Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Traction force microscopy is used to measure cell generated forces. This protocol simplifies the collection analysis of traction force data, in order to make it more accessible to inexperienced users. The main advantage of this technique over existing reference-free traction force microscopy platforms, is that multiphoton lithography allows a quick and easy patterning setting redesign according to individual experimental needs.

There are many key steps in the fabrication and implementation of this new reference-free traction platform that are easier to understand through visual representation rather than text alone. For photopolymerization of the base hydrogel, first add three microliters of the pre-polymer solution onto a thin, flat sheet of perfluoroalkoxy alkanes, and place flat 150 micrometer thick PDMS strips surrounding, but not in contact with, the drop of pre-polymer solution. Place an acrylate silanized coverslip on the PDMS, with the pre-polymer droplet centered under the coverslip to flatten the pre-polymer droplet to the thickness of the PDMS spacers.

Expose the sandwiched pre-polymer solution to UV light for about one minute. When a hydrogel is fully formed, carefully separate the coverslip from the PDMS spacers. Use high performance double-sided acrylic adhesive to attach an open bottom Petri dish to the coverslip.

Take special care when adhering the hydrogel-laden coverslip to the Petri dish. A wet dish or too little pressure during the application will allow leaks to form, ruining the sample. Apply pressure to the adhesive contact surface to create a complete seal between the coverslip, adhesive, and Petri dish, taking care not to crack the glass.

Use sterile filtered PBS to rinse the hydrogel. Then add eight microliters of NVP to 800 microliters of the lab solution prepared for the hydrogel synthesis. To convert a binary image to a digital mask, open MATLAB and open and run the runscript.

m MATLAB script. Select the binary TIF file for conversion and select a folder to save the region's files. Input the desired final size in microns of the selected image.

The input image will be scaled and dimensioned to match these parameters. To create a single pixel array, in the Region of Interest Generator Options, uncheck the Remove tic box under Small Regions/Single Pixels, check the Squares tic box and uncheck Use under Horizontal Break Lines. Then, click OK.The OVL file will be found in the previously specified folder.

Open the file in the microscope software and load the desired regions that controlled the laser shutter during two photon laser scanning lithography. The following steps should be performed in conditions with minimal light. For fabrication of fiducial marker arrays under low light conditions, thoroughly mix 200 microliters of the previously prepared NVP lab solution with 20 milligrams of Alexa Fluor 633 and remove all of the PBS from the dish containing the hydrogel.

Add the PEG-633 mixture to the hydrogel as a droplet that completely encompasses the base hydrogel and load the Petri dish onto the sample holder of the microscope stage. Then cover the dish and allow the patterning solution to soak into the hydrogel for at least 30 minutes, protected from light. To configure the microscope for imaging, select the appropriate lasers and filters for visualizing Alexa Fluor 488.

Locate the hydrogel using the PEG-488 signal and block the collection of longer emission wavelengths from the detector. Use vertical and horizontal tile scans to locate the center of the hydrogel in the XY plane and zero the stage in this position. Use line scans in the Z-stack function to locate the surface of the hydrogel and zero the focus in this position.

Level the hydrogel by repeating the line scans away from the XY center to identify the surface position, adjusting the set screws for the microscope stage, as necessary. Within the microscope software workspace, create a separate experiment file for photopatterning and set the multi-photon power to 1.8%and the scanning speed to six. Adjust the size of the image frame and pixels to achieve a pixel size of 0.1 micrometer per pixel and an aspect ratio of 100 to one and load the regions file into the region's tab.

Use a macro to set all of the regions to acquire and turn on the Z-stack function. Set the spacing to 3.5 micrometers for a total depth of 28 micrometers and total number of Z slices of nine. Then use the Positions function to set specific locations on the hydrogel where the fiducial marker arrays will be photopatterned.

As the soak time approaches the 30 minute mark, acquire successive Z-stack line scans of the surface of the hydrogel every five minutes to check for swelling based on changes in the location of the surface relative to the zeroed focus position. If no change in the surface position has occurred over the five minute interval, run the patterning settings and use the 488 nanometer laser to verify that the surface of the hydrogel did not move during the patterning. Then remove the hydrogel from the microscope, aspirate the PEG-633 solution from the Petri dish, and rinse the dish with sterile filtered PBS.

To acquire a cell and fiducial marker images, return the Petri dish to the sample holder to locate the patterned areas. Locate a cell of interest and acquire a transmitted or fluorescent image of the cell. Then acquire a Z-stack of the patterned array beneath the cell as demonstrated, and acquire a second set of transmitted or fluorescent images of the cell.

When collecting an image stack, the resulting images should display a regular array of patterned features that oscillate in intensity as a function of the Z position within the image stack. The tracking. m script provides several diagnostic images, including a Z-projection of the fluorescent fiducial markers to assess the pre-processing quality, a plot of the detected centroids, color coded as a function of the Z position to assess the object detection quality, and a plot of the tracks representing the detected marker centroids that have been linked into columns in the Z direction to assess the object tracking quality.

The DISP 3D. m script provides a plot of intensity as a function of the Z position for each column of detected features in a given image stack to assess the quality of the fiducial marker intensity profiles. Both DISP 3D.

m and DISP shear. m together provide histograms of the measured displacement noise in each of the Cartesian coordinate dimensions as well as interpolated heat maps of displacement. In addition, the interp final3D_2.

m provides heat maps of the surface tractions calculated using the outsourced code. The most important thing to remember during this procedure is that solutions containing LAP are sensitive to light and should be protected as much as possible from ambient light sources. Remember that NVP is a volatile organic compound and should always be handled in a chemical flow hood.