Atomic force microscopy (AFM) is being used in a great variety of force measurement applications1, including investigating the unfolding pathways of native membrane proteins2, probing the structure of single polysaccharide molecules3, and monitoring the response of living cells to biochemical stimuli4. All of these techniques rely on the accurate determination of the cantilever spring constant in order to yield quantitative results. For although the cantilever deflection can be measured with great accuracy and sub-Angstrom sensitivity, converting these measurements to units of force via Hookes law, F = k • x, requires that the spring constant, k, be determined for each cantilever. DI Multimode V