C2 confocal scan head

Confocal images were captured with a Nikon C2+ confocal scan head attached to a Nikon Eclipse Ti-E microscope. Confocal images were captured using a high NA, PLAN 60X objective (Nikon CFI Plan Apo Lambda 60X Oil with NA = 1.4), which enabled us to create high resolution images with a larger number of cell nuclei in the FOV throughout the experiments than during dSTORM measurements. The setup and data acquisition process were controlled by the Nikon NIS-Elements 5.02 software and the captured images were postprocessed in MATLAB. The Nikon Laser Unit was used to set the wavelengths and the power of the applied lasers operated at 405 nm (P_max = 60 mW; Nichia) and 647 nm (2RU-VFL-P-300-647-B1, P_max = 300 mW, MPB Communications Ltd).
All DSBs induced in the cell nuclei (N = 49–125) were displayed and counted in 3D.

Mouse embryonic fibroblasts were plated onto collagen-coated glass coverslips and cultured overnight to 80% confluency in DMEM high glucose, GlutaMAX, with pyruvate (Thermofisher), supplemented with 15% hyclone fetal calf serum (Thermofisher). Cultures were fixed for 10 min at room temperature with 4% paraformaldehyde in 1 X PBS and washed three times with 1 X PBS. Cells were then blocked for 1 h with 10% normal donkey serum diluted in 1 X PBS also containing 0.25% TritonX-100, followed by overnight incubation at 4°C with Monoclonal Antibody 414 (Mab414; AbCam ab24609) diluted 1:50 in the same solution. The following day coverslips were briefly rinsed in 1 X PBS, washed 3 x 10 min with 1 X PBS, and then incubated for 1 h at room temperature with Alexa Fluor 488 anti-mouse (Jackson Immunoresearch) in the 10% normal donkey serum solution. A second set of washes were applied and then coverslips were inverted and mounted onto slides with Vectashield containing DAPI (Vector Laboratories). For imaging a Nikon (Tokyo, Japan) C2 confocal scanhead attached to a Nikon TiE inverted microscope outfitted with a APO 60x 1.4 NA oil objective lens was used. Images of different genotypes were processed identically using ImageJ and Photoshop.

Confocal images were captured using a Nikon C2 + confocal scan head attached to a Nikon Eclipse Ti-E microscope. Confocal and superresolved dSTORM images were captured sequentially using the same microscope objective (Nikon CFI Apochromat TIRF, NA = 1.49, × 100) throughout the experiments to minimize spatial drift and reduce image registration issues. The setup and data acquisition process were controlled using the Nikon NIS-Elements 5.02 software, and the captured images were postprocessed in ImageJ-Fiji (https://fiji.sc/). The Nikon Laser Unit was used to set the wavelengths and the power of the applied lasers operated at 405 and 647 nm.

Confocal images were captured using a Nikon C2+ confocal scan head attached to a Nikon Eclipse Ti-E microscope equipped with a high NA objective (Nikon CFI Apo TIRF 100XC Oil, NA = 1.49). The setup and data acquisition process were controlled by Nikon NIS-Elements 5.02 software, and images were postprocessed in MATLAB. The Nikon Laser Unit was used to set the wavelength and power of the following applied lasers: Sapphire 488 LP-200 (Pmax = 200 mW, Coherent, Santa Clara, CA, USA) for 488 nm excitation; Cobolt Jive (Pmax = 300 mW, Cobolt, Kassel, Germany) for 561 nm excitation; 2RU-VFL-P-300-647-B1 (Pmax = 300 mW, MPB Communications, Montreal, Canada) for 647 nm excitation.

Then, 2 × 10⁵ cells (A549-luc) were cultured on a glass bottom plate in F-12K with 200 μg/mL geneticin. After 24 h, the 0.5 mM polythiophene solution was added to the cell culture medium (final concentration was 0.25 μM) and incubated for 48 h. Confocal microscopy was performed on a Nikon Eclipse Ti-E inverted confocal fluorescence microscope (Nikon Instruments, Tokyo, Japan) using 60x oil immersion Plan Apo VC and 1.4-numerical aperture objective. Samples were excited with 488 and 561 nm solid-state lasers, and the emission was captured with a Nikon C2 confocal scan head (Nikon) interfaced to a PC running NIS-Elements C software. Three-dimensional stacks were generated from a series of confocal plane images with 1.0 μm steps.

The home-built experimental setup (Supplementary Fig. S1) is based on a Chameleon Ultra II (Coherent Inc., Santa Clara, CA, USA) femtosecond laser source with the following parameters: 80 MHz, 200 fs at 860 nm. The output beam is split into two parts with a 70/30 beam splitter (BS), both of which are collimated. The reflected portion propagates via a vortex phase plate in order to generate a doughnut shaped beam. Subsequently, both parts are recombined using a polarizing beam splitter, and circularly polarized with a combination of half and quarter wave plates. The laser lines are coupled with a commercial Nikon C2 confocal scan head (Nikon Instruments, Tokyo, Japan) and focused on the sample with an oil immersion objective, 100x NA 1.32. The backward signal is collected with a focusing objective and passes through the dichroic mirror, in order to reject the excitation wavelength, and then, in case of SHG, is guided to a non- descanned photomultiplier tube (PMT1 in Supplementary Fig. S1), while for two-photon fluorescence microscopy the signal is collected by the internal PMT after a pinhole that we kept open. The forward signal is collected with an air objective (10x) that is coupled with a PMT (PMT2 in Supplementary Fig. S1). The images are acquired by Nis-Elements software (Nikon Instruments, Tokyo, Japan).