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.
