Supplementary Figure 6: Improved displacements quantification via 3D-MTC with STED.

A living CHO cell was plated on collagen-1 coated dish overnight. A magnetizing field was applied along Z-axis and a twisting field was applied along X-axis (the short axis of the cell). The top panel is the data of GFP-H2B of the nucleus via a confocal microscope. The bottom panel is the data of the same cell under the same stress conditions quantified with a STED. Parameters for STED are: STED laser power (90 %) is 810 mW; the STED laser is CW (continuous wave) phase pattern; the excitation laser wavelength is 488nm; the STED depletion laser wavelength is 592 nm; scanning method is beam scanning. The scanning speed is 1000 Hz (1000 lines per sec). The 90 % STED power was estimated to be reduced to ~64.8 mW at the objective lens. (a) The GFP-H2B image of whole nucleus. The white box is the enlarged area. The top left of confocal image is the brightfield image of the same cell, the black dot is an RGD-coated magnetic bead. (b) Enlarged GFP-H2B images. (c) Full Width at Half Maximum (FWHM) of the fluorescence images. Confocal FWHM is 251 nm; STED FWHM is 118 nm. (d) Histograms of the fluorescence counts (with a Gaussian fit) at various FWHMs using confocal or STED. FWHM of confocal is 251.3 ± 1.03 nm, of STED is 118.04 ± 0.55 nm; mean ± s.e.m., 100 GFP-H2B spots were measured that were near the GFP-LacI spots next to the DHFR gene locus¹⁹. P<0.001 when comparing FWHM of confocal with that of STED. (e) A 15-Pa stress was applied at 0.3 Hz and peak displacement map was computed (displacement). The white box is the enlarged area. (f) Enlarged displacement maps. The black arrow (150 nm) represents the scale for the displacement magnitude. Image acquisition time is 320 millisecond per image. Comparing the white box of H2B fluorescence via STED with that via confocal, one could see better spatial resolution of the GFP-H2B images. FWHM of the PSF is 118 nm, better than the ~185 nm resolution using the re-scan confocal microscopy¹. Comparing the white box of the displacement map via STED with that via confocal, improved displacements were also noticed. Several other cells showed similar trends.