Uplfln 40

We used a custom-built MT setup wherein a pair of 5 × 5 × 5 mm³ permanent magnets (W-05-N50-G, Supermagnete, Switzerland) oriented in vertical configuration^{32 (link)} and with a gap size of 1 mm was employed to topologically constrain the DNA tethers. A DC-Motor (M-126.PD2, PI, Germany) controlled the distance between the flow cell and magnets, another DC-Motor (C-150.PD, PI, Germany) controlled rotation of the magnets. A ×40 oil-immersion objective (UPLFLN ×40, Olympus, Japan) was employed to image the beads onto a CMOS sensor camera (12 M Falcon2, Teledyne Dalsa, Canada) with a field of view of 400 μm by 300 μm. Images were recorded at 58 Hz and transferred to a frame grabber (PCIe 1433, NI, USA). A custom-written tracking software analysed the images to extract the (x,y,z) coordinates of all beads in real time^{33 (link)}. A LED (69647, Lumitronix LED Technik GmbH, Germany) was used for illumination. For tracking of the bead z-position, a look-up table (LUT) is required to translate the defocused pattern of the bead to its height. The LUT was generated over a range of 10 μm, with a step size of 100 nm, by moving the objective using a piezo stage (Pifoc P-726.1CD, PI, Germany).