Plan apochromat oil objective

RAW264.7 cells were seeded at 2 × 10⁵ cells per well in a 24-well plate with 10-mm coverslips and allowed to attach overnight at 37°C and 5% CO₂. Infection with Syto9 fluorescent-labeled E. faecalis V583 was performed with MOI of 10 for 1 hour. The coverslips seeded with cells were then fixed with 4% PFA at 4°C for 15 min, permeabilized with 0.1% Triton X-100 for 15 min at room temperature, and washed thrice in PBS. Cells were then blocked with PBS supplemented with 0.1% saponin and 2% BSA. For actin labeling, the phalloidin–Alexa Fluor 555 conjugate (Thermo Fisher Scientific, USA) was diluted 1:40 in PBS and incubated for 1 hour. Coverslips were then washed three times in PBS with 0.1% saponin. They were then subjected to a final wash with PBS, thrice. Last, the coverslips were mounted with SlowFade Diamond Antifade (Thermo Fisher Scientific) and sealed. Confocal images were then acquired on a 63×/numerical aperture (NA) 1.4, Plan Apochromat oil objective fitted onto Elyra PS.1 with LSM 780 confocal unit (Carl Zeiss) using the Zeiss Zen Black 2012 FP2 software suite. Laser power and gain were kept constant between experiments. Z-stacked images were processed using Zen 2.1 (Carl Zeiss). Acquired images were visually analyzed using ImageJ (52 (link)).

Infected macrophages, following 1 or 24 hr of infection were washed with PBS, fixed in 2% paraformaldehyde for 30 min, washed once with PBS and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Nucleic acids were stained with 4′,6-diamidino-2-phenylindole (1 μg/mL DAPI, Sigma) and the cells were washed three times with PBS. The slides were observed using an inverted Zeiss LSM 880 Axio-observer Z1 confocal laser scanning microscope with Airyscan detector. Cells were visualized using Zeiss Plan-Apochromat oil objective lens of X 63 and numerical aperture of 1.4. Z-stacked images were acquired with a digital zoom of X 5.58 (X 1.8 for broad fields), using the Zen lite software (Carl Zeiss microscopy). Images were processed using Image J software package. A single representative section of the compiled Z-projections produced by Image J software is presented in all the figures.

Cells were seeded into chambered cover glasses (LabTEK: Thermo Fisher Scientific), and the lids of the chambers were sealed with baysilone paste (Neolab). Approximately 30 min before imaging, culture medium was exchanged to prewarmed CO₂-independent medium without phenol red, containing 20% FCS, 2 mM glutamine, and 100 mg/ml penicillin–streptomycin. Live cell microscopy was performed in 37°C microscope incubators (EMBL GP106) on a Zeiss 780 confocal microscope with a 63× PlanApochromat oil objective (NA 1.4, Carl Zeiss) and in-house temperature controller, controlled by the Zen 2010 Software. Six z stacks with 2.00-μm intervals were used for each position. Images were acquired with 5-min time resolution. When applicable, automated quantitative analysis of cells was pursued to monitor for mitotic progression in single cells. To this end, nuclei were detected in the H2B-mCherry channel and classified as previously described (Held et al, 2010 (link); Walter et al, 2010 (link)) with an overall accuracy of > 90.0%. Cells were tracked with a constrained nearest-neighbor tracking procedure, and mitotic onset was detected as interphase–prophase or interphase–prometaphase transition. To reduce the effect of classification errors on phase length measurements, classification results were corrected using hidden Markov models (Held et al, 2010 (link)).

The FRAP measurements were performed
on a Zeiss LSM 800 confocal microscope equipped with a 63× (NA
= 1.4) Plan-Apochromat oil objective (Carl Zeiss AG, Germany). The
dsGUVs were sealed within a BSA-coated observation chamber and placed
in a thermostatic chamber at 25 °C. Two circular areas of 2.5
μm radius were defined as the probed area at the bottom of each
dsGUVs determinated by z-stack profiling beforehand:
(1) as bleaching spot and (2) as reference spot (unbleached) for data
correction. Using bleaching, experimental regions, and time series
options in Zeiss Zen software (Zen v2.3), 10 images (laser power,
1.0%) were recorded prior to bleaching (100 iteration; laser intensity,
100%) and 100 images after bleaching (laser intensity, 1.0%) as depicted
in Figure 1B. A 561
nm laser (excitation of Liss Rhod B) was used for the FRAP measurements,
with a pinhole aperture set to one Airy Unit. The 247 × 247 pixels
images were recorded with an integration of 71.85 ms per image. The
diffusion coefficient was extracted from the acquired images using
an adapted MATLAB (MathWorks, Inc.) code as described previously,^{3 (link)} where a nonlinear least-square fit was applied
to the normalized fluorescence intensity from the recovery phase.
Details are presented in the Supporting Information note 4.