Fig. 2: The distribution of diameters measured from MINFLUX imaging of labeled microtubules for each reporter.

a–c Confocal and MINFLUX images obtained using different labeling strategies in fixed BSC-1 cells stably expressing tubulin. The first two lines are confocal imaging and the last two lines are MINFLIUX imaging. a Direct immunofluorescence labeling with anti-ALFA nanobodies, i.e., S reporter. b Direct immunofluorescence labeling with rabbit anti-tubulin antibodies, i.e., M reporter. c Indirect immunofluorescence labeling with mouse anti-tubulin antibodies and secondary mouse antibodies, i.e., L reporter. d–f Experiments and simulations of the probability distribution for the diameter d around the center of microtubule. The top row is the experiment, the bottom row is the simulation. The five circles represent the minimum value, first quartile, average value, second quartile, and the maximum value, respectively. The minimum and maximum values are defined as the average of the top 10 smallest and the top 10 largest values, respectively. d Direct immunofluorescence labeling with anti-ALFA nanobodies, i.e., S reporter. e Direct immunofluorescence labeling with rabbit anti-tubulin antibodies, i.e., M reporter. f Indirect immunofluorescence labeling with mouse anti-tubulin antibodies and secondary mouse antibodies, i.e., L reporter. g Lateral intensity distribution of individual microtubule segments calculated from MINFLUX images, with peak-to-peak distances (reflecting the diameter value, d) calculated. h Measurement of peak-to-peak distances from at least 1000 microtubule segments across three kinds of reporters. The MINFLUX images have pixel sizes of 4 nm (a3–c3) and 1 nm (a4–c4, close-up), and the localization accuracy is visualized by a Gaussian distribution with a standard deviation of 2 nm (Fig. S1). Normal dist. S: defined as the artificial normal distribution of the same mean and the same standard deviation as the experimental data for S reporter; Normal dist. M: defined as the artificial normal distribution with the same mean and standard deviation as the experimental data for M reporter; Normal dist. L: defined as the artificial normal distribution with the same mean and standard deviation as the experimental data for L reporter.