a, Top, example images of DNA in a Kc 167 cell, visualized with the viable DNA dye Hoechst 33342, both in the live cell before fixation and in the same cell after applying our fixation buffer (osmotically balanced methanol-free formaldehyde in PBS). Bottom, same as the top panels but for a Kc 167 cell before and after fixation with methanol, a fixative that is known to cause a shrinkage effect. b, Quantifications of the distances between chromatin features in live and fixed cells. Corresponding chromatin features were identified in the live and corresponding fixed cell images through scale-invariant feature transform (SIFT) registration. We measured distances between pairs of identified SIFT features in each cell, and calculated the ratio between the median inter-feature distances before and after fixation for each cell. Plotted here are the histograms of ratios determined from many cells for fixation with our osmotically balanced methanol-free formaldehyde fixation buffer (magenta), for a ‘mock fixed’ condition in which the growth media was replaced with fresh media without any fixation reagent (cyan), and for fixation with methanol (red), n ≈ 80 cells in each cases. The average ratios are 1.009 ± 0.003 and 1.008 ± 0.003 for fixation with our fixation buffer and the mock fixation, respectively, indicating a lack of shrinkage effect. In contrast, the average ratio for the methanol-fixation case (0.868 ± 0.005) is appreciably less than one, indicating a chromosome shrinkage induced by methanol. c, STORM images of TRF1-mMaple3 labelled telomeres in live and fixed HEK293 cells. Cells are fixed with our osmotically balanced methanol-free formaldehyde in PBS. Two examples of telomere STORM images are shown for each condition. d, Quantifications of the radius of gyration of the telomeric domains in live and fixed cells. We determined the radius of gyration for each telomere structure and plotted here are the histograms of the radii of gyration across about 150 telomeres from around 30 cells for live (cyan) and fixed (magenta) cells. The average radius of gyration is 77 ± 3 nm for live cells and 78 ± 2 nm for fixed cells, again indicating that there is no significant chromatin shrinkage effect upon fixation. The telomere size measurement is not limited by our image resolution with mMaple3 (~30 nm).