The colloidal probes are normally fabricated by gluing a colloidal particle to a tip-less cantilever with a micromanipulator in air. The subsequent rewetting of the probe may lead to the formation of nanosized bubbles on the probe surface. This problem can be avoided by attaching the colloidal particles under wet conditions in AFM fluid cell to appropriately functionalized cantilevers. While the colloidal probe technique is mostly used in the sphere-plane geometry, it can be also used in the sphere-sphere geometry. The latter geometry further requires a lateral centering of the two particles, which can be either achieved with an optical microscope or an AFM scan. The results obtained in these two different geometries can be related with the Derjaguin approximation.
The force measurements rely on an accurate value of the spring constant of the cantilever. This spring constant can be measured by different techniques. The thermal noise method is the simplest to use, as it is implemented on most AFMs. This approach relies on the determination of the mean square amplitude of the cantilever displacement due to spontaneous thermal fluctuations. This quantity is related to the spring constant by means of the equipartition theorem. In the added mass method one attaches a series of metal beads to the cantilever and each case one determines the resonance frequency. By exploiting the relation for a harmonic oscillator between the resonance frequency and the mass added one can evaluate the spring constant as well. The frictional force method relies on measurement of the approach and retract curves of the cantilever through a viscous fluid. Since the hydrodynamic drag of a sphere close to a planar substrate is known theoretically, the spring constant of the cantilever can be deduced. The geometrical method exploits relations between the geometry of the cantilever and its elastic properties.
The separation is normally measured from the onset of the constant compliance region. While the relative surface separation can be determined with a resolution of 0.1 nm or better, the absolute surface separation is obtained from the onset of the constant compliance region. While this onset can be determined for solid samples with a precision between 0.5–2 nm, the location of this onset can be problematic for soft repulsive interactions and for deformable surfaces. For this reason, techniques have been developed to measure the surface separation independently (e.g., total internal reflection microscopy, reflection interference contrast microscopy).
By scanning the sample with the colloidal probe laterally permits to exploit friction forces between the probe and the substrate. Since this technique exploits the torsion of the cantilever, to obtain quantitative data the torsional spring constant of the cantilever must be determined.
A related technique involving similar type of force measurements with the AFM is the single molecular force spectroscopy. However, this technique uses a regular AFM tip to which a single polymer molecule is attached. From the retraction part of the force curve, one can obtain information about stretching of the polymer or its peeling from the surface.