li><li>Brinkmann, F., 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=Interdigitated+Multicolored+Bioink+Micropatterns+by+Multiplexed+Polymer+Pen+Lithography.">Interdigitated Multicolored Bioink Micropatterns by Multiplexed Polymer Pen Lithography.</a> Small. 9, 3266-3275 (2013).</li><li>Senesi, A. J., Rozkiewicz, D. I., Reinhoudt, D. N., Mirkin, C. 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The alignment algorithm and software were developed by, and are proprietary to, the University of Manchester. It is available for download at http://www.click2go.umip.com/.

This protocol serves to provide users with a convenient methodology to rapidly carry out nanolithographic patterning with high uniformity and controllable feature size over large (cm2) areas. Substrates bearing these large area nanopatterns can then be further elaborated for a variety of applications. One major application of this technology is in the generation of nanofabricated surfaces for cell-surface interaction studies. This report shows some illustrative examples of cell culture on these materials, demonstrating control of hMSC morphology by nanofabricated substrates.
The key enabler of this protocol is the automation of the alignment procedure (step 4) that allows highly uniform and high-throughput production of features on surfaces, down to nanoscale resolution, which enables the rapid turnover of cell culture experiments. The polymer pen lithography carried out using this alignment algorithm is able to generate nanoscale features within approximately 30 min. The reproducibility and accuracy of automated alignment, and thus the uniformity of the patterned features, is however critically dependent on the quality of the probe arrays that are produced (step 1 and 2). Any flaws in their preparation that result in blunt, broken or missing probes; such as trapped air bubbles (step 1.5) or improper separation of the probes from the master (step 1.8) can result in inaccurate alignment and poor quality lithography.
This reported method shares a limitation in common with other alignment methods that rely on force feedback. The accurate determination of when the probes are in contact with the surface is constrained by the need to account for background vibrations caused by the ambient environment and the movement of the sample stage. In general, the sensors have a force sensitivity in the &#181;N regime (2 &#181;N in this case), but the alignment algorithm is designed to only register a force of at least 490 &#181;N as definitive contact between the probes and the surface, in order to avoid any &#39;false positives&#39; resulting from background noise.13 Thus, this method tends to produce large features (1-2 &#181;m) since the probes must extended a large distance on the z-axis (with a consequent higher force) in order to register contact. In order to compensate, smaller features can be generated by reducing the z-axis distance travelled during the lithography step (e.g., entering the &#39;Black&#39; setting in step 5.2.3.2 as 3 &#181;m instead of 5 &#181;m).
Nevertheless, even with this limitation, the automation algorithm is able to address a critical aspect in the application of parallelized scanning probe lithography methods, as alignment was previously the most time demanding and imprecise step in the implementation of these techniques. This automation now shifts the rate-limiting step of the fabrication process from the alignment to the lithographic writing itself. While this protocol demonstrates the application of this alignment procedure to PPL, the framework could be applied to a number of SPL techniques such as lipid-DPN26 and matrix-assisted lithography27 as well as potential future catalytic probe systems.28

Progress in nanotechnology is dependent on the development of techniques capable of efficiently and reliably fabricating nanoscale features on surfaces.1,2 However, generating such features over large areas (multiple cm2) reliably and at relatively low cost is a non-trivial endeavor. Most existing techniques, derived from the semiconductor industry, rely on ablative photolithography to fabricate &#39;hard&#39; materials. More recently, lithographic techniques derived from scanning probe microscopy (SPM) have emerged as a convenient and versatile approach for the rapid prototyping of nanoscale designs.3 SPM-based techniques are able to conveniently and rapidly &#39;write&#39; any user-defined pattern. The most well-known of these is dip-pen nanolithography (DPN), pioneered by Mirkin et al.,4 where a scanning probe is used as a &#39;pen&#39; to transfer a molecular &#39;ink&#39; to the surface producing features in a fashion analogous to writing. Under ambient conditions, as a probe is scanned across a surface the &#39;ink&#39; molecules are transferred to the surface via a water meniscus that forms between the probe and the surface (Figure 1). DPN thus allows the nanolithographic deposition of a wide range of materials, including &#39;soft&#39; materials such as polymers and biomolecules.5 Related techniques using probes engineered with channels for fluid delivery, variously referred to as &#39;nanopipettes&#39; and &#39;nano-fountain pens&#39;, have also been reported.6,7,8
The main obstacle to the wider application of SPM-derived lithography is throughput, as it requires an excessively long time to pattern centimeter-scale areas with a single probe. Early efforts to address this issue focused on the parallelization of cantilever-based DPN, with both &#39;one- dimensional&#39; and &#39;two-dimensional&#39; (2D) probe arrays being reported for the lithography of centimeter-sized areas.5,9 However, these cantilever arrays are produced through relatively complex multistep fabrication methods and are relatively fragile. The invention of polymer pen lithography (PPL) addressed this issue by replacing the standard SPM cantilevers with a 2D array of soft siloxane elastomer probes bonded to a glass slide.10 This simple probe setup significantly decreases the cost and complexity of patterning large areas, opening up nanolithography to a wider range of applications. This cantilever-free architecture has also been expanded to hard-tip soft-spring lithography,11 which provides a hybrid of soft elastomeric backing with hard silicon tips giving improved resolution in comparison to patterns produced using soft elastomer tips.
A crucial factor in the execution of these 2D array technologies is that the probe array must be exactly parallel to the surface substrate so that when lithography is utilized, all the probes come into contact with the surface simultaneously. Even a small misalignment can cause a large difference in feature size from one side of the array to the other, since some probes will come into contact with the surface earlier during the descent of the array, while others will come into contact later or not at all.12 Exact alignment is especially important with PPL due to the deformability of the soft elastomer probes, where the probes contacting the surface earlier will be compressed, leaving a larger footprint on the surface.
The early work on PPL employed purely visual inspection to guide the alignment process, using a camera mounted above the array to observe the deformation of the pyramidal probes as they were brought into contact with the surface.10 Alignment was judged by observing which side of the probes came into contact with the surface first, then adjusting the angle and repeating the procedure in an iterative manner until the difference in contact on each side of the probe was indistinguishable to the eye. As this alignment procedure relies on subjective visual inspection by the operator, reproducibility is low.
Subsequently, a more objective approach has been developed, consisting of a force sensor mounted beneath the substrate to measure the force applied upon contact of the probes on the surface.12 Alignment was thus achieved by adjusting the tilt angles to maximize the force exerted, which indicated that all the probes were simultaneously in contact. This method showed that alignment to within 0.004&#176; of the surface parallel was possible. This &#39;force feedback levelling&#39; has now been implemented into fully automated systems in two independent reports.13,14 Both use a triad of force sensors mounted either beneath the substrate or above the array and measure the amount of force exerted upon contact between the probe arrays and surface. These systems give high precision, reporting misalignments of &#8804;0.001&#176; over a 1 cm length scale,14 or &#8804; 0.0003&#176;over 1.4 cm.13 These automated alignment systems also provide major savings in operator time and overall time taken to complete the lithography process.
One major application of high-throughput surface fabrication enabled by this technology is the generation of cell culture substrates. It is now well established that cell phenotype can be manipulated by controlling the initial interaction between cells and surface features, and that this can be enhanced at the nanoscale.15 Specifically, scanning probe lithography methods have been shown to be a facile method to produce a variety of nanofabricated surfaces for such cell culture experiments.16 For example, surfaces presenting nanoscale patterns of self-assembled monolayers and extracellular matrix proteins templated by PPL and DPN have been used to study the potential of nano-modified materials in material induced differentiation of stem cells.17
This protocol describes the utilization of a modified atomic force microscope (AFM) system that enables large-area PPL. We detail the detection of force using multiple force sensors as the means of determining probe-surface contact, together with an algorithm that automates the iterative alignment process. Subsequent functionalization of these patterns with the extracellular matrix protein fibronectin and the culture of human mesenchymal stem cells (hMSC) are described, as a demonstration of PPL-fabricated surfaces applied for cell culture.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FlexAFM mounted on a motorised 5-axis (XYZ&amp;Theta;&amp;Phi;) translation and goniometer stage</td><td>NanoSurf</td><td>P40008</td><td></td></tr><tr><td>AFM control software</td><td>NanoSurf</td><td>C3000</td><td></td></tr><tr><td>Engraving pen</td><td>Sigma-Aldrich</td><td>Z225568</td><td></td></tr><tr><td>Plasma Cleaner</td><td>Harrick plasma</td><td>PDC-32G-2</td><td></td></tr><tr><td>PlasmaFlo</td><td>Harrick plasma</td><td>PDC-FMG-2</td><td></td></tr><tr><td>Economy Dry Oxygen Service Pump</td><td>Harrick plasma</td><td>PDC-OPE-2</td><td></td></tr><tr><td>Tube Rotator</td><td>Stuart</td><td>SB3</td><td></td></tr><tr><td>Vacuum Desiccator</td><td>Thermo Fisher Scientific</td><td>5311-0250</td><td></td></tr><tr><td>Milli-Q Water Purification System</td><td>Merck Millipore</td><td>ZRXQ015WW</td><td></td></tr><tr><td>Modular Humidity Generator</td><td>proUmid</td><td>MHG32</td><td></td></tr><tr><td>Proline Plus Pipette 100-1000 &amp;micro;L</td><td>Sartorius</td><td>728070</td><td></td></tr><tr><td>Silicon masters</td><td>NIL Technology</td><td>custom-made</td><td></td></tr><tr><td>Upright snapshot fluorescence microscope</td><td>Olympus</td><td>BX51</td><td></td></tr><tr><td>Microscope objectives</td><td>Olympus</td><td>10x and 60x UPlan FLN &amp;infin;/-/FN 26.5</td><td></td></tr><tr><td>Upright bright field microscope</td><td>Leica</td><td>DM 2500M</td><td></td></tr><tr><td>Ultrasonicator</td><td>Ultrawave Ltd.</td><td>U95</td><td></td></tr><tr><td>Spreadsheet for recording and intepreting automated alignment results</td><td>Microsoft</td><td>Excel</td><td></td></tr><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-propanol</td><td>Sigma-Aldrich</td><td>34863</td><td>FLAMMABLE</td></tr><tr><td>Microscope Sildes, Clear, Ground</td><td>Thermo Fisher Scientific</td><td>451000</td><td></td></tr><tr><td>(7&amp;ndash;8% vinylmethylsiloxane)-dimethylsiloxane copolymer, trimethylsiloxy-terminated</td><td>Gelest</td><td>VDT-731</td><td></td></tr><tr><td>1,3,5,7-tetramethyl-1,3,5,7-tetravinylcyclotetrasiloxane</td><td>Gelest</td><td>SIT7900.0</td><td></td></tr><tr><td>Platinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex solution</td><td>Sigma-Aldrich</td><td>479527</td><td>HARMFUL, TOXIC</td></tr><tr><td>(25&amp;ndash;35% methylhydrosiloxane)-dimethylsiloxane copolymer, trimethylsiloxane-terminated</td><td>Gelest</td><td>HMS-301</td><td></td></tr><tr><td>Weigh Boat 100 mL</td><td>Scientific Laboratory Supplies</td><td>BALI828</td><td></td></tr><tr><td>Pasteur pipette</td><td>Appleton Woods</td><td>KS230</td><td></td></tr><tr><td>Petri dish</td><td>SARSTEDT</td><td>82.1473</td><td></td></tr><tr><td>Razor blade</td><td>Thermo Fisher Scientific</td><td>ST10-031T</td><td></td></tr><tr><td>Adhesive Carbon Tape</td><td>Agar scientific</td><td>AGG3939</td><td></td></tr><tr><td>16-Mercaptohexadecanoic acid</td><td>Sigma-Aldrich</td><td>448303-1G</td><td>HARMFUL, TOXIC</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>34852</td><td>FLAMMABLE</td></tr><tr><td>Gold coated microscope slide</td><td>Sigma-Aldrich</td><td>643203</td><td>Once opened gold will remain reactive to thiols for at least 1 month</td></tr><tr><td>Thiourea</td><td>Sigma-Aldrich</td><td>T8656</td><td>HARMFUL, TOXIC</td></tr><tr><td>Iron(III) nitrate nonahydrate</td><td>Sigma-Aldrich</td><td>529303</td><td>HARMFUL, TOXIC</td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>84415</td><td>HARMFUL, TOXIC</td></tr><tr><td>(11-Mercaptoundecyl)hexa(ethylene glycol)</td><td>Sigma-Aldrich</td><td>675105</td><td>HARMFUL, TOXIC</td></tr><tr><td>Fibronectin from human plasma</td><td>Sigma-Aldrich</td><td>F0895</td><td></td></tr><tr><td>Cobalt(II) nitrate hexahydrate</td><td>Sigma-Aldrich</td><td>203106</td><td>HARMFUL, TOXIC</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>MSCGM Mesenchymal Stem Cell Growth Medium</td><td>Lonza UK</td><td>PT-3001</td><td></td></tr><tr><td>Human Mesenchymal Stem Cells</td><td>Lonza UK</td><td>PT-2501</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich</td><td>T4174</td><td></td></tr><tr><td>Heraeus Multifuge X1 Centrifuge</td><td>Thermo Fisher Scientific</td><td>75004210</td><td></td></tr><tr><td>CELLSTAR Centrifuge Tubes</td><td>Greiner Bio-One</td><td>188261</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>P/0840/53</td><td>HARMFUL, TOXIC</td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>Thermo Fisher Scientific</td><td>A12379</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>&quot;Detergent&quot; in manuscript</td></tr><tr><td>VECTASHIELD Antifade Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>Rabbit anti-fibronectin antibody</td><td>Abcam</td><td>ab2413</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594</td><td>Thermo Fisher Scientific</td><td>R37117</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A3912</td><td></td></tr><tr><td>12-well plate</td><td>Thermo Fisher Scientific</td><td>10253041</td><td></td></tr><tr><td>T75 tissue culture flask</td><td>Thermo Fisher Scientific</td><td>10790113</td><td></td></tr><tr><td>cantilever</td><td>BudgetSensor</td><td>ContAl-G</td><td></td></tr></tbody></table>

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.

To check whether the automated alignment had been successful, the graphs plotted from the alignment data (in the spreadsheet from step 4.8) were examined. Where the alignment process had been successful the two plots, corresponding to the angle by which the sample stage has been tilted along the &#952; and &#966; axes, showed a series of rising and descending data points. In each of the plots, two linear fits of the data points showed a well-defined intersect &#34;peak&#34; indicating the maximum z-extension and the corresponding angle at which alignment was achieved (Figure 4A and 4B). This process is repeated four times (i.e., twice for each axis) and plotted as a set of four coordinates. The intersection of each pair of coordinates thus shows the overall optimum angles (Figure 4C).13 In cases where the alignment was not successful, it can be observed that their corresponding &#952; and &#966; angle plots do not give good quality linear fits, or do not intersect (Figure 5). Such failed alignments are typically as a result of the arrays being improperly trimmed or mounted to the probe holder (steps 1.7, 1.8, and 2.2). In these cases, the arrays were discarded and a new one prepared and mounted (steps 1 and 2), and the alignment process repeated (step 4).
Upon successful alignment and lithography with MHA by PPL, patterned gold substrates were then imaged using lateral force microscopy to examine whether deposition had taken place. A larger area examination of the printed surfaces was also conducted by optical microscopy of the substrates after etching of the gold not protected by the deposited thiol (Figure 6 and Figure 7). However, the etched patterns cannot be used for further functionalization and should only be used to confirm patterning on representative samples of a batch of printed surface substrates. If the etched patterns show blank areas corresponding to individual pens (Figure 8), this result indicates that the production of probe arrays has not been done successfully, and that some probes are damaged or missing. This inhomogeneity of the probes may be due to the use of an old master where the perfluorinated coating has worn away (step 1.3), resulting in some probes being torn away when the array is separated from the master. In these cases, a new master should be used. The result may also be due to the presence of air bubbles trapped between the glass backing and the master (step 1.5), or if the probe array was not cleanly separated from the master after curing (step 1.8).
Florescent microscopy images of the fibronectin functionalized surfaces incubated hMSCs were also collected (Figure 9). In general, all substrates were stable within the in vitro culture environment and the cells adhered and adapted their morphology to the printed patterns in case of smaller isolated 20 x 20 array of features.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56967/56967fig1.jpg" /> 
Figure 1. Schematic representation of polymer pen lithography showing molecular ink transport via a water meniscus on probe tip. (A) Side view and (B) top view of the polymer pen array indicate that when the probe array and surface substrate are fully aligned, all the probes come into contact with the surface simultaneously, resulting in parallelized lithography. <a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56967/56967fig2.jpg" /> 
Figure 2. Schematic diagram of polymer pen lithography set up. (A) Expanded side view of experimental set up where the prepared probe array is attached to probe holder and mounted to AFM scanner. The substrate is placed on the stage, below which are located the three force sensors. (B) A representation of the assembled instrumentation, showing the AFM scan head relative to the sample stage. (C) Bottom view showing force sensor location. <a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56967/56967fig3.jpg" /> 
Figure 3. Schematic representation depicting the spectroscopy program for the alignment procedure. The AFM scanner is set to move the probes toward sample by a distance of 10 &#181;m within 100 ms, held at position for 250 ms, followed by a retraction of 10 &#181;m within 100 ms, and then held for 250 ms at the retracted position. The motion is then repeated throughout the alignment process. <a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56967/56967fig4.jpg" /> 
Figure 4. Graphs illustrating a successful alignment. Graphs of z position against the tilt angles (A) &#952; and (B) &#966; for a successful alignment, where &#9679; indicates the actual values measured and + indicates the best fit with the least-squares method. (C) Graph of &#966; against &#952; fitted angles with the four points where the maximum z-position was reached. The intersection point marked is the final optimum tilt angle across both axes. <a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56967/56967fig5.jpg" /> 
Figure 5. Graphs illustrating an unsuccessful alignment. Graphs of z position against the tilt angles (A) &#952; and (B) &#966; for an unsuccessful alignment, where &#9679; indicates the actual values measured and + indicates the best fit with the least-squares method. (C) Graph of &#966; against &#952; fitted angles with the four points where the maximum z-position was reached. No clear optima or intersection point are observed and therefore the optimum alignment angles are not resolved. <a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56967/56967fig6.jpg" /> 
Figure 6. Illustrative optical microscopy and atomic force microscopy images of gold substrates that were patterned with MHA by the aligned PPL arrays and then etched. (A) and (B) are sequentially magnified optical microscopy images of the etched patterns; (C) is an AFM topography image of a single grid of patterns. <a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56967/56967fig7.jpg" /> 
Figure 7. Illustrative optical microscopy images of gold substrates that were patterned with MHA by the aligned PPL arrays and then etched. (A) and (B) are sequentially magnified optical microscopy images of etched patterns and (C) is a lower magnification image that shows large area homogeneous patterns. <a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56967/56967fig8.jpg" /> 
Figure 8. Illustrative optical microscopy image of a gold substrate that was unevenly patterned with MHA and then etched. The intended patterns (shown in the inset) were repeating grids of 20 dot lines arranged in 20 lines, with every two lines produced by increasing the z-axis extension by 1 &#181;m (ranging from 5 to -5 &#181;m). It can be seen that in some areas no patterns are generated, due to missing probes in those locations. In the areas where only two lines of dots are produced, this result indicates that a probe is present but it is not of the same height as the fully functioning probes, so only deposit features when the array is extended to the full z-axis distance. In this image, the contrast has been deliberately altered to enable observation of the partially printed areas. <a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56967/56967fig9.jpg" /> 
Figure 9. Epifluorescence microscopy images of hMSCs cultured on the fibronectin arrays templated by PPL. (A) and (B) are high magnification images showing individual cells. (C) Shows an example pattern of the fibronectin array without an adherent cell and (D) is a wide field image of the cells cultured in a grid arrangement (a schematic of the printed pattern is also shown in the inlay). The cells are stained to show fibronectin (red), F-actin (green) and cell nuclei (blue). <a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies at JoVE.com

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

large-area-scanning-probe-nanolithography-facilitated-automated

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.6K Views.  University of Manchester. 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.

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Atomically Traceable Nanostructure Fabrication

Reducing the scale of etched nanostructures below the 10 nm range eventually will require an atomic scale understanding of the entire fabrication process being used in order to maintain exquisite control over both feature size and feature density. Here, we demonstrate a method for tracking atomically resolved and controlled structures from initial template definition through final nanostructure metrology, opening up a pathway for top-down atomic control over nanofabrication. Hydrogen depassivation lithography is the first step of the nanoscale fabrication process followed by selective atomic layer deposition of up to 2.8 nm of titania to make a nanoscale etch mask. Contrast with the background is shown, indicating different mechanisms for growth on the desired patterns and on the H passivated background. The patterns are then transferred into the bulk using reactive ion etching to form 20 nm tall nanostructures with linewidths down to ~6 nm. To illustrate the limitations of this process, arrays of holes and lines are fabricated. The various nanofabrication process steps are performed at disparate locations, so process integration is discussed. Related issues are discussed including using fiducial marks for finding nanostructures on a macroscopic sample and protecting the chemically reactive patterned Si(100)-H surface against degradation due to atmospheric exposure.

We report a protocol for combining the atomic metrology of the Scanning Tunneling Microscope for surface patterning with selective Atomic Layer Deposition and Reactive Ion Etching. Using a robust process involving numerous atmospheric exposures and transport, 3D nanostructures with atomic metrology are fabricated.

Reducing the scale of etched nanostructures below the 10 nm range eventually will require an atomic scale understanding of the entire fabrication process ...

Engineering

Single-Digit Nanometer Electron-Beam Lithography with an Aberration-Corrected Scanning Transmission Electron Microscope

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by employing an aberration-corrected scanning transmission electron microscope as the exposure tool. Here, we present results of single-digit nanometer patterning of two widely used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. The method achieves sub-5 nanometer features in poly (methyl methacrylate) and sub-10 nanometer resolution in hydrogen silsesquioxane. High-fidelity transfer of these patterns into target materials of choice can be performed using metal lift-off, plasma etch, and resist infiltration with organometallics.

We use an aberration-corrected scanning transmission electron microscope to define single-digit nanometer patterns in two widely-used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. Resist patterns can be replicated in target materials of choice with single-digit nanometer fidelity using liftoff, plasma etching, and resist infiltration by organometallics.

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

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. ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...