Interference Reflection Microscopy for Label-Free Visualization of Microtubule Dynamics

Interference reflection microscopy enables imaging microtubules that dynamically grow and shrink.

Fix paraffin strips on one coverslip and place a slightly smaller coverslip on top. Heat to melt the paraffin, forming a sealed channel. Pipette an antibody-containing solution through the channel. The antibodies attach to the coverslip surface. Add a blocking solution, blocking the antibody-unbound regions and preventing non-specific protein binding.

Place the coverslip on the microscope stage. Set the sample heater to a temperature facilitating microtubule growth.

Pipette microtubule seeds stabilized with a non-hydrolyzable guanosine triphosphate, GTP, analog. The seeds bind to the antibody-coated coverslip. Add a buffer containing unlabeled tubulin, GTP, and reducing agents.

At optimum temperature and buffer conditions, the seeds act as nucleation sites, allowing unlabeled tubulin binding and microtubule growth.

Begin imaging. The incident light focuses through an aperture diaphragm onto a beam splitter that partially reflects light to the objective, illuminating the sample. The coverslip's glass-buffer interface partially reflects the incident light. The remaining incident light passes through and gets reflected at the buffer-microtubule interfaces.

Based on the microtubule-coverslip distance, the reflected beams from the two interfaces interfere, producing constructive interference — beams combining to produce a bright signal, or destructive interference — beams canceling to produce a dark signal.

Visualize the microtubules as high-contrast images. Capture time-lapse images, detecting microtubule growth and shrinkage.

To begin, insert a 50/50 mirror into the filter wheel of a fluorescent microscope using an appropriate filter cube. Handle the mirror with care as often they have an anti-reflection coating. Turn to a high magnification objective that also has a high numerical aperture. The one shown here is a 100x oil objective with a numerical aperture of 1.3.

Next, use a razor blade and a microscope slide as a straight edge to cut 3-millimeter-wide strips of plastic paraffin film. Place two of the plastic paraffin film strips 3 millimeters apart on a clean 22 by 22-millimeter coverslip. Then, place an 18 by 18-millimeter coverslip on top of the strips to form a channel.

Transfer the coverslip to a heat block at 100 degrees Celsius for 10 to 30 seconds for the paraffin film to form a sealed channel. Using a pipette, flow in 50 micrograms per milliliter of an anti-rhodamine antibody by perfusion, and incubate the slide for 10 minutes.

Following incubation, wash the channel five times using filtered BRB80. Then, flow in 1% poloxamer 407 in the filtered BRB80 to block the surface against nonspecific binding, and incubate the slide for 10 minutes. Again, wash the channel five times using filtered BRB80.

To prevent the sample from drying out, add two droplets of the filtered BRB80 at the ends of the channel and add more buffer as needed. Place the sample on the microscope stage and turn on the epi-illumination light source. Focus on the paraffin film edge to find the sample surface, and then, move the field to set the view to the center of the chamber. You will observe multiple surfaces as the objective is moved up and down due to back reflection of light from optics within the optical path.

Next, center the field diaphragm in the field of view by closing it halfway and using the adjustment screws Once the diaphragm is properly aligned, reopen it. Then, slide in the Bertrand lens to view the back focal plane, also known as the exit pupil of the objective.

Close the aperture diaphragm beyond the edges of the exit pupil, and use the adjustment screws to center the aperture diaphragm with respect to the exit pupil. Double-check by opening the aperture diaphragm and matching its edges with those of the exit pupil. Then, set the aperture diaphragm to about 2/3rds of the numerical aperture of the objective.

To image microtubule dynamics using brain tubulin, start by setting the sample heater temperature to 37 degrees Celsius. Using a pipette, flow in 10 microliters of GMPCPP-stabilized microtubule seeds, and monitor them binding to the surface by imaging the surface live. Once 10 to 20 seeds are bound within the field of view, wash the sample using twice the channel volume of prewarmed and filtered BRB80.

Next, flow in 10 microliters of the polymerization mix.

To measure microtubule growth, set up a time-lapse using the acquisition software to acquire an image every 5 seconds for 15 minutes. Enhance the contrast by acquiring an average image of 10 at each time point. Acquire background images as shown in the previous section. Calculate the median by going to Image, selecting Stack, then Z Project, then Median. Subtract the corresponding background by going to Process, going to Image Calculator, and choosing Subtract from the dropdown menu. Make sure the 32-bit float result option is checked.

For microtubule shrinkage, acquire images at 100 frames per second by setting the time delay to zero and keeping the exposure time at 10 milliseconds.