The significance of this method is that it allows label-free microtubules to the imaged simultaneously with fluorescently labeled proteins at high frame rates. The main advantage of this technique is its easy implementation and minimal demands on optical components. It also circumvents the need for fluoro-4 labeling of microtubules.
To begin, prepare the PDMS polymer by combining the curing agent and the base elastomer in a 1:10 mass ratio. Mix them for two minutes and then degas the mixture in a vacuum chamber until all bubbles disappear. Afterward, pour the mixture onto the master mold in an approximately 0.5-centimeter-thick layer, taking care to avoid creating bubbles, and bake the mixture in a preheated oven at 70 degrees Celsius for 40 minutes.
Then, cut out the structured regions of the polymer and punch holes at each end of the channel using a PDMS puncher. Later, clean the structured side of the PDMS block. Subsequently, rinse thrice with isopropanol and methanol followed by ultrapure water and blow dry the surface.
Next, plasma clean the PDMS using oxygen or air plasma and place the plasma-cleaned PDMS on an appropriately cleaned cover glass. Heat it a hot plate at 80 degrees Celsius for 15 minutes. Insert appropriately sized low-density polyethylene tubing into the holes and connect the outlet tubing to a 0.5-milliliter syringe.
Then, flow the solutions into the microchannels by immersing the inlet tube in the solution and drawing the required volume with the syringe. Place the sample on the microscope stage and turn on the epi-illumination light source for IRM imaging. To focus the microscope, look for the correct focal plane near the PDMS solution interface and pick a field-of-view near the center of the channel.
Next, prepare 0.1-micromolar solution of fluorescent microbeads in BRB80 and flow in at least one channel volume of the microbead solution. Monitor the reaction via IRM or TIRF. When the desired density of microbeads is achieved, wash out the excess with BRB80.
Flow diluted seeds into the reaction chamber and monitor the reaction through IRM. When the desired density of seeds is achieved, wash out the excess with BRB80. Next, prepare a microtubule extension mixture containing 12-micromolar unlabeled tubulin, one-millimolar GTP, five-millimolar DTT in BRB80 buffer.
Then, flow in at least one channel volume of the extension mixture while ensuring that the reaction temperature is 28 degrees Celsius. To begin, set the imaging settings on the microscope software. Start the imaging and flow the kinesin solution into the chamber.
Then, visualize the GFP labeled kinesin 1 unshrinking microtubules. After the measurement is complete, record a short video in which the stage is slowly translated in a circular or lateral motion. The median projection of this video will serve as a background image and will be subtracted from the raw measurements.
Create a median projection in ImageJ from the background recording by clicking on Image. Then, select the option Stacks and click on Z Project. Next, subtract the median background projection from the raw image data by clicking on Process and then select the option Image Calculator while ensuring to check the 32-bit float result option.
For image registration, pick a collection of microbeads near the microtubule of interest and use them to align the TIRF and IRM images. For each bead in this collection, use the multipoint selection tool to mark the approximate location in the TIRF channel and then the corresponding location in the IRM channel. Then, run the provided ImageJ macro.
Microbeads appear as dark spots in the IRM channel on the right and as bright spots in the TIRF channel on the left. The alignment procedure correctly overlays the two channels by localizing a selection of beads. A kymograph of EGFP-labeled kinesin molecules walking towards the shrinking ends of microtubules was also obtained using this protocol.
When performing this procedure, it's important to choose high numerical aperture objectives in order to achieve total internal reflection and also to maximize the collection efficiency and image contrast. This method can be used for high-resolution imaging of single fluorescent molecules simultaneously within the macromolecular structure massive enough to be visualized by IRM, such as cell membrane or actin filament. This protocol also provides an easily adaptable procedure for simultaneous imaging with any combination of IRM, TIRF, and epifluorescence.
