This assay protocol outlines the procedures for sample preparation of gold and the determination of nanoparticle size using atomic force microscopy (AFM). An AFM utilizes a cantilever with a sharp probe to scan a specimen surface. The cantilever beam is attached at one end to a piezoelectric displacement actuator controlled by the AFM. At the other end of the cantilever is the probe tip that interacts with the surface. At close proximity to the surface, the probe experiences a force (attractive or repulsive) due to surface interactions, which imposes a bending moment on the cantilever. In response to this moment, the cantilever deflects, and this deflection is measured using a laser beam that is reflected from a mirrored surface on the back side of the cantilever onto a split photodiode. A schematic diagram of the system is shown in Figure 1. The cantilever deflection is measured by the differential output (difference in responses of the upper and lower sections) of the split photodiode. The deflections are very small relative to the cantilever thickness and length. Thus, the probe displacement is linearly related to the deflection. The cantilever is typically silicon or silicon nitride with a tip radius of curvature on the order of nanometers.

Based on the nature of the probe-surface interaction (attractive or repulsive), an AFM can be selected to operate in various modes, namely contact mode, intermittent contact mode, or non-contact mode. In contact mode, the interaction between the tip and surface is repulsive, and the tip literally contacts the surface. At the opposite extreme, the tip interacts with the surface via long-range surface force interactions; this is called non-contact mode. In intermittent contact mode, the cantilever is oscillated close to its resonance frequency perpendicular to the specimen surface, at separations closer to the sample than in non-contact mode. As the oscillating probe is brought into proximity with the surface, the probe-surface interactions vary from long range attraction to weak repulsion and, as a consequence, the amplitude (and phase) of the cantilever oscillation varies. During a typical imposed 100 nm amplitude oscillation, for a short duration of time, the tip extends into the repulsive region close to the surface, intermittently touching the surface and thereby reducing the amplitude. Intermittent contact mode has the advantage of being able to image soft surfaces or particles weakly adhered to a surface and is hence preferred for nanoparticle size measurements.

A microscope feedback mechanism can be employed to maintain a user defined AFM set point amplitude, in the case of intermittent contact mode. When such feedback is operational, constant vibration amplitude can be maintained by displacing the built-in end of the cantilever up and down by means of the piezo-actuator. (Operation of an AFM with feedback off enables the interactions to be measured and this is known as force spectroscopy.) This displacement directly corresponds to the height of the sample. A topographic image of the surface can be generated by rastering the probe over the specimen surface and recording the displacement of the piezo-actuator as a function of position.

Unlike electron microscopes, which provide a two-dimensional projection or a two-dimensional image of a sample, AFM provides a three-dimensional surface profile. Although the lateral dimensions are influenced by the shape of the probe, the height measurements can provide the height of nanoparticles with a high degree of accuracy and precision. If the particles are assumed to be spherical, the height measurement corresponds to the diameter or size of the particle. In this assay protocol, procedures for dispersing gold nanoparticles on various surfaces such that they are suitable for imaging and height measurement via intermittent contact mode AFM are first described. Generic procedures for AFM calibration and operation to make such measurements are then discussed. Finally, the procedures for data analysis and reporting are provided. The nanoparticles used to exemplify these procedures are National Institute of Standards and Technology (NIST) Au nanoparticle Reference Materials, RM 8011 (nominally 10 nm), RM 8012 (nominally 30 nm), and RM 8013 (nominally 60 nm), all of which contain citrate-stabilized negatively charged Au nanoparticles in an aqueous solution.