Static Atomic Force Microscopy to Visualize and Characterize Assembled Nucleosomes
Transcrição
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.
