There is significant interest in quantifying force production inside cells, but since conditions in vivo are less well controlled than those in vitro, in vivo measurements are challenging. In particular, the in vivo environment may vary locally as far as its optical properties, and the organelles manipulated by the optical trap frequently vary in size and shape. Several methods have been proposed to overcome these difficulties. We evaluate the relative merits of these methods and directly compare two of them, a refractive index matching method, and a light-momentum-change method. Since in vivo forces are frequently relatively high (e.g., can exceed 15 pN for lipid droplets), a high-power laser is employed. We discover that this high-powered trap induces local temperature changes, and we develop an approach to compensate for uncertainties in the magnitude of applied force due to such temperature variations.

Intra-cellular transport via the microtubule motors kinesin and dynein plays animportant role in maintaining cell structure and function. Often, multiple kinesinor dynein motors move the same cargo. Their collective function dependscritically on the single motors’ detachment kinetics under load. Single Kinesin’sand Dynein’s super-force off rates have been measured using anoptical-trap based method. We rapidly increased the force on a moving beadand measured the time to detachment. From such events, detachment time distributionsfor specific super-force values have been measured. In contrast toa possible constant off-rate kinesin has an off-rate increasing with force. Atlow loads, dynein is sensitive to load; detaching easily but at higher load it exhibiteda catch-bond type behavior, with off rate decreasing with load. Thesuper-force experiments also allowed us to determine the probability of backwardstepping for the motors. Kinesin and dynein can back-step under load, butthis was relatively rare in both directions (<20%), and the typical backwardtravel distance was short.