Plan apo 100x 1.4 na dic h objective

The OT system consists of an infrared Nd:YVO4 Osprey laser (λ = 1064 nm) (Quantronix, USA) that illuminates the back focal plane of a PLAN APO 100X 1.4 NA DIC H Nikon objective lens attached to an inverted Nikon Eclipse Ti-S microscope (Nikon, Melville, NY) to create the optical trap. A homemade temperature/CO₂ chamber is coupled to the microscope, allowing cells to remain in optimal culture conditions throughout the experiments (37 ± 0.5 °C; 5.0 ± 0.5 % of CO₂ and pH 7.0 - 7.5). A piezoelectric stage E-710 (PI, Germany) is also coupled to the microscope in order to control the sample position with nanometric precision.
To calibrate the OT, a sample, containing polystyrene beads with radius a = (1.52 ± 0.02) μm (Polysciences, Warrington, PA), is placed on the microscope. A single bead is trapped and the sample is set to move with different controlled velocities. Movies of the entire process are recorded and analyzed to obtain the bead position displacements. The trap transverse stiffness per unit power P at the objective entrance is k/P = (0.12 ± 0.02)pN μm^-1 mW^-1. The trapped bead displacement multiplied by the value of the trap stiffness gives the optical force on the bead. The trap stiffness values can be increased or decreased by changing the laser beam power [50 (link)].

The optical tweezers (OT) system employed an infrared Ytterbium linearly polarized and collimated laser beam with a wavelength of 1064 nm and maximum power of 5 W (model YLR-5-1064-LP) (IPG Photonics, NY, USA). The laser was coupled to an inverted Nikon Eclipse TE300 microscope (Nikon, Melville, NY, USA) equipped with a PLAN APO 100X 1.4 NA DIC H Nikon objective, used to create the trap. The OT system was calibrated using the same procedure previously described [13 (link),25 (link),26 (link)]. The trap stiffness (k_OT) per unit power (P) at the objective entrance was $\left(0.25\pm 0.01\right) \mathrm{pN}\mathsf{\mu }{\mathrm{m}}^{-1}{\mathrm{mW}}^{-1}.$