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- Image processing and quantitative analysis
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SR SIM and WF included diode laser 488-100 (488nm). Images were captured with an EM-CCD 608 camera (Andor iXON EM+; 1004x1002 px, cooled at -64°C, 16-bit) at typical exposure times 609 varying between 80-200 ms and with gain values between 20-25. High performance SR-SIM 610 setup included 5 rotations and 5 phases of the grated pattern for each image layer. Gratings for 611 patterned illumination were spaced by 42 µm for the 100x/NA1.57 oil immersion objective. Up 612 to 7 (usually 3) Z-stacks were acquired per image with a slice thickness of 110nm for the 613 100x/NA1.57 objectives. Light source for CLSM included Argon-Neon Laser (458, 488 and 514 614 nm). Images were captured using GaAsP spectral detector, scanning speed 4, line averaging 4 615 and pinhole: 1airy unit. CLSM was setup to meet the SR-SIM magnification (1008x and 1600x 616 depending on the objective) in the optimum pixel resolution (according to Nyquist sampling 617 criteria). 618 For time lapsed imaging with SIM the Alpha Plan Apochromat 100x/NA1.57 oil objective was 619 exclusively used and images were acquired from a single optical section. Grid rotations were 620 reduced from 5 to 3 and the exposure time of the EM-CCD was reduced to minimum. In this 621 way, time interval was 2.6 seconds and this value was used as a standard for WF (acquired 622 simultaneously with SIM), CLSM, SD and TIRF in order to ensure similar temporal resolution 623 employing the same objective. 624 SIM, WF, CLSM and TIRF platforms are integrated in the Zeiss Elyra PS.1 system used hereby, 625 therefore it was possible to acquire sequential time-lapsed series exactly from the same cell with 626 the aforementioned microscopies. Spinning disc microscopy was conducted using an 627 independent microscopy platform equipped with the Zeiss Axio Observer inverted microscope 628 combined with a Yokogawa CSU-X1 scanning head. The same Alpha Plan Apochromat 629 100x/NA1.57 oil objective, cover slips and immersion oil (Nexterion high precision coverslips 630 and high refractive index Immersol HI as used for acquisition of SIM images) were used for 631 bioimaging with this system. Images were acquired by high resolution camera Evolve 512 black- 632 thinned EM-CCD (Photometrics). 633
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Raw CLSM, WF and SR SIM images were acquired with Zeiss Zen 11 software (Zen Blue 635
version, Carl Zeiss Microscopy GmbH, Jena). Measurements involving intensity profiles of 636
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individual microtubules or microtubule bundles were directly conducted in Zen 11. For multiple 637 measurements, the original line selected for intensity profiling was cloned with the appropriate 638 Zen 11 tool to different positions of the same or other individual or bundled microtubules in the 639 image. To alleviate for differences in absolute intensity values between WF, SIM and CLSM 640 images, raw values were exported to MS Excel, normalized to a range between 0 and 1 and 641 plotted against distance. Scatter plots of normalized intensity values vs distance were used to 642 measure FWHM with Image J. 643 To quantitatively address bundle structure by means of maximum absolute fluorescence 644 intensity, line profiles were drawn perpendicular to cortical microtubule bundles acquired by 645 SIM (as depicted in Figs. 2A, D, G), based on visual inspection of the actual individual 646 microtubules converging to such bundles, to avoid user bias on placing profiles based on 647 intensity values. The respective profiles were then exactly copied to the corresponding WF 648 image and the sequentially acquired CLSM image (appx. 2 min after the simultaneous 649 acquisition of the SIM/WF image). This was done because absolute fluorescence intensities of 650 individual microtubules visualized by WF and CLSM showed broad variability, thus absolute 651 fluorescence intensity could not serve as a predictor to decide the placement of the profile as to 652 correspond the microtubule number independently for WF and CLSM. 653 Kymographs of dynamic microtubules were generated by three alternative ways: either by using 654 the time profile tool of the Zen 2011 blue version, or in Image J, on line selections of time stacks 655 subsequently resliced, or by using the MultipleKymograph plugin for Image J 656 (http://www.embl.de/eamnet/html/body_kymograph.html) developed by J. Rietdorf and A. Seitz 657 (European Molecular Biology Laboratory, Heidelberg, Germany). Quantitative analysis of 658 kymographs was done again in ImageJ using the Kymoquant plugin 659 (http://cmci.embl.de/downloads/kymoquant) written by Koto Miura (European Molecular 660 Biology Laboratory, Heidelberg, Germany). 661 Catastrophe and rescue frequencies were calculated according to previously published work 662 (Dhonukshe and Gadella, 2003). Briefly, total number of events (summed from as many 663 individual microtubules as reported in text) was divided by total amount of time required for 664
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