Static Atomic Force Microscopy to Visualize and Characterize Assembled Nucleosomes

Nucleosomes, the basic repeating unit of eukaryotic chromatin, comprise DNA turns wrapped around a core of histone proteins.

To visualize assembled nucleosomes by static atomic force microscopy, AFM, begin with a thin mica strip, functionalized to render the surface positively-charged. Cut the strip into small squares. Pipette an assembled nucleosome suspension.

The negatively-charged DNA in the nucleosome binds to the functionalized mica strip's positively-charged group through electrostatic interactions. Wash with buffer to prevent nucleosome overcrowding.

Secure the mica strip on a specimen support disc. Mount it on the AFM instrument stage.

Attach the probe holder to the instrument's optical head.  The holder contains a pre-mounted AFM cantilever with a tip at its apex.

Align the laser beam onto the cantilever back for accurate deflection measurement. Position the tip in direct contact with the nucleosome-containing surface.  

During contact mode imaging, as the cantilever tip scans across the nucleosome surface, repulsive forces arise following interactions between atoms of the tip and sample. This causes the cantilever to bend. The laser beam is reflected differently and directed to the position-sensitive photodetector.

The feedback loop maintains constant cantilever deflection by vertical scanner movement during the scan. This feedback signal is further used to generate topographic nucleosome images. The nucleosome core appears as bright blobs, with thin arms representing the flanking DNA.

Prepare the mica surface for static AFM imaging. To do this, first prepare a 50-millimolar APS stock solution in deionized water. Store 1 milliliter aliquots of the solution at 4 degrees Celsius, until it is needed.

From the stock solution, prepare a working APS solution for mica modification by dissolving 50 microliters of the 50-millimolar APS stock in 15 milliliters of distilled deionized water. Mix the solution and then, fill a cuvette with the solution.

Next, cut 1 by 3-centimeter strips of mica from high-quality mica sheets. Check that the piece fits when place diagonally in a cuvette. Then, cleave layers of the mica until both sides are freshly cleaved and the piece is as thin as 0.1 millimeter.

Immediately place the mica piece into the APS-filled cuvette, and incubate the mica for 30 minutes. Transfer the mica piece to a cuvette filled with distilled deionized water and soak it for 30 seconds. Then, use argon to completely dry both sides of the APS-mica strip.

Apply double-faced adhesive tape to several magnetic pucks, and place them to the side. Then, cut the APS-mica substrate to 1-centimeter by 1-centimeter squares and cover them in a clean Petri dish. Next, prepare three dilutions of the assembled nucleosomes using a 0.22-micron filtered buffer containing 10-millimolar HEPES and 4-millimolar magnesium chloride at a pH of 7.5.

To limit the loss of nucleosomes at the low final concentration, the dilution should be done one at a time, immediately prior to deposition on the APS-mica.

Deposit 5 to 10 microliters of each nucleosome sample at the center of an APS-mica piece, and let them incubate for 2 minutes. Then, gently rinse the sample with 2 to 3 milliliters of distilled deionized water to remove the buffer components, and dry the deposited sample under a light flow of argon.

To begin, mount a tip on the tip holder of the AFM setup. Then, mount the first sample on the AFM stage, being careful not to contact the sample surface. Position the laser over the cantilever until its sum is at the maximum and adjust the vertical and lateral deflection values to near 0. Then, tune the AFM probe to find its resonance frequency. Adjust the drive amplitude, and set the image size to 100 by 100 nanometers. Once set up, click the Engage button to begin the approach.

When the approach is complete, gradually optimize the amplitude set point until the surface of the sample is clearly seen. Then, increase the scan size to 1 micron by 1 micron and the resolution to 512 by 512 pixels. Finally, click the Capture button to begin image acquisition.