High-Speed Time-Lapse Atomic Force Microscopy to Capture Nucleosome Dynamics

Nucleosomes — eukaryotic DNA repeating units — consist of DNA segments wrapped around histone protein cores. DNA unwrapping around the histone core is crucial for nucleosome dynamics.

To capture nucleosome dynamics, begin with a smooth, functionalized mica substrate mounted to an atomic force microscope, AFM, scanner stage. Overlay the substrate with a nucleosome-containing buffer.

The negatively charged nucleosome DNA interacts with positive charges on the functionalized mica, immobilizing them on the surface. Wash the immobilized mica substrate with a buffer to prevent the nucleosomes overcrowding.

Transfer the nucleosome-containing scanner stage to an imaging chamber containing a pre-mounted cantilever, with a tip at the edge and facing up toward the nucleosomes.

Fill the chamber with an imaging buffer. Align the laser beam onto the cantilever back to ensure accurate deflection measurements.

The AFM tip oscillates close to the fully wrapped nucleosomes and scans over the surface to measure the nucleosomes' height at regular intervals.

Spontaneous DNA unwrapping increases nucleosome height, alternating the tip's oscillations and leading to cantilever deflection. This cantilever deflection shifts the reflected laser beam position, which is detected by a position-sensitive detector and produces topographic nucleosome images.

In AFM images, the histone core appears as a glowing blob with unwrapped DNA of increasing heights over time, indicative of nucleosome dynamics.

Use glass rod-scanner glue to attach a glass rod to the AFM scanner stage. Allow this to dry for a minimum of 10 minutes. In the meantime, make 0.1-millimeter thick circular pieces of mica with a 1.5-millimeter diameter by punching them from a larger mica sheet. Use the high-speed-AFM mica-glass rod glue to attach this mica piece to the glass rod on the high-speed-AFM, and allow it to remain untouched for a minimum of 10 minutes while it dries.

Then, cleave layers from the mica using a pressure-sensitive tape until a well-cleaved layer is seen on the tape. Next, dilute 1 microliter of 50-millimolar APS stock in 99 microliters of distilled deionized water to make a 500-micromolar APS solution. Deposit 2.5 microliters of the solution on the freshly cleaved mica surface, and let the surface functionalize for 30 minutes.

Following the incubation, rinse the mica several times with distilled deionized water by applying 3-microliter rinses. Remove the water completely following each rinse by placing a non-woven wipe at the edge of the mica. After the final rinse, place 3 microliters of distilled deionized water on the surface, and let it sit for a minimum of 5 minutes to remove any non-specifically bound APS.

Next, place the probe in the high-speed-AFM holder, and position the holder on the AFM stage with the tip facing up. Rinse the holder using 100 microliters of distilled deionized water, followed by two 100-microliter rinses of 0.22-micron filtered nucleosome-imaging buffer. With the rinses done, fill the chamber with 100 microliters of nucleosome-imaging buffer, submerging the tip.

Adjust the cantilever position until it is hit with the laser. Then, rinse the APS-mica five times with filtered nucleosome-imaging buffer, using 4 microliters per rinse. Dilute 1 microliter of the nucleosome assembly stock into 250 microliters of filtered nucleosome-imaging buffer for a final nucleosome concentration of 1 nanomolar.

Deposit 2.5 microliters of this dilution on the surface, and let it sit for 2 minutes. Rinse the surface twice with 4 microliters of nucleosome-imaging buffer to avoid overcrowding. After the final rinse, leave the surface covered in imaging buffer. Set the scanner and sample on top of the tip holder so that the sample is face down.

To begin the approach, use the auto-approach function with a set point amplitude close to the free oscillation amplitude. Adjust the set point until the surface is being well-tracked. Then, set the image area around 150 by 150 nanometers to 200 by 200 nanometers with a data acquisition rate of around 300 milliseconds per imaging frame.