This work was supported by Connaught Fund New Investigator Award to S.P., Canada Foundation for Innovation, NSERC Discovery Grant Program (grants RGPIN-2015-05114 and RGPIN-2020-05881), University of Manchester and University of Toronto Joint Research Fund, and University of Toronto XSeed Program. C.T. was supported by NSERC USRA fellowship.

1. Coverslip preprocessing
<ol>
	<li>Prepare squeaky-clean coverslips as described in Waterman-Storer, 199825.</li>
	<li>Prepare 1% (3-aminopropyl)trimethoxysilane (APTMS) solution and incubate the coverslips in the solution for 10 min with gentle agitation. Make sure that coverslips move freely in the solution.</li>
	<li>Wash coverslips twice with dH2O for 5 min each.</li>
	<li>Prepare 0.5% glutaraldehyde (GA) solution and incubate the coverslips in the solution for 30 min on a shake...

<ol>
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The results above demonstrate that the described IR laser assisted micropatterning (microphotopatterning) protocol provides reproducible adherent patterns of various shapes that enables the manipulation of cell shape and cytoskeletal architecture. Although numerous micropatterning methods have been developed both prior to and after the debut of microphotopatterning, this method possesses several advantages. First, it does not require specialized equipment and cleanrooms that are usually only found within Engineering depa...

Cell shape is a key determinant of fundamental biological processes such as tissue morphogenesis1, cell migration2, cell proliferation3, and gene expression4. Changes in cell shape are driven by an intricate balance between dynamic rearrangements of the cytoskeleton that deforms the plasma membrane and extrinsic factors such as external forces exerted on the cell and the geometry of cell-cell and cell-matrix adhesions5. Migrating mesenchymal cells, for instance, polymerize a dense actin network at the leading edge that pushes the plasma membrane forward and creates a wide lamellipodia6, while actomyosin contractility retracts the cell&#39;s narrow trailing edge to detach the cell from its current position7,8. Disrupting signaling events that give rise to such specialized cytoskeletal structures perturbs shape and polarity and slows cell migration9. In addition, epithelial sheet bending during gastrulation requires actomyosin-based apical constriction that causes cells and their neighbors to become wedge-shaped10. Although these studies highlight the importance of cell shape, the inherent heterogeneity in cell shape has encumbered efforts to identify mechanisms that connect morphology to function.
To this end, numerous approaches to manipulate cell shape have been developed over the past three decades. These approaches achieve their goal by either constraining the cell with a three-dimensional mold or controlling cellular adhesion geometry through patterned deposition of extracellular matrix (ECM) proteins onto an antifouling surface, a technique termed micropatterning11. Here we will review a number of techniques that have gained popularity throughout the years.
Originally pioneered as an approach for microelectronic applications, soft lithography-based microcontact printing has unequivocally become a cult favorite12. A master wafer is first fabricated by selectively exposing areas of a photoresist-coated silicon substrate to photoirradiation, leaving behind a patterned surface13. An elastomer, such as PDMS, is then poured onto the master wafer to generate a soft &#34;stamp&#34; that transfers ECM proteins to a desired substrate11,14. Once fabricated, a master wafer can be used to cast many PDMS stamps that give rise to highly reproducible micropatterns12. However, the patterns cannot be readily adjusted due to the lengthy photolithography process. This process also requires highly specialized&#160;equipment and cleanrooms that are not typically available in Biology departments.
More recently, direct printing using deep UV&#160;has been reported to circumvent limitations posed by traditional lithography-based approaches. Deep UV light is directed through a photomask to selective areas of a glass coverslip coated with poly-L-lysine-grafted-polyethylene glycol. Chemical groups exposed to deep UV are photoconverted without the use of photosensitive linkers to enable binding of ECM proteins15. The lack of photosensitive linkers enables patterned coverslips to remain stable at room temperature for over seven months15. This method avoids the use of cleanrooms and photolithography equipment and requires less specialized training. However, the requirement for photomasks still poses a substantial hurdle for experiments that require readily available changes in patterns.
In addition to methods that manipulate cell geometry through controlled deposition of ECM proteins on a 2D surface, other seek to control cell shape by confining cells in 3D microstructures. Many studies have adapted the soft lithography-based approach described above to generate 3D, rather than 2D, PDMS chambers to investigate shape-dependent biological processes in embryos, bacteria, yeast and plants16,17,18,19. Two-photon polymerization (2PP) has also taken the lead as a microfabrication technique that can create complex 3D hydrogel scaffolds with nanometer resolution20. 2PP relies on the principles of two-photon adsorption, where two photons delivered in femtosecond&#160;pulses are absorbed simultaneously by a molecule - photoinitiator in this case - that enables local polymerization of photopolymers21. This technique has been heavily employed to print 3D scaffolds that mimic the native ECM structures of human tissue and has been shown to induce low photochemical damage to cells22.
The debut of microphotopatterning 10 years ago gave researchers the opportunity to fabricate micropatterns while avoiding inaccessible and specialized equipment. Microphotopatterning creates patterns on the micron scale by thermally removing selective regions of poly-vinyl alcohol (PVA) coated on activated glass surfaces using an infrared&#160;laser23,24. ECM proteins that attach only the underlying glass surface and not PVA then serve as biochemical cues to enable controlled spreading dynamics and cell shape. This method also offers superior flexibility since patterns can be readily changed in real time. Here, we provide a step-by-step protocol of microphotopatterning by using a commercial multi-photon imaging system. The described protocol is designed for rapid and automated fabrication of large patterns. We demonstrated that these patterns efficiently control cell shape by constraining the geometry of cell-ECM adhesions. Finally, we demonstrate that the described patterning technique modulates the organization and dynamics of the actin cytoskeleton.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(3-Aminopropyl)trimethoxysilane</td>
			<td>Aldrich</td>
			<td>281778</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Cell Culture Dish</td>
			<td>VWR</td>
			<td>10062-880</td>
			<td>Polysterene, TC treated, vented</td>
		</tr>
		<tr>
			<td>25X Apo LWD Water Dipping Objective</td>
			<td>Nikon</td>
			<td>MRD77225</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5 cm Cell Culture Dish</td>
			<td>VWR</td>
			<td>10861-586</td>
			<td>Polysterene, TC treated, vented</td>
		</tr>
		<tr>
			<td>4&#39;,6-Diamidino-2-Phenylindole (DAPI)</td>
			<td>Thermo</td>
			<td>62248</td>
			<td>1mg/mL dihydrochloride solution</td>
		</tr>
		<tr>
			<td>Bovine Serine Albumin</td>
			<td>BioShop</td>
			<td>ALB005</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Wisent</td>
			<td>311-425-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>E9508</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>FC010</td>
			<td>1mg/mL in pH 7.5 buffer</td>
		</tr>
		<tr>
			<td>Fibronectin Antibody</td>
			<td>BD</td>
			<td>610077</td>
			<td>Mouse</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>ImageJ</td>
			<td></td>
			<td>Version 1.53c</td>
		</tr>
		<tr>
			<td>Fluorescent Phalloidin</td>
			<td>Invitrogen</td>
			<td>A12380</td>
			<td>568nm</td>
		</tr>
		<tr>
			<td>Glass Coverslip</td>
			<td>VWR</td>
			<td>16004-302</td>
			<td>22 &#215; 22 mm</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>16220</td>
			<td>25% aqueous solution</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Caledon</td>
			<td>6025-1-29</td>
			<td>37% aqueous solution</td>
		</tr>
		<tr>
			<td>IR Laser</td>
			<td>Coherent</td>
			<td>Chameleon Vision</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimal Essential Medium &#945;</td>
			<td>Gibco</td>
			<td>12561-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>Sigma</td>
			<td>F4680</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Secondary Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4408S</td>
			<td>Goat, 488nm</td>
		</tr>
		<tr>
			<td>Multi-Photon Microscope</td>
			<td>Nikon</td>
			<td>A1R MP+</td>
			<td></td>
		</tr>
		<tr>
			<td>Myosin Light Chain Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>3672S</td>
			<td>Rabbit</td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Version 5.21.03</td>
		</tr>
		<tr>
			<td>Nitric Acid</td>
			<td>Caledon</td>
			<td>7525-1-29</td>
			<td>70% aqueous solution</td>
		</tr>
		<tr>
			<td>Photoshop</td>
			<td>Adobe</td>
			<td></td>
			<td>Version 21.2.1</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma</td>
			<td>P2443</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Poly(vinyl alchohol)</td>
			<td>Aldrich</td>
			<td>341584</td>
			<td>MW 89000-98000, 98% hydrolyzed</td>
		</tr>
		<tr>
			<td>Rabbit Secondary Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4412S</td>
			<td>Goat, 488nm</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>VWR</td>
			<td>10127-876</td>
			<td>Alsoknown as analog rocker</td>
		</tr>
		<tr>
			<td>Sodium Borohydride</td>
			<td>Aldrich</td>
			<td>452882</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Sigma</td>
			<td>S5136</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic</td>
			<td>Sigma</td>
			<td>S5011</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Spyder</td>
			<td>Anaconda</td>
			<td>4.1.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Wisent</td>
			<td>325-042-CL</td>
			<td>0.05% aqueous solution with 0.53mM EDTA</td>
		</tr>
	</tbody>
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control of cell geometry through infrared laser assisted micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The quality of the experimental data obtained through&#160;micropatterning is largely dependent on the quality of the patterns. To determine the quality of patterns generated with the method above, we first used reflectance microscopy to assess the shape and size of the photo ablated areas of the coverslip. We found that each individual pattern looked very similar to the ablation mask and displayed clear boarders and a surface that reflected light uniformly (Figure 2B). A variety of shapes a...

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Control of Cell Geometry through Infrared Laser Assisted Micropatterning

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Micropatterning is a powerful technique employed by scientists to understand connections between cell shape and the activity of intercellular machines. Although several techniques allow researchers to modulate cell shape, these techniques often require specialized equipment inaccessible to some biology labs. This video will guide you through the steps necessary to automate micropatterning on a commercially available multiphoton imaging system.One advantage of this protocol is that it avoids the use of specialized equipment. Patterns can also be readily changed without refabricating photomasks, which is typically the most time-consuming process in micropatterning. Our image processing tool generates average cell images that reflects a representative distribution of proteins in an unbiased and automated manner.Although we use coverslips in our experiments, the automated workflow also allows you to pattern on slides and glass-bottom dishes. Visual demonstration of this method is important since proper microscope setup is critical for the system to image, identify the correct focal plane, and pattern in an automated fashion. Prepare PVA solution as instructed.Add one part HCL to eight parts PVA. Invert the tube carefully a few times to mix. Pour two milliliters of the solution into a small Petri dish and submerge a clean preprocessed coverslip into the liquid.Incubate at room temperature for five minutes on a shaker. Carefully remove the coverslip from the solution. Use the coverslip spinner to spin coat for 40 seconds.In the meantime, clean the tweezers. Transfer the coverslip to a box and dry at four degrees overnight. Turn on the microscope software.First, set the laser line to 750 nanometers. Then set up a new optical configuration called, Image. This is the baseline optical configuration that allows us to image the coverslip through reflectants.Leave other options as default and select the appropriate objective. Configure the hardware settings under this optical configuration. Set the infrared laser as our stimulation laser.Place the beam splitter into the light path to use the IR laser for imaging. Select the Galvano scanner unit and the appropriate D scan detector. Select a scan size and dwell time that is sufficient to capture small features on the coverslip.Make sure the Use IR Laser box is checked. Adjust the acquisition laser power and detector sensitivity to obtain a bright, but not saturated image, of the coverslip surface. In the scan area window, set zoom to one to capture the entire field of view.Save the optical configuration. Now we'll set up a series of optical configurations that will allow the microscope to focus, load the ROI mask and generate patterns in an automated fashion. The first optical configuration allows us to print a fiduciary marker used to auto-focus on the current field of view.Duplicate the image optical configuration and rename it, Print fiduciary marker. Since we are printing in this step, move the beam splitter out of the light path and replace it with an appropriate dichroic mirror. Select the smallest scan size to save time.Since no imaging is required in this step, set detector sensitivity to zero. Increase the laser power to enable printing. In the scan area window, set zoom to maximum and place the scan area in the middle of the field of view.Now we will set up a Z-stack experiment to account for any unevenness in the PVA surface. Set movement at the Z position to relative. Select the appropriate Z device.Next, duplicate the image optical configuration and rename it, Auto focus. Select the smallest scan size and decrease the zoom factor in the scan area window so the field of view is slightly larger than the fiduciary marker. This ensures that other small features on the coverslip will not interfere with autofocusing.In the Devices menu, select Auto Focus setup. Set the scan thickness to that in the Z-stack experiment. The microscope will scan through this range and find the best focal plane using the fiduciary marker.Next, duplicate the image optical configuration and rename it, Load ROI. Set the scan size identical to that of the ROI mask because the mask will be loaded onto an image taken with this optical configuration. We used 2048 pixels to achieve an optimal balance between resolution and speed.Now we will set up the optical configuration used to pattern the coverslip. Duplicate the print fiduciary marker optical configuration and rename it, Micropattern. Set zoom factor to one.Increase the stimulation laser power to ablate PVA. Select an appropriate scan speed. In the ND stimulation window, set up an ND stimulation experiment.Add a few phases to the time schedule and set each at stimulation. Ensure stimulation area and duration are correct. In the same window, enable the stage move main Z function before each phase.This again accounts for any deviations in the Z direction. Finally, set up an optical configuration to make a visible label on the coverslip that can help us locate the patterns. Duplicate print fiduciary marker and rename it, Label surface.Increase laser power significantly and set zoom to one. Transfer the PVA-coated coverslip onto a holder. For an upright microscope, ensure the PVA surface faces down.Add water to the corners to stabilize the coverslip. Mount the holder onto the microscope stage. Lower the objective and add water in between.In the microscope software, turn on the IR laser shutter and perform auto alignment prior to patterning. Switch to the image optical configuration. Scan the field of view while slowly moving the objective closer to the coverslip.At first, the image will appear extremely dim. Move the objective closer to the coverslip until the image brightness increases slightly. This is the coverslip surface that is facing the objective.Continue to move the objective until the brightness decreases and increases again. This is the PVA surface that we will pattern. Focus on any small feature, such as coverslip imperfections or dust, and set zero on the Z drive.Switch to the label surface optical configuration. Click on Capture. Return to imaging and scan.Glass damage shows that the label has been successfully printed. Move the stage by one to two fields of view to avoid glass particles. Scan to confirm.In the devices menu, open the stage movement macro. Set the variables M and N to the desired number of field of views that will be patterned. Save and run the macro.After patterning is complete, switch to the image optical configuration and scan through the patterns. Pattern islands in each field of view should have clear borders and reflect light evenly. Patterns that appear darker and uneven suggest incomplete PVA removal.This is usually due to insufficient laser power or incorrect focal plane. If laser power is set too high, it can also cause the PVA to bubble. After plating cells on coverslips, cells should spread and take on the shape of the pattern.This is an immunofluorescence image of a human fibroblast plated on a crossbow pattern. The cell displays an actin-rich rim of lamellipodia. Thick, ventral stress fibers along the two sides and dorsal stress fibers connected by transverse arcs.Myosin light chain sat behind the dense lamellipodia rim and displayed a striated pattern along actin bundles. Using our custom image processing tool, we generated an averaged image of our proteins of interest. This image shows that the actin structures described above are consistent across a large number of patterns.Although we lose individual structures of myosin after averaging, it's localization along actin bundles is conserved. After watching this video, you should be able to set up a micro patterning workflow using a commercially available multiphoton system with minimal manual work. It's important to optimize laser power for your own system to avoid generating incomplete patterns or PVA Maximum efficiency can be achieved by adjusting the parameters in autofocusing and micropatterning steps.In addition to investigating cellular pathways involving cell morphology, this technique can also be applied to drug screening and other applications that are sensitive to variations in cell shape.

control-cell-geometry-through-infrared-laser-assisted

Research

JoVE Journal

Bioengineering

3.3K visualizações.  University of Toronto. Micropatterning é uma técnica poderosa empregada pelos cientistas para entender as conexões entre a forma celular e a atividade de máquinas intercelulares. Embora várias técnicas permitam aos pesquisadores modular a forma celular, essas técnicas muitas vezes requerem equipamentos especializados inacessíveis para alguns laboratórios de biologia. Este vídeo irá guiá-lo através das etapas necessárias para automatizar a micropatterning em um sistema de imagem multifon disponível co...