Encyclopedia of Experiments: Biological Techniques
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Transcript
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.
