Zen black edition

Immunolabeled cells were imaged by laser-scanning confocal microscopy (LSM-710, Zeiss, Jena, Germany) configured around an AxioObserver 21 (inverted) stand with a motorized XY stage. Image acquisition was performed using the Zen Black edition (Carl Zeiss, 2011; 64 bit, version 8.1.5.484) software package. 16-bit images were acquired with a 20x/0.8 NA plan apochromat objective lens, with a pixel dwell time of 0.99 μsec and a pixel size of 0.13 μm². Images were acquired using 4x line averaging, with simultaneous scanning of the 405 Diode and 633 HeNe lasers, and a sequential scan for the 488 Argon and 561 DPSS lasers. The 488 laser line was also used to generate a transmitted light DIC image. At least 5 fields of view (424.84 μm²) were acquired for each condition (EF strength x time), and each experiment was repeated at least 3 times. Detector windows for each channel were adjusted to assure no cross talk between channels as follows: 405 nm (410–483 nm), 488 nm (492–560 nm), 561 nm (580–629 nm), and 633 nm (637–735 nm).

For continuous monitoring of the initial steps of virus infection, the motility of Acanthamoeba cells was reduced using 1% low melting agarose. Briefly, Acanthamoeba cells were infected with SYBR Green I-stained virus and then cultured with 1% low melting agarose containing PYG medium. To monitor the lysis of virus-infected cells and examine the morphology of cells infected with UV- and heat-inactivated virus particles, cells were cultured in PYG medium without low-melting agarose. The differential interference contrast (DIC) images were captured using a Carl Zeiss LSM 780 microscope (Zeiss, Germany). Videos of Acanthamoeba cell lysis during infection were recorded using a combination of the ‘tile scanning’ and ‘time series’ options available in the Zeiss microscopy software (ZEN black edition). The tiling and stitching options were used to generate high-resolution images covering a larger area of multiple continuous fields. The videos were processed with Zeiss microscopy software (ZEN blue edition, available online). For this, a ‘region of interest’ was drawn in the video using the ‘ROI’ tool under ‘Graphics’ to create a subset of the selected video, which helps display the lysis of a single cell in an image (or video). ImageJ was used to generate the movies, and the videos were further converted into images using ZEN blue software.

Fixed samples (γ-tubulin staining) were imaged using an inverted Zeiss 880 microscope fitted with an Airyscan detector. The system was equipped with Plan-Apochromat 63x/1.4-NA oil lens. The laser excitation lines used were 405 nm diode, 488 nm argon and 561 nm diode laser. Stacks of 25 slices with 0.14-μm-thick intervals were collected with pixel size (xy) of 0.035 μm, using a piezo-driven z-positioner stage. Images were Airy-processed in 3D with a strength value of ‘auto’ (∼6). The software used to acquire images and process the images taken in super-resolution Airyscan mode was ZEN (black edition, Zeiss).

Wide-field microscopy was performed using an AxioImager 2 (Zeiss) employing either a 63× or 100× objective. Confocal microscopy was performed with an LSM 880 (Zeiss). SIM was performed using an Elyra superresolution microscopy (Zeiss), using five rotations of the illumination pattern. Image processing (reconstruction and channel alignment) was conducted using Zen Black edition (Zeiss), and 3D rendering was performed using Volocity (Perkin-Elmer).

The two image sets generated using dual-side acquisition were fused into one image, using a discrete Fourier transformation with Zeiss Zen Black Edition (version 9.2.8.54).

FISH stained roots and bacterial cells were investigated at the Zeiss confocal laser scanning microscope LSM880 (Zeiss, Oberkochen, Germany) with argon ion laser and helium neon laser for excitation of FITC (488 nm), Cy3 (561 nm) and an unlabeled control channel (633 nm). Cells were observed with a 64x C-Apochromat water immersion objective. Micrographs were recorded using the software Zen Black Edition (Zeiss, Oberkochen, Germany).