Ultrastable Atomic Force Microscopy for Biophysics

Churnside, Allison Beth

Graduate Thesis Or Dissertation

Ultrastable Atomic Force Microscopy for Biophysics Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/9019s247x

Abstract

Atomic force microscopy (AFM) is a multifunctional workhorse of nanoscience and molecular biophysics, but instrumental drift remains a critical issue that limits the precision and duration of experiments. We have significantly reduced the two most important types of drift: in position and in force. The first, position drift, is defined as uncontrolled motion between the tip and the sample, which occurs in all three dimensions. By scattering a laser off the apex of a commercial AFM tip, we locally measured and thereby actively controlled its three-dimensional position above a sample surface to <0.4Å(Δf = 0.01-10 Hz) in air at room temperature. With this enhanced stability, we demonstrated atomic-scale (~1 Å) tip-sample stability and registration over tens of minutes with a series of AFM images. The second type of drift is force drift. We found that the primary source of force drift for a popular class of soft cantilevers is their gold coating, even though they are coated on both sides to minimize drift. When the gold coating was removed through a simple chemical etch, this drift in deflection was reduced by more than an order of magnitude over the first 2 hours after wetting the tip. Removing the gold also led to ~10-fold reduction in reflected light, yet short-term (0.1-10 s) force precision improved. With both position and force drift greatly reduced, the utility of the AFM is enhanced. These improvements led to several new AFM abilities, including a five-fold increase in the image signal-to-noise ratio; tip-registered, label-free optical imaging; registered tip return to a particular point on the sample; and dual-detection force spectroscopy, which enables a new extension clamp mode. We have applied these abilities to folding of both membrane and soluble proteins. In principle, the techniques we describe can be fully incorporated into many types of scanning probe microscopy, making this work a general improvement to scanning probe techniques.

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