Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Summary

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.

Abstract

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.

Introduction

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's narrow trailing edge to detach the cell from its current position^7,8. 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 "stamp" that transfers ECM proteins to a desired substrate^11,14. 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 equipment and cleanrooms that are not typically available in Biology departments.

More recently, direct printing using deep UV 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 plants^16,17,18,19. 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 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 laser^23,24. 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.

Protocol

1. Coverslip preprocessing

1. Prepare squeaky-clean coverslips as described in Waterman-Storer, 1998²⁵.
2. 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.
3. Wash coverslips twice with dH₂O for 5 min each.
4. Prepare 0.5% glutaraldehyde (GA) solution and incubate the coverslips in the solution for 30 min on a shaker. For 25 coverslips, use 50 mL of glutaraldehyde solution. Make sure that coverslips move freely in the solution.
5. Wash coverslips three times with dH₂O for 10 min each.
6. Spin dry coverslips for 30 s using a custom-built coverslip spinner. A detailed description of the coverslip spinner has been published²⁶ and is available online (https://mullinslab.ucsf.edu/home-built-coverslip-drierspinner/). Activated coverslips can be stored for up to one month at +4 °C in a box with dividers so that they stand apart from each other.

2. PVA coating

1. Mix PVA (98% hydrolyzed PVA, MW 98000) with dH₂O to make a 5.6% solution.
2. Solubilize the mixture in a 90 °C water bath and immediately filter through a 0.2 µm filter in a biosafety cabinet while hot. Filter the stock solution again if it precipitates. PVA solution can be stored at room temperature for 3 months.
3. In a 15 mL tube, add one part HCl to eight parts PVA. Invert the tube carefully a few times to mix.
4. Pour 2 mL of the mixed solution into a 35 mm Petri dish and submerge a clean, preprocessed coverslip into the liquid, taking care that the coverslips do not stick to the bottom. Incubate at room temperature for 5 min on a shaker.
5. Carefully remove the coverslips from the solution. Use the coverslip spinner to spin coat for 40 s. In the meantime, clean the tweezers. Transfer the coverslip to a box and dry at +4 °C overnight. PVA-coated coverslips can be stored at +4 °C for up to two weeks.

3. Configuring the Multiphoton Microscope

NOTE: The described protocol is tuned up to create cell adhesive micropatterns of desired shape and size on upright or inverted multi-photon imaging systems, especially the ones that are not equipped with a hardware autofocus. Thus, for every field of view (FOV), the patterning script ablates a small area to create a fiduciary marker on the coverslip, uses a software autofocus to focus the microscope on the coverslip surface, and ablates the desired pattern. Running this script in a loop for adjacent FOVs robustly creates a large array of micropatterns (5 x 5 mm or larger) that constrain cell shape and modulate the activity of intracellular biological processes. The described protocol was developed for Nikon A1R MP+ imaging system controlled by NIS-Elements software. If an imaging system from another vendor is used for patterning, the optical configurations and patterning script should be adjusted according to the manufacturer's instructions.

1. Turn on the microscope software. Ensure that the "Apo LWD 25X/1.10W DIC N2" objective is mounted on the microscope.
   NOTE: The protocol described here is optimized for a 25x/1.1 NA water immersion objective, but other objectives can also be used for patterning. Readers should be aware that pattering with a high-magnification objective (e.g., 40x and 60x) takes longer time as it significantly decreases the number of patterns ablated in each FOV. Low magnification objectives can be used for patterning as long as they provide uniform illumination across the FOV and laser power sufficient to ablate the PVA layer.
2. In the A1plus MP GUI window, set the laser line to 750 nm.
3. Setting up the "Image" optical configuration
   NOTE: NIS-Elements provides users with several tools (graphical interface, an acquisition workflow builder JOBS, optical configurations, and macros) to control the microscope hardware. In this protocol, most of the hardware control is achieved through various optical configurations as well as ND Acquisition and ND Stimulation modules.
   1. In the Calibration tab, click on New Optical Configuration. Name the optical configuration "Image". This is the baseline optical configuration that allows imaging of the coverslip through reflectance. Leave all other options as default and select the appropriate objective.
   2. In the A1plus MP GUI window, click on the Settings button to configure the hardware settings under this optical configuration. Set the Stimulation Laser to IR stim, place the Beam Splitter (BS 20/80) in the light path, set the Scanner Unit to Galvano and select the appropriate descanned detector.
   3. In the A1plus Compact GUI window, select a scan size and dwell time that is sufficient to capture small features on the coverslip(1.1 ms and 1024 pixels on our system, respectively). Click on the Normal button so no line averaging or line integration is performed. Make sure the Use IR Laser box is checked. Set up unidirectional scanning for simplicity.
      NOTE: If scan speed is of concern, bidirectional scanning can be used.
   4. In the same window, adjust laser power and detector sensitivity to obtain bright but not saturated image of the coverslip surface. Setting laser power to 3 - 5% of the maximum power and detector sensitivity ("HV" slider) to 15 V is a good starting point.
   5. In the A1plus Scan Area window, set Zoom to 1 to capture the entire FOV.
   6. Save the optical configuration.
4. Setting up the "Print_Fudiciary_Marker" optical configuration
   1. Duplicate the "Image" optical configuration and rename it "Print_Fudiciary_Marker".
   2. In the A1plus Compact GUI window, select the smallest scan size and dwell time (64 pixels and 80.2 ms on our system, respectively).
   3. Since no imaging is required in this step, set detector sensitivity to 0 and increase laser power to 30%. Higher laser power will thermally remove the PVA layer on the coverslip in the desired regions.
   4. In the A1plus Scan Area window, set Zoom to maximum (15.87 for our system) and place the scan area in the middle of the FOV.
   5. In the ND Acquisition window, set up a Z-stack experiment. Set movement in the Z position to Relative and select the appropriate Z device. Set the step size to 2 µm and the stack depth to 10 µm above and below to account for any unevenness in the microscope stage or the PVA surface between adjacent FOVs.
5. Setting up the "Autofocus" optical configuration
   1. Duplicate the "Image" optical configuration and rename it "Autofocus".
   2. In the A1plus Scan Area window, decrease the Zoom factor so the FOV is slightly larger than the fiduciary marker. This ensures that other small features on the coverslip will not interfere with autofocusing.
   3. In the Devices menu, select Autofocus Set Up. Set the scan thickness to that of the Z-stack in the Z-stack experiment (see step 3.5.5). The microscope will scan through this range and find the best focal plane using the fiduciary marker. Leave the step size as default.
6. Setting up the "Load_ROI" optical configuration
   1. Duplicate the "Image" optical configuration and rename it "Load_ROI".
   2. In the A1plus Compact GUI window, set scan size identical to that of the ROI mask. This optical configuration will be used to capture an image onto which the ROI mask will be loaded. We used 2048 pixels to achieve an optimal balance between resolution and speed.
      NOTE: It is essential that this captured image is identical in size to the ROI mask.
7. Setting up the "Micropattern" optical configuration
   1. Duplicate the "Print_Fiduciary_Marker" optical configuration and rename it "Micropattern".
   2. Set the Zoom factor to one.
   3. In the A1plus MP GUI window, increase the stimulation laser power to ablate PVA and select an appropriate scan speed (40% and 32 s/frame in our experiments, respectively).
   4. In the ND Stimulation window, set up a ND stimulation experiment. Add a few phases to the Time Schedule and set each as Stimulation. Ensure stimulation area and duration are correct.
      NOTE: The number of phases can be adjusted according to the thickness and smoothness of the PVA layer as well as the laser power used in this optical configuration.
   5. In the same window, enable the the StgMoveMainZ(-1.000,1) function before each phase. This again accounts for any deviations in the Z-direction.
      NOTE: The distance and direction in which the objective moves can be adjusted according to the thickness and smoothness of the PVA layer.
8. Setting up the "Label_Surface" optical configuration
   1. Duplicate the "Print_Fudiciary_Marker" optical configuration and rename it "Label_Surface".
   2. In the A1plus Compact GUI window, increase laser power significantly and set Zoom to one. High laser power used here will physically damage the glass coverslip and produce a label visible to the naked eye. This aids in locating the patterns in further experiments.

4. Generating the ROI mask and Setting up the macro

1. Generating the ROI mask
   1. Use Adobe Photoshop or other available software to generate a 2048 × 2048 RGB image. The image corresponds to a FOV under the microscope (i.e., 532.48 × 532.48 µm, 0.26 µm per pixel). The image should have a black (0, 0, 0) background.
      ​NOTE: A number of commercial and free image editors can be used to generate ROI masks for laser assisted micropatterning. Although we use Adobe Photoshop to generate the masks, GIMP and ImageJ/Fiji are also available as alternative free options.
   2. Draw 8 - 12 white (255,255,255) patterns on the black background. Pattern size varies depending on cell type (~200 pixels in diameter). Leave a 500 × 500 blank area in the center of the image for autofocusing. Leave sufficient space between adjacent patterns (>200 pixels) and at the boarder of the FOV for optimal ablation and cell attachment. Patterns presented in this protocol are available on Github (https://github.com/PlotnikovLab/Micropatterning).
2. Setting up the macro
   1. Click on the Macro menu and select New Macro.
   2. Import the "Pattern_Stimulation" code available on Github (https://github.com/PlotnikovLab/Micropatterning) into the New Macro window. Save this piece of code to an appropriate folder.
   3. Open another New Macro window and import the "Stage_Movement" code available on Github (http://github.com/PlotnikovLab/Micropatterning). Ensure that the “Stage_Movement” working directory in this code is identical to that in step 4.2.2. Save the “Stage_Movement” code to the same folder in step 4.2.2.

5. Generating micropatterns using photo ablation

1. Turn on the microscope and its accessories. Ensure that the IR laser has warmed up sufficiently prior to this.
2. Transfer the PVA-coated coverslip onto a holder. For an upright microscope, ensure the PVA surface to be ablated faces down.
3. Add water to the corners to stabilize the coverslip and mount the holder onto the microscope stage.
4. Lower the objective and add water onto the coverslip.
   NOTE: The protocol described here is optimized for a 25x/1.1 NA water immersion objective. If a dry or oil immersion objective is used, water should be replaced with an appropriate immersion medium. When a water immersion objective is used to generate a large micropattern array, evaporation might become an issue. If this is the case, water should be replaced with GenTeal, an over-the-counter eye lubricant available from pharmacies.
5. In the microscope software, turn on the IR laser shutter in the A1plus MP GUI window. Click on the Autoalignment button to align the laser prior to patterning.
   NOTE: It is crucial to perform laser autoalignment at the beginning of each patterning session as small deviations in the laser path will significantly affect the quality of the patterns.
6. Switch to the "Image" optical configuration. In the A1plus Compact GUI window, click Scan to scan the FOV while slowly moving the objective closer to the coverslip.
7. Carefully monitor the image. At first, the image will appear extremely dim. Move the objective closer to the coverslip until the image brightness increases. 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 to be patterned. Focus on any small feature (coverslip imperfections, dust, etc.) on this surface and set zero on the Z drive. Always set zero after focusing.
   NOTE: Although both surfaces of the coverslip are coated with PVA, the optical properties of glass are not significantly altered by PVA coating. We routinely image such coverslips with dry, water- and oil-immersion objectives and did not find the non-patterned PVA surface to interfere with imaging.
8. Switch to the "Label_Surface" optical configuration. Click on Capture. Return to the "imaging" optical configuration and scan. The glass surface should be damaged and appear amorphous. The damaged area is visible to the naked eye, indicating the location of patterns in further experiments.
   NOTE: To increase the size of the visible label, repeat step 5.8 in multiple adjacent FOVs.
9. Check again that the objective is roughly in the center of the coverslip. Ensure that there is sufficient water between the objective and the coverslip. Move the stage by 1 to 2 FOVs to avoid glass particles and scan to confirm.
10. In the Devices menu, open the Stage_Movement macro. Check that the Pattern_Stimulation working directory is correct; if not, stimulation will not occur. Set the variables "PatternLength" and "PatternHeight" to the desired number of FOVs that will be patterned. Save and run the macro.
11. After patterning is complete, switch to the "Imaging" optical configuration. Again, check that the IR laser shutter is open in the A1plus MP GUI window.
12. Move the stage to view the patterns. Scan through the patterns to check their quality.
13. Transfer the coverslip to the grid box, with patterns facing up.
14. Store patterned coverslips at +4 °C for up to one week before use.

6. Fibronectin adsorption

1. Make fresh 1 M NaBH₄ in 1 M NaOH solution. Add at a 1:100 ratio to pH 8.0 phosphate buffer containing 0.2 M ethanolamine.
2. Transfer patterned coverslips to a 35 mm tissue culture dish. Incubate each coverslip with 1 mL of the solution above for 8 min to quench autofluorescence, and then rinse 3 times with PBS.
3. Dilute fibronectin (FN) in PBS to a final concentration of 10 μg/mL. Incubate the coverslip in FN for 1 h at +37 °C.
   NOTE: If substantial nonspecific binding of ECM protein to the substrate is observed, FN can be diluted in PBS containing 0.1% Pluronic F-127²⁴.
4. Wash the coverslip 2 times with PBS. If not immediately used, store in PBS at +4 °C overnight.

7. Cell attachment

NOTE: The following protocol is optimized for primary human gingival fibroblasts.

1. Culture a 10 cm dish of cells to 70% confluency.
2. Warm up cell culture media, PBS and 0.05% trypsin in a +37 °C water bath.
   NOTE: For cells that adhere weakly to the substrate, non-proteolytic dissociation with versene (0.48 mM EDTA) or a proprietary enzyme-free buffer may increase cell attachment to the patterns and should be considered.
3. Prior to seeding cells, relocate the patterned coverslip to a clean 35 mm tissue culture dish containing 1 mL warm PBS.
4. Aspirate the cell culture media from the 10 cm tissue culture dish and wash once with PBS.
5. Add 700 µL of 0.05% trypsin/EDTA to the dish and incubate cells in a +37 °C, 5% CO₂ incubator for 1 min.
6. In the meantime, aspirate the PBS covering the patterned coverslip and add 1 mL of cell culture media.
7. Confirm that cells have detached. Then resuspend the trypsinized cells with 10 mL of cell culture media and add 1 mL to the patterned coverslip.
8. Culture cells in the incubator for 2 - 3 h and check if a sufficient number of cells have attached to the patterns. If so, change media once to remove unattached cells. This minimizes the chance of multiple cells landing on the same pattern.
   NOTE: Attachment time may vary depending on cell type.
9. After another 3-4 h, or when a sufficient number of cells have spread on patterns, cells are ready for further experiments. Do not wait for too long to avoid cell division.

8. Data acquisition

1. Fix cells with 4% PFA in cytoskeleton buffer²⁷ for 10 min at room temperature.
2. Follow immunofluorescence protocols established for the proteins of interest. PVA-coated coverslips work well with any immunofluorescence protocol.
3. Acquire images of cells using an appropriate microscope. Depending on the goal of the experiment, image either one or multiple cells per FOV.

9. Image analysis

NOTE: The following protocol allows users to obtain the average fluorescence signal of the protein of interest over a large number of cells from Z-stacks of microscope images.

1. Open the acquired images in NIS Elements and crop the images. Ensure that each image contains only one well-spread cell. Detailed instructions on image processing can be found in the script below.
2. Install Anaconda and launch Spyder through Anaconda Navigator.
   NOTE: The .py scripts can be run in any environment with the appropriate packages identified in the requirements.txt. We recommend Anaconda because it contains most of the required packages for the codes below.
3. Download the script from Github (https://github.com/PlotnikovLab/Micropatterning) and open in Spyder (“Pattern_Averaging_3Channels.py” or “Pattern_Averaging_4Channels.py” for images with 3 Channels or 4 Channels, respectively).
4. Set parameters based on the acquired images. See the script for detailed descriptions.
5. Press F5 to run the script. The progress can be tracked in the Console panel.
6. Retrieve the output files saved in the same folder. The output images are the average of each channel for all samples. The excel sheet shows the mean intensity of each channel within a sample cell, which enables further quantitative analysis.

Representative Results

The quality of the experimental data obtained through 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 and sizes can be printed depending on the desired cytoskeleton architecture, but we used the crossbow shape that best suits our purposes. Atomic force microscopy (AFM) revealed that such patterns were approximately 140 nm in height and had a smooth surface with minimal topological variation throughout (Figure 2F). Suboptimal patterning settings, such as low laser power and incorrect focal plane, resulted in incomplete removal of the PVA surface that manifests as darker, partial patterns with uneven topology (Figure 2C). Setting laser power too high resulted in PVA "bubbling" and when extreme, coverslip surface damage that is also undesirable (Figure 2D).

In preliminary experiments we attempted to increase the patterning area by abating multiple FOVs on a single coverslip. We found that this approach, although works in principle, is unreliable and tedious due to Z-drift of the microscope stand and slight tilt of the coverslip. To achieve high-quality patterns over a large number of FOVs in an automated fashion, we implemented a customized macro that improved the precision of microscope focusing during the patterning process. The multiphoton microscope employs a pulse laser that efficiently degrades PVA with high power in a narrow focal plane, making the patterning process sensitive to any unevenness or tilt in the sample. As a result, it is important to identify the precise focal plane in each FOV. This is even more problematic for systems lacking a perfect focus module, as deviations as little as one to two microns can render the patterning process fruitless. To address this problem, the customized macro script first patterns a small square in the center of each new FOV that need only be roughly in focus by scanning through a relatively thick Z-stack (≈20 μm). The microscope then quickly scans through the stack of images and uses NIS Elements autofocus function to identify the optimal focal plane. The pattern mask is then loaded and set as stimulation ROI for IR stimulation to occur. The stage subsequently moves to the next FOV and repeats this process. In addition, the square-wave shaped path of the microscope stage movement ensured minimal error accumulation between sequential FOVs. By using this protocol, we routinely fabricate patterns composed of 49 microscope FOVs covering 3.5 x 3.5 mm area of the coverslip in less than 3 h.

To test if patterned areas, not unperturbed PVA, could adsorb ECM proteins, we coated patterned coverslips with 10 μg/mL FN and stained them with anti-FN antibody. Using wide field fluorescent microscopy, we found that FN uniformly adsorbed to the patterned areas where PVA had been removed by laser ablation (Figure 2E).

To determine if cytoskeleton architecture and tension distribution could be modified as expected on the patterns, we seeded cells on patterned coverslips and visualized the distribution of myosin light chain, a marker of contractility, through fluorescence microscopy. After initial seeding, cells gradually gravitated towards the coverslip. Those that landed on patterned areas attached and spread into the shape of the pattern over time. Those that landed on PVA only loosely attached and were removed after several washes and media changes. We found that cells spread on patterns displayed phenotypical fibroblast structures including an actin-dense rim of lamellipodia, thick ventral stress fibers along the two sides, and dorsal stress fibers emanating from the lamellipodia connected by transverse arcs (Figure 3A). Myosin light chain (MLC) sat behind the dense lamellipodia rim and displayed a striated pattern along actin bundles. As indicated by averaged images of many cells, this phenotype was consistent across a large number of patterns (Figure 3B).

Figure 1. Schematic of IR laser assisted micropatterning (microphotopatterning). (A) Glass coverslips are chemically conjugated with APTMS, GA and PVA, in the respective order. The PVA surface is non-adhesive to cells and proteins. (B) PVA is removed in preset patterns by an IR laser. (C) Patterned coverslips are coated with an ECM protein that will only adsorb to patterned areas. (D) Cells are plated on coverslips, fixed, and immunostained for proteins of interest. Cells that land on patterned islands spread into the shape of the pattern that gives rise to characteristic cytoskeletal structures, while those that land on PVA remain spherical. Please click here to view a larger version of this figure.

Figure 2. Micropattern validation and characterization. (A) IR laser assisted micropatterning (microphotopatterning) setup on a microscope stage. The IR laser thermally ablates PVA in multiple FOVs. (B) A reflectance microscopy image of micropatterns that have clear boarders and are identical to the ROI masks. (C) A reflectance microscopy image of incomplete removal of PVA resulting from suboptimal laser power. (D) A reflectance microscopy image of PVA bubbling resulting from excess laser power. (E) Immunofluorescence images of FN coated patterned coverslips stained with anti-FN primary antibodies and fluorophore-conjugated secondary antibodies. (F) An AFM topology scan line and a representative AFM image of a crossbow pattern. To measure the topology of the micropattern, contact mode imaging was performed using a Bruker AFM probe (MLCT-B) mounted on a NanoWizard 4 atomic force microscope. Please click here to view a larger version of this figure.

Figure 3. Representative images of cells plated on micropatterns. (A) Representative images of a primary human gingival fibroblast plated on crossbow patterns immunostained for actin and MLC. Images were acquired with a confocal microscope equipped with a 100x objective. (B) Averaged immunofluorescence images of actin and MLC in cells plated on crossbow patterns generated by a custom Python script. Please click here to view a larger version of this figure.

Discussion

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 departments. In fact, as multiphoton microscopes are becoming a more common sight in Biology departments, microphotopatterning expands the applications of the multiphoton microscope and adds to the potential pool of users. Patterns can also be changed on demand and printed immediately, instead of having to fabricate a new master wafer that entails a lengthy lithography process.

Compared to the pre-existing microphotopatterning protocol, one improvement in our protocol is the elimination of several time-consuming curing steps during coverslip preparation. We show that the quality of PVA-coverslip attachment remains unperturbed as the patterns were still intact and could bind cells even two weeks after fabrication. More importantly, we improved automation of the patterning process by eliminating the need to preset the position of each FOV²⁴. Instead, we implement an understandable macro that allows patterning of a large area while precisely identifying the optimal focal plane of each FOV.

Although the macros in the protocol enable automation of patterning on our system, we understand that every commercial laser scanning microscope comes with their own proprietary software that is rarely compatible with others, making it difficult to implement our exact protocol on other systems. However, the overall workflow can be well adapted to other commercial systems to facilitate automated micropatterning, namely the process of focusing on each individual FOV, loading the mask, ablating PVA, and moving the microscope stage to a new FOV.

Several steps in the patterning protocol should be undertaken with great care to ensure efficient patterning. The most critical step is to optimize stimulation conditions before generating patterns. In multi-photon microscopy, two or more photons must arrive almost simultaneously at a fluorescent molecule and combine their energy to excite fluorescence. This low probability event creates an extremely thin optical section that increases signal-to-noise ratio²⁸. Although beneficial for imaging, this feature makes the removal of the thin PVA coating extremely sensitive to the sample's Z-position. Several measures can be implemented to counter this. First, laser power should be fine-tuned to ensure thorough removal of PVA without "boiling" the polymer or damaging the glass coverslip. If PVA removal is consistently incomplete, we recommend checking laser alignment as this usually increases laser power. Second, the microscope stage should be leveled to avoid sample tilt, which could result in incomplete patterns. IR stimulations in multiple Z-positions should also be set up to ensure that the PVA layer is targeted. Alternatively, if the microscope is equipped with a perfect focus module, preliminary tests can be conducted to determine the optimal offset in Z-position for PVA targeting. Another critical step is to set up a microscope macro that allows pattern stimulation in an automated fashion. The macro should find the focal plane of each new FOV to avoid complications from sample tilt or surface unevenness. It should also allow the stage to move in an S-shape from row to row, analogous to the path taken in bi-directional scanning, to minimize deviations in Z between consecutive FOVs.

One limitation to the protocol described is the time required to produce a large number of patterned coverslips, an unparalleled advantage offered by lithography-based microcontact printing^29,30. As a result, this protocol is best suited for experiments in which a limited number of conditions are required, or those that require readily available adjustments in pattern shape and size. Furthermore, for systems lacking a hardware autofocus module, we integrate a series of stimulation events in different Z-planes to ensure automated and effective PVA removal. Since IR stimulation is the most time-consuming step, the addition of each stimulation event (~30 sec) significantly lengthens the patterning process. If time is of concern, we suggest fine tuning autofocus by decreasing step size. This facilitates the identification of the best focal plane which will decrease the number of IR stimulation events required. In our experiments, decreasing the number of stimulation events from five to two reduces the time by half (1.5 h).

In conclusion, the IR laser assisted micropatterning (microphotopatterning) protocol we describe can be used in any lab that has access to an IR laser-equipped microscope. In addition to studying cytoskeletal architecture and signaling pathways that connect form to function, this technique can also be applied to drug screening and other applications that are sensitive to cell-to-cell variability.

Materials

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