Predicting sample heating induced by cantilevers illuminated by intense light beams

Hybrid microscopy based on Atomic Force Microscopy (AFM) and fluorescence microscopy represents a commonplace experimental approach to study cell biology processes in liquid media at physiological temperature. However, many types of experimental artifacts can arise depending on the fluorescence illumination and detection technique utilized. For example, fluorescence excitation light gets absorbed by AFM cantilevers inducing local heating provoking undesirable as well as uncontrollable cantilever deflections. Here we present a numerical modelling approach based on a Finite Element Model (FEM) to predict sample heating in liquid media quantitatively, depending on illumination wavelength, illumination pattern, and cantilever shape and composition. Modelling results indicate substantial local temperature increases in-line with temperature increases derived from experimental cantilever deflections induced by fluorescence excitation light. We predict temperature increases of ∼0.05 – 0.5 °C for wide-field illumination and ∼5 – 15 °C for confocal illumination within the boundary conditions established, which could, for example, induce local protein conformational changes. We conclude that sample heating is an important issue requiring consideration in experimental set-ups involving intense light illumination of AFM cantilevers, especially when conducting single molecule investigations.