Elyra s 1

Images were captured by confocal microscopy (LSM780, LSM880, both from Zeiss, Oberkochen, Germany), stereo microscopy (SZX16, Olympus), and SIM (ELYRA S.1, Zeiss). Image analyses were performed using ZEN software for merging, 3D images, and SIM processing. For the randomization of statistical analysis, the images were randomly taken from the samples and individual cells in the wide field of image were analyzed. Quantitative analyses of the average ciliary length and number of multiciliated or RPE1 cells were performed with the ZEN and ImageJ programs. Statistical analysis including error bars with mean ± SD and P values was calculated using a two-tailed t-test, one-way ANOVA, or two-way ANOVA using Prism 9.

Images from two commercially available SIM microscopes were analysed, obtained on a Delta-Vision|OMX v4 by GE Healthcare (Issaquah, WA, USA) and on an Elyra S1 by Zeiss (Jena, Germany). Also, raw images were acquired on a home-built, SLM-based two-beam interference illumination SR-SIM microscope. This system consists of a 60 × , 1.2 numerical aperture water immersion objective (Olympus, Hamburg, Germany), a 642 nm, 85 mW fiber-coupled diode laser for excitation, a charge-coupled device camera (Coolsnap HQ, Photometrics, Tuscon, AZ, USA) and a liquid crystal display-based SLM for light modulation (LC-R 1920, Holoeye Photonics, Berlin, Germany). A sketch of the set-up can be found in Fig. 2e. The TIRF SR-SIM set-up is documented by Kner et al.5 (link).

A Zeiss Elyra S.1 equipped with a 63x NA 1.4 lens was used to acquire 16-bit 3D SIM images with 3 rotations and 5 phases. Double-colour labelling was with various combinations of either Alexa 488 or ATTO 488, and either Alexa 594, Alexa 568, or ATTO 565, on cells seeded on high-precision 170-nm glass coverslips (Ibidi μ-slide, 80827). Reconstruction was using Zen Blue software, using automatic parameters. The median (± median absolute deviation) lateral and axial resolution of the system using these settings was measured at 114 ± 4 nm and 352 ± 15 nm, respectively (full-width at half-maximum). Channel alignment was performed in Zen Blue, using a double-colour bead calibration standard.

Cells were seeded on Geltrex-coated coverslip. After completed differentiation, the medium was aspirated, and cells were carefully washed twice with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde (PFA) for 20-30 minutes at room temperature. Wells were then washed three times with PBS to remove PFA completely. Cells were blocked using Supermix (200 ml TBS, 0.5 g gelatin, 1 ml Triton X-100) for 2 h and incubated with primary antibodies at 4°C overnight (Table 1). On day two, cells were washed three times with PBS and incubated with secondary antibody diluted in the Supermix for 2 h. Coverslips were washed twice with PBS and were mounted using ProLong Gold antifade reagent with DAPI (Thermo Fisher Scientific, Cat. No. P36931) and kept at 4°C until visualization. Images were either acquired at the SciLifeLab BioVis Facility (Uppsala University, Sweden) using confocal Zeiss ELYRA S.1 and Zen black software (Zeiss, Oberkochen, Germany) or taken using an Olympus microscope BX53 with an Olympus DP73 camera and the CellSens dimension software. Images were handled using ImageJ (Fiji edition, https://imagej.net/Fiji/).

Nuclei were isolated from iPSNs and postmortem human motor and occipital cortex tissue using the Nuclei Pure Prep Nuclei Isolation Kit (Sigma Aldrich) following manufacturer protocol with slight modifications as previously described [4 , 5 (link)]. About 10 million iPSNs or 100 mg of frozen postmortem motor cortex tissue (obtained from the Target ALS Human Postmortem Tissue Core (see Additional file 2: Table 2 for demographic information) was used for nuclei isolation. A 1.85 M sucrose gradient was used to enrich for neuronal nuclei. Following isolation, nuclei were centrifuged onto collagen coated (1 mg/mL; Advanced Biomatrix) slides with a CytoSpin 4 centrifuge (Thermo Fisher Scientific) and immunostained as previously described [4 , 5 (link)] (see Additional file 2: Table 3 for antibody information). Isolated nuclei were subsequently imaged by super resolution structured illumination microscopy (SIM) using a Zeiss ELYRA S1 as previously described [4 , 5 (link)]. All images were acquired using identical imaging parameters (e.g. laser power, gain) and subjected to default SIM deconvolution and processing in Zeiss Zen Black 2.3 SP1. Representative images are presented as 3D maximum intensity projections generated in Zeiss Zen Black 2.3 SP1. Images were faux colored green for contrast and display.

In order to investigate the ultrafast dynamics of compound 1in cellulo, laser scanning microscopy and pump-probe spectroscopy were combined in a transient absorption microscope45 . The technological specifications of this setup can be found in ref. 44 and is schematically shown in Supplementary Fig. 6. In the transient-absorption microscope the samples are scanned in a raster pattern by means of a galvanometer-based scanner system (Cambridge Technologies) that together with the focusing objective (Nikon, CFI Plan Apo Lambda 20X, NA = 0.75, WD = 1 mm) provide diffraction limited optical resolution of the microscope. With this system it is possible to obtain linear absorption as well as transient absorption images and information on the local excited state dynamics by recording pump-probe kinetics in a fixed position of the sample with a temporal resolution of about 1 ps. The samples can be moved along the perpendicular plane of the laser beams with 10 nm precision using a scanning stage (SCAN 75 × 50, Märzhäuser).
Additionally the cellular uptake and cellular distribution of compound 1 in the cells was assessed by confocal laser scanning (using a Carl Zeiss, LSM 510 Meta) and structured illumination microscopy (using a Carl Zeiss, ELYRA-S.1).