A novel on-the-fly calibration method of optical tweezers is presented, which enables in situ control and measure of absolute temperature and viscosity at nanoscale dimensions. Such noncontact measurement and control at the nanoscale are challenging as the present techniques only provide off-line measurements that do not provide absolute values. Additionally, some of the present methods have a low spatial resolution. We simultaneously apply the high temporal sensitivity of position autocorrelation and equipartition theorem to precisely measure and control in situ temperature and the corresponding microrheological property around the focal volume of the trap at high spatial resolution. The femtosecond optical tweezers (FOTs) use a single-beam high repetition rate laser for optical trapping to result in finer temperature gradients in comparison to the continuous-wave laser tweezers. Such finer temperature gradients are due to the additional nonlinear optical (NLO) phenomena occurring only at the nanoscale focal plane of the FOTs. Because NLO processes are laser peak power-dependent, they promote an effective study of physical properties occurring only at the focal plane. Using FOTs at optically benign near-infrared wavelengths, we demonstrate microrheological control and measurement in water by adding a highly absorbing yet low fluorescent dye (IR780).
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