Orca flash4.0 lt camera

Images from preparations were acquired using a Leica DMi8 fluorescence microscope equipped with a Leica HCX PL APO 100×/1.40–0.70 Oil objective, a Hamamatsu ORCA Flash 4.0 LT camera and the LAS X software. Images from the green, blue, red and differential interference contrast (DIC) channels were processed using the Icy software (http://icy.bioimageanalysis.org/) and Adobe Photoshop software packages.

Primary neurons were imaged with Olympus IX81 inverted epifluorescence microscope, operated with a SlideBook v.6.0.8 software (Intelligent Imaging Innovations). Primary fibroblasts were imaged using Olympus IX81 microscope operated with μManager software^{44 (link)}. Images were taken using Orca Flash 4.0 LT camera (Hamamatsu). The following LED light sources were used for fluorescence excitation (all from Mightex Systems): 480 nm for EGFP and 570 nm for mCherry.

Quantitative imaging was performed as described [44] (link) with the following exceptions. Images were captured on a fully-automated Zeiss AxioObserver with Definite Focus at Oregon Health and Science University's Advanced Light Microscopy Core. The images were captured with a Hamamatsu Orca-flash 4.0 LT Camera using a Plan-Apochromat 20x NA 0.8 objective. Quantification of fluorescence intensities was performed using CellProfiler. Briefly, the DAPI staining was used to create a nuclear mask, and fluorescence intensities of the stained markers (FOXO4, CDKN2A, CDKN1A, and TP53) and FOXO4 foci were measured within the nuclear mask. DAPI fluorescence intensity was also quantified and used for DNA content measurements. Cytometry scatterplots were generated using Tibco Spotfire. For labelling ES2, a cysteine was added to the N terminus and BODIPY FL maleimide (ThermoFisher, B10250) was used to conjugate the fluorophore to the peptide following the manufacturer's protocol. ES2 was incubated with the A375 cells for 2 hours at varying concentrations. Images of ES2 and FOXO4 were captured with a Yokogawa CSU-X1 on Zeiss AxioObserver spinning disk confocal using a Plan_Apochromat 63x oil, NA 1.4 objective.

Image acquisition was carried out under a Zeiss AxioObserver Z1 epifluorescence microscopy system with 40 × /1.3 oil Plan-Apochromat, 63 × /1.4 oil Plan-Aprochromat and 100 × /1.4 oil Plan-Aprochromat objectives and a Hamamatsu ORCA-Flash4.0 LT camera. The system is calibrated and aligned by using 200 nm-diameter TetraSpeck microspheres (ThermoFisher). Z-stack images were acquired at 0.2 μm intervals covering a range from 3–8 μm by using ZEN blue software.
Deconvolution was carried out using Huygens Professional deconvolution software (SVI) with a measured point-spread-function generated by 200 nm-diameter TetraSpeck microspheres. Classical maximum likelihood estimation method with iterations of 40–60 and signal-to-noise of 20–40 was applied.

Slices and resectioned tissue were imaged on either a Nikon TE2000‐U inverted microscope (10X Plan‐Fluor and 20X Plan‐Apo objectives) with a UniBlitz shutter system (Vincent Associates, Rochester, NY) and an Orca‐flash 4.0 LT camera (Hamamatsu, Hamamatsu City, Shizuoka Prefecture, Japan), or a Zeiss LSM 880 confocal microscope with an Axiocam 503 mono camera (Carl Zeiss, Inc., Thornton, NY). Data in GalNAz‐DBCO‐Cy3 fluorescent cell counting and the EdU/ GalNAz colocalization experiments were gathered via confocal Z‐stack with 30 planes, 1 µm apart being captured through the center of the tissue. A max intensity Z‐projection was performed using FIJI (ImageJ, v1.0; NIH) and cells were manually counted by a researcher blinded to treatment. Data for GalNAz‐DBCO‐Cy3 fluorescent cell proximity to peripherin immunoreactive fibers was performed by a researcher blinded to treatment who randomly sampled 3‐ Z‐planes throughout the tissue section based on GalNAz‐DBCO‐Cy3 fluorescence only. Analysis was performed in FIJI using a 3 µm dilation around all GalNAz‐DBCO‐Cy3 fluorescent cells before the analyzing particles tool to quantify peripherin immunoreactive fibers within the 3 µm dilations.

Cells at different infectious stages (uninfected, ring, and trophozoite stage) were resuspended in RPMI 1640 medium (pH 7.4) supplemented with 1 mg mL⁻¹ bovine serum albumin (Sigma-Aldrich) at a hematocrit of ~0.1% and incubated for 30 min at 37 °C in petri dishes with glass bottom. Note that RPMI 1640 contains 2 mg mL⁻¹d-glucose needed to keep a constant ATP level to preserve the activity of the cytoskeleton^{62 (link)}. The infectious stage was confirmed for each individual cell by microscopic examination (Supplementary Fig. 4). Samples were set inside a temperature-controlled chamber (37 °C) mounted on an Axio Observer Z1 microscope (Zeiss) equipped with a ring aperture, a ×100 oil-immersion objective lens (N.A. = 1.4), and an ORCA-Flash4.0 LT camera (Hamamatsu). Five hundred phase-contrast images of erythrocytes were collected by setting the exposure time and time interval at 25 ms each. Compared with the phase-contrast images, the gradient images give a much better contrast to visualize both parasites surrounded by vacuole membranes and cell membranes (Supplementary Fig. 5).