Ps 1 microscope system

Widefield fluorescence microscopy for signal detection followed Cao et al. (2016 (link)). The images were processed (brightness and contrast adjustment only) and merged using Adobe Photoshop software ver.12 × 32 (Adobe Systems).
To analyze the ultrastructure and spatial arrangement of signals and chromatin at a lateral resolution of ~ 120 nm (super-resolution, achieved with a 488 nm laser), 3D-structured illumination microscopy (3D-SIM) was applied using a Plan-Apochromat 63×/1.4 oil objective of an Elyra PS.1 microscope system and the software ZENblack (Carl Zeiss GmbH). Image stacks were captured separately for each fluorochrome using the 405, 488, and 561 nm laser lines for excitation and appropriate emission filters (Weisshart et al. 2016 (link)). Maximum intensity projections of whole cells were calculated via the ZEN software. Zoom-in sections were presented as single slices to indicate the subnuclear chromatin structures at the super-resolution level.

To analyse the substructure of chromatin beyond the classical Abbe/Raleigh limit (super-resolution) spatial Structured Illumination Microscopy (3D-SIM) was applied using a C-Apo 63 × /1.2 W Korr objective of an Elyra PS.1 microscope system and the software ZEN (Carl Zeiss GmbH). Images were captured using 405, 488, and 561 nm laser lines for excitation (42, 34, and 28 μm grids for 561, 488, and 405 nm excitations; 5 rotations) and the appropriate emission filters. 3D-SIM stacks with a step size of 110 nm were acquired consecutively for each fluorophore starting with the highest wavelength dye to minimize bleaching. SIM image stacks were used to produce 3D movies by the Imaris 8.0 (Bitplane) and ZEN 2012 software.

The fluorescence signals of Topo II were imaged by wide-field (WF), deconvolution (DCV) of WF, and super-resolution 3D-SIM, using an Elyra PS.1 microscope system equipped with a 63×/1.4 Oil Plan-Apochromat objective and the software ZENBlack (Carl Zeiss GmbH, Jena, Germany). Images were captured separately for DAPI and Alexa488 using the 405 nm and 488 lasers for excitation and appropriate emission filters [44 (link)]. Ca. 20 slices were captured within a ~2 µm Z-stack. Reconstruction of SIM images was done with the ZENBlack software structured illumination processing module. Wiener deconvolution was used as implemented in the Zeiss SIM module [46 (link)]. In a first step, the Wiener filter was set free and automatically determined, followed by a systematic alteration of its value. The minimal strength, where just no structured noise was visible, was selected. PALM was also performed with the 405 nm and 488 lasers [44 (link)]. PALM images were processed with the ZENBlack software PALM processing module. Routinely method-based drift correction was performed, and grouping was applied. Localization events were fitted by a Gauss function. Either centroids or the Gauss function was plotted. 3D rendering of SIM and PALM image stacks to produce movies was performed with the Imaris 9.6 software (Bitplane AG, Zurich, Switzerland).

For conventional wide-field fluorescence microscopy, we used either a Nikon Eclipse 600 microscope equipped with a DS-Qi1Mc cooled camera or a Zeiss AxioImager.Z2 microscope with an Axiocam 506 mono camera. Images were generated using the NIS Elements 3.0 software program (Laboratory Imaging, Praha, Czech Republic) or ZEN pro 2012 (Carl Zeiss GmbH). Depending on the material used, we inspected at least 100 mitoses, nuclei, or isolated chromosomes per experiment. To analyse the substructure of chromatin beyond the classical Abbe/Raleigh limit (super-resolution), spatial Structured Illumination Microscopy (3D-SIM) was applied using a C-Apo 63 × /1.2 W Korr objective of an Elyra PS.1 microscope system and the software ZEN 2012 black (Carl Zeiss GmbH). Images were captured using the 405, 488, and 561 nm laser lines for excitation and the appropriate emission filters. The degree of co-localization between CenH3-1 and -2 was measured in SIM image stacks and calculated using the Imaris 8.0 (Bitplane) software. SIM image stacks were also used to produce 3D movies with the Imaris 8.0 (Bitplane) and ZEN 2012 black softwares.

To analyse the ultrastructure of immunosignals and chromatin beyond the classical Abbe/Raleigh limit at a lateral resolution of ~120 nm (super-resolution, achieved with a 488 nm laser) spatial structured illumination microscopy (3D-SIM) was applied using a 63 × /1.4 Oil Plan-Apochromat objective of an Elyra PS.1 microscope system and the software ZENblack (Carl Zeiss GmbH). Images were captured separately for each fluorochrome using the 642, 561, 488, and 405 nm laser lines for excitation and appropriate emission filters (Weisshart et al., 2016 (link)). Maximum intensity projections of whole meiocytes were calculated via the ZEN software. Zoom in sections were presented as single slices to indicate the subnuclear chromatin and protein structures at the super-resolution level. 3D rendering and CENH3 volume measurements based on SIM image stacks was done using the Imaris 8.0 (Bitplane) software.

For standard microscopy, slides were analyzed using a BX61 microscope equipped with a DP72 CCD camera (Olympus, Japan). Images were captured in black and white, pseudo-colored separately, and merged into multilayer RGB images using Adobe Photoshop (Adobe Systems, San Jose, California). To achieve super-resolution, spatial structured illumination microscopy (3D-SIM) was performed with an Elyra PS.1 microscope system equipped with a 63×/1.4 Oil Plan-Apochromat objective using the ZENBlack software (Carl Zeiss GmbH). Image stacks were captured separately for each fluorochrome using 561-, 488-, and 405-nm laser lines for excitation and appropriate emission filters (Weisshart et al. 2016 (link)). The CENH3 immuno-signal volumes, nuclear volumes, and the signal intensity per individual CENH3 volume were generated and measured with the Imaris 9.7 (Bitplane) software tool “Surface” (Randall et al. 2022 ).