Imaging Polysomes Isolated from a Mouse Brain Using Atomic Force Microscopy

Begin with a mica sheet with adhered polysomes isolated from mouse brain tissue.

A polysome is a complex of multiple ribosomes attached to an RNA strand.

Using tape, secure the mica sheet to the sample holder.

Place the sample holder into the atomic force microscope stage.

Position the premounted cantilever. Align the laser and detector to ensure accurate signal detection during imaging.

Adjust the oscillation frequency of the cantilever to optimize the detection of surface features while preserving sample integrity.

Begin imaging by oscillating the cantilever tip over the sample surface.

As the tip encounters an elevated surface of the polysome, the laser is deflected.

These deflections of the laser are captured by the detector, which generates an image with nanoscale resolution.

Use appropriate software to correct arbitrary tilt and drifting effects, enabling the visualization of the polysome.

Attach the prepared sample to the sample holder of the AFM using double-sided tape. Then, insert the sample holder in the AFM stage in accordance with the manufacturer's instructions. Following calibration, approach the sample until the cantilever tip engages the surface.

Select a scan area of 2 by 2 microns, a resolution of at least 512 by 512 pixels. Choose live background subtraction mode and select a Z-scale of 20 to 25 nanometers. Then, begin image acquisition. Inspect the image, looking for the presence of round objects characterized by a height between 10 and 15 nanometers when acquiring images in the air.

Next, adjust the set point and feedback parameters until sharp objects are visualized. The background should appear relatively flat in good samples with some 2 to 4 nanometer high objects. Once the image looks good, acquire several 2 by 2 micron scans at different sample areas.