F8807

We performed axial calibration using red fluorescent nanospheres (F8807, Thermo Fisher). We scanned the nanospheres across a 2-µm axial range with an interval of 20 nm. Then, we used maximum likelihood estimation [28 (link)] to fit an anisotropic 2D Gaussian function to the image of the nanosphere in each frame. Finally, we plotted a calibration curve of the Gaussian sigma as a function of the nanosphere’s axial position relative to the objective lens (Fig. S1B.)

A thin silicone gel substrate embedded with fluorescent microspheres was prepared, as described in detail elsewhere [35 (link)] with some modifications. Briefly, a pair of liquid silicones (CY52-276A and B; Dow Corning Toray, Japan) were mixed at a weight ratio of 6 : 5 and degassed. The mixture was spread on a 22 × 22 mm coverslip (Matsunami) using a spin coater (LH-D7; MIKASA, Japan). The thickness of the silicone layer was less than 50 μm. A 35 mm plastic dish with a hole (14 mm in diameter) at the bottom (Matsunami) was assembled with the silicone-coated coverslip by curing the silicone at 70°C for 30 minutes. Assembled silicone substrate-bottom dishes were kept in a hermetically sealed case with a 100 μL aliquot of liquid silane (3-aminopropyl triethoxysilane; Sigma-Aldrich Japan, Japan) for 1 h to attach the silane to the surface of the silicone substrate by vapor deposition. A 250 μL aliquot of a solution containing dark red fluorescent microspheres (0.2 μm in diameter, with peak excitation and emission wavelengths of 660 and 680 nm, resp., F-8807; Invitrogen, USA) diluted 400 times with distilled water was added to the solidified silicone. After approximately 10 minutes, the substrate was washed with distilled water. Young's modulus of the silicone substrate was typically 1.0 kPa [35 (link)].

The best focus planes for isolated microtubules were manually selected from z-stacks. 2.5 μm lineplots were drawn across isolated microtubules in this single plane image using Fiji⁵³ . Small rectangular background areas were manually drawn to obtain the background values around selected microtubule lines. Average background was subtracted from the lineplot values. Gaussian curves were fit to the background-subtracted values and FWHM was calculated based on the fits and average values were obtained by analyzing 30–45 lineplots from 5 images per condition using a Python script. A similar calculation was performed for 200 nm dark red fluosphere beads (Invitrogen #F8807). Distributions were displayed as box plots. Lineplots with multiple discernible peaks were discarded. Two-sample t-test was performed to check for statistical significance.