Optical tweezers have become a powerful tool for basic and applied research in cell biology. Here, we describe an experimentally verified theory for the trapping forces generated by optical tweezers based on first principles that allows absolute calibration. For pedagogical reasons, the steps that led to the development of the theory over the past 15 years are outlined. The results are applicable to a broad range of microsphere radii, from the Rayleigh regime to the ray optics one, for different polarizations and trapping heights, including all commonly employed parameter domains. Protocols for implementing absolute calibration are given, explaining how to measure all required experimental parameters, and including a link to an applet for stiffness calculations.
Keywords: Absolute calibration; Optical trap forces; Optical tweezers.