Yanni Zhao

The traction forces exerted by an adherent cell on a substrate have been studied only in the two-dimensions tangential to substrate surface. We developed a novel technique to measure the three-dimensional traction forces exerted by live bovine aortic endothelial cells on polyacrylamide deformable substrate. On 3D images acquired by confocal microscopy, displacements were determined with image-processing programs, and traction forces in tangential and normal directions were computed by finite element method. BAECs generated traction force in normal direction with an order of magnitude comparable to Txy. Tz is upward at the cell edge and downward under the nucleus, changing continuously with a sign reversal between cell edge and nucleus edge. The method was evaluated regarding accuracy and precision of displacement measurements, effects of FE mesh size, displacement noises, and simple bootstrapping. These results provide new insights into cell-matrix interactions in terms of spatial and temporal variations in traction forces in 3D. This technique can be applied to study live cells to assess their biomechanical dynamics in conjunction with biochemical and functional activities, for investigating cellular functions in health and disease.

Background: Optical Doppler tomography combines laser Doppler flowmetry with optical coherence tomography to obtain high-resolution images of blood flow in human skin in situ and in real time. Observations: We present a case in which ODT was used on a patient with a port-wine stain birth-mark to document the change of blood flow in response to laser therapy. It might be possible to use ODT blood flow measurements in situ to assist in assessing the efficacy of laser PWS therapy. If partial restoration of flow occurs immediately or shortly after laser exposure, indicative of reperfusion due to inadequate blood vessel injury, the PWS can be retreated using higher light dosages. Retreatment is continued until the measured Doppler shift is zero due to a permanent reduction in blood flow, indicative of irreversible microthrombus formation in the PWS vessels. Conclusions: We have demonstrated that ODT may be used for noninvasive imaging of blood vessels in PWS skin. Moreover, ODT will potentially allow laser therapy to be optimized on an individual patient basis by providing a fast, semiquantitative evaluation of the efficacy of PWS laser therapy in situ and in real time.

The mechanical environment crucially influences many cell functions 1. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin-cytoskeleton interaction2, which is essential for the transduction of mechanical stimuli3-5. Using fluorescent resonance energy transfer, here we develop a genetically encoded Src reporter that enables the imaging and quantification of spatio-temporal activation of Src in live cells. We introduced a local mechanical stimulation to human umbilical vein endothelial cells by applying laser-tweezer traction on fibronectin-coated beads adhering to the cells. Using the Src reporter, we observed a rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane. This wave propagated away from the stimulation site with a speed of 18.1 ± 1.7 nm s-1. This force-induced directional and long-range activation of Src was abolished by the disruption of actin filaments or microtubules. Our reporter has thus made it possible to monitor mechanotransduction in live cells with spatio-temporal characterization. We find that the transmission of mechanically induced Src activation is a dynamic process that directs signals via the cytoskeleton to spatial destinations.