Introduction. This data provides the calibration of an optical tweezers within the linear Hookian region. Once the laser was aligned, the power at the objective measured and a bead trapped the instrument was calibrated. Data Collection. Calibration was performed by moving a trapped bead by supplying a periodic square wave train input (amplitude: −800 to +800 nm, period: 80 ms) to an acousto-optical device, AOD and monitoring the trajectory of the bead in the 𝑋𝑌 plane with a quadrant photo diode, QPD for ~ one minute. Data was collected at 200 kHz from three (3) QPD channels: displacement in the 𝑋 (∆𝑋𝑀) (i) and 𝑌 directions (∆𝑌𝑀) (ii) and the sum of the fluorescent intensity (∑𝐿) (iii). Typically, each bead was stimulated with a train of square waves of constant amplitude in the 𝑋 direction. This stimulation was performed four (4) times at each of the different amplitudes i.e., −800, +800, −500, and +500 nm. The QPD signal in the dark, ∆𝑋𝐷, ∆𝑌𝐷, ∑𝐷 was recorded and subtracted in real time, and the gain, 𝐺 of the QPD noted. The Stokes-Faxen coefficient, 𝛽 was calculated having recorded the height of the bead above the Petri dish, ℎ and the viscosity, 𝜂 of the saline solution. The detected fluorescent bead was excited either with a Xenon lamp or TLED transmitted light source. Some calibrations were performed with a TLED light that was borrowed from the manufacturer for testing, and the signal to noise of these measurements was significantly decreased. Data Transformation. For each of the two (2) channels the measured signal was normalized by the sum, and the ~750 waves were averaged for each pulse train stimulus. The reciprocal of the time constant (s-1) was determined from the exponential rise or decay of the back-ground subtracted averaged response. Spring constant (pN/nm) was determined from the product of this reciprocal time constant and the Stokes-Faxen coefficient (pNs/nm). The net displacement of the bead (nm) was calculated by determining the resultant vector of the 𝑋 and 𝑌 components of the QPD (V/V). For the four (4) stimuli determined for each bead the normalized displacement (V/V) was plotted as a function of net bead displacement and the slope (V/V/nm) calculated from best linear fit. For each calibration the mean spring constant, reciprocal time constant, and mean slope are provided. Data Format. The data was saved in LabView with the proprietary TDMS format (National Instruments, NI, Austin, TX). It was transformed to a text file and imported into MATLAB (The Mathworks, Natick, MA) and stored as struct and analyzed with a Python script. The collection contains raw and transformed results from 69 days for a total 153 beads. The data is provided with annotated descriptions in HDF5, a standard non-proprietary container storage format. Each file is about 1.1 GBs (HDF5). The package contains: 1. Standard Container HDF5 with custom organization format of data with annotations. 2. Python Script (calibrateopticaltweezers.py file) that describes how data is analyzed and written to HDF5 and .MAT formats. (1 file)

Funding: NIH NCI R21CA152779 and NIH NIDCD RO1DC00354