Elyra ps 1 microscope system

Widefield fluorescence images were captured using an epifluorescence microscope BX61 (Olympus Europa SE &Co. KG, Germany) equipped with an Orca ER CCD camera (Hamamatsu, Japan) and pseudo-colored by the Adobe Photoshop 6.0 software. To analyze chromatin ultrastructures, we applied super-resolution spatial structured illumination microscopy (3D-SIM) using a 63×/1.40 Oil Plan-Apochromat objective of an Elyra PS.1 microscope system (Carl Zeiss GmbH, Germany). Image stacks were captured separately for each fluorochrome using the 561, 488, and 405 nm laser lines for excitation and appropriate emission filters. Maximum intensity projections from image stacks were calculated via the Zeiss ZENBlack software (Carl Zeiss GmbH, Germany). Zoom-in sections were presented as single slices to indicate the subnuclear chromatin structures at the super-resolution level. 3D rendering to produce spatial animations was done based on SIM image stacks using the Imaris 9.6 (Bitplane, UK) software. The volumes of CENH3 signals and DAPI-labeled whole G1 nuclei were generated and measured via the Imaris tool ‘Surface’, and the number of signals was counted. The percentage of colocalized immunolabeling and FISH signals were calculated via the Imaris tool ‘Coloc’ and the number of signals was detected using the Imaris tool ‘Spots’.

Polytene chromosomes were fixed and stained with aceto-orcein by the method of Zhimulev et al. [39 (link)]. Phase contrast images were acquired with an Olympus BX51 microscope using a 100×/1.30 Uplan FI objective and a DP52 camera (Olympus, Tokyo, Japan). The same fixation procedure was used to prepare polytene chromosomes for three-dimensional, structured illumination microscopy (3D-SIM), which was performed with a Zeiss Elyra PS.1 microscope system using a Plan-Apochromat 63×/1.4 oil objective and the ZENBlack 2.1 software (Carl Zeiss GmbH, Jena, Germany). Image stacks for each fluorochrome were generated with 561, 488 and 405 nm laser excitation and appropriate emission filters [40 (link)].

To image fixed and live cell preparations an Olympus BX61 microscope (Olympus) and a confocal laser scanning microscope LSM 780 (Carl Zeiss GmbH), respectively, were used.
To analyze 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 × 1.4NA Oil Plan-Apochromat objective of an Elyra PS.1 microscope system and the software ZEN (Carl Zeiss GmbH). Images were captured separately for each fluorochrome using the 561, 488, and 405 nm laser lines for excitation and appropriate emission filters (Weisshart et al., 2016 (link)).

3D-SIM images were acquired using ElyraPS1 microscope system (Carl Zeiss) which is located at the National Center for Inter-University Research Facilities (Seoul National University). The SIM system achieves a resolution of 100 nm along the x-y axis and 300 nm along the z axis. Laser lines at 405, 488 and 561 nm were used for excitation. For channel alignment, we used multispec calibration slide (170 nm beads, cat. No. 1783–455, Carl Zeiss). The SIM images (15 images with five different phases for 3 different rotation of illumination grid for each SIM image) were acquired with an EMCCD camera (Andor Technology) (512x512 pixels) and processed with Zen (Carl Zeiss) software.

SIM was used to obtain high-resolution images using an ElyraPS1 microscope system (Zeiss) with a 100x oil-immersion lens (alpha Plan Apochromat 100x, NA 1.46, oil immersion) and a resolution of 120 nm along the x-y axis and 500 nm along the z-axis (PSF measured on 100 nm beads; Sampling voxel size: 0,050μm*0,050μm*0,150 μm). Three lasers (405, 488, and 561 nm) were used for excitation. SIM images were acquired with an EMCCD camera (Andor Technology Ltd, UK) and processed with ZEN software, where exposure times varied between 100 and 150 ms. Three-dimensional images were generated using a z-step of 150 nm (total thickness ~5 μm). The acquisition was done sequentially using Zeiss Filter Sets 43HE, 38HE and BP 420–480. 15 frames were acquired to reconstruct one image (5 rotations x 3 phases, with a SIM Grating period of 51μm for the blue channel, 42 μm for the green channel, 34μm for the red channel). 100 nm beads were imaged to measure the chromatic mis-alignment of our system (fit procedure by the Zen software); this parameter enabled correcting the alignment on each acquired multi-channel stack. Image reconstructions and co-localization quantification were determined with IMARIS software (Bitplane).