2.4 AFM imaging and data processing
AFM images were acquired in tapping mode at room temperature in air using a TT-AFM (manufactured by AFM Workshop) equipped with long cantilevers (AppNano ACLA, tip radius < 10 nm). In a typical experiment, 256 pixel x 256 pixel images were collected from a 1 µm x 1 µm area and scanned at a line rate of 0.7 Hz. The AFM was initially calibrated with a square grid step height standard (AppNano SHS-0.1). For work involving 560 bp DNA, a cross line grating standard was subsequently used for calibration (Ted Pella 677-AFM, line spacing 500 nm ± 1%).
All images were analyzed using the open-source software package Gwyddion, which is designed for scanning probe microscopy data visualization and analysis.21 AFM images were processed by plane leveling, removal of background using a polynomial in the x and y directions, alignment of rows using the median, and 1D FFT filtering, as needed, to remove some of the high frequency electronic noise.
In our experiments, the DNA heights were 0.1-.45 nm above background, often resulting in low signal-to-noise. In a study that used a tip with a similar spring constant to ours, the mean height of DNA was 0.6-0.8 nm.22 It is possible that our tip deformed the DNA. An area of exploration is to choose a cantilever with a smaller spring constant, as suggested by Asylum Research. DNA molecules could also be embedded in a layer comprised of water and salts on the mica surface leading to a short height.22 The average surface roughness of a mica surface treated with 10 mM NiCl2 is .3 nm peak-to-peak.20
Many articles do not state what is in the DNA and protein stock solutions used to prepare the solutions for deposition onto mica. A long period of experimentation is possibly needed to develop a suitable sample preparation protocol for first-time AFM studies of DNA and proteins. It is useful to have a readily accessible AFM for the protocol development stage.