Axio observer

The device combining the photothermal actuation of microcantilevers and optical microscopy was used as described^{15 (link)}. The device was supplemented with a z-piezo stage (CellHesion^® module, JPK Instruments), which allowed precisely confining the cell between both cantilevers. The lower, slave cantilever bearing chip was fixed with vacuum grease onto a CNC milled peek wedge with a 10° angle and dimensions of 3.30 × 3.00 × 0.75 mm³ (W × L × H). The angled wedge allowed the readout laser to be reflected from the slave cantilever into the photodiode. The wedge was fixed with vacuum grease to a Petri dish. The device was combined with an inverted optical microscope (Zeiss, Axio Observer) equipped with a OrcaFlash 4.0 camera (Hamamatsu) and a 20x plan apochromat objective with a 0.8 NA (Zeiss). All experiments were conducted in a controlled environmental system^{16 (link)} to maintain cell culture conditions, temperature control (37.0 °C), and pH adjustment using a humidified gas mixture based on synthetic air containing 5% CO₂.

Immunofluorescence microscopy was performed as previously described^{125 (link)}, with modifications. Bacteria were prepared as described above under “Bacterial Survival Assays in Blood” and incubated with 10% (v/v) NHS or HI-NHS for 45 min at 4 °C under shaking. The samples were washed twice with PBS supplemented with 1% BSA (Sigma) (PBS/BSA). Next, the bacteria were fixed by PBS/BSA containing 4% paraformaldehyde (PFA, Alfa Aesar) for 20 min at RT. Bacteria were washed once with PBS/BSA, blocked in PBS/BSA for 1 h at RT, and incubated with mouse anti-C5b-9 (1 μg ml⁻¹, aE11, Santa Cruz) followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (1 μg ml⁻¹, Life Technologies) for 45 min. Bacteria were washed once with PBS/BSA and then with Hanks Balanced Salt Solution (HBSS, Gibco). Ultimately, the pellet was resuspended in HBSS supplemented with the lipophilic dye FM5‐95 (Invitrogen, 10 μg ml⁻¹) to label bacterial membranes. Labeled bacterial cells were mounted onto glass slides (covered with a thin layer of agarose gel) and secured with coverslips. Fluorescence microscopy was performed on a Zeiss AxioObserver equipped with an ORCA‐Flash4.0 V2 Digital CMOS camera (Hamamatsu Photonics) and ZEN Blue software. Images were acquired through a ×100 phase-contrast objective and analyzed by ImageJ/Fiji (v 2.1.0/1.53c).

Samples were immobilized by poly-L-lysine-coated slides. Epifluorescence microscopy was performed on a Zeiss Axio Observer equipped with a Hamamatsu Orca-Flash 4.0 V3 Digital CMOS camera, Colibri 7, and an oil-immersion phase-contrast objective Plan Apochromat 100x/1.45 N.A. (Nikon). Phase contrast and epifluorescence exposures were 100 ms and 500 ms, respectively.