Uplsapo 60

The spin state was measured by fluorescence readout in a high-NA confocal microscope (excitation 532 nm, ~1 mW power, detection in the > 650 nm band by an objective lens Olympus UPLSAPO 60 × 1.35O). In total, 4–8×10⁵ readout repetitions per trace were made, corresponding to a measurement time of 15–30 min for each trace. All sequences were recorded twice, with and without an additional π pulse before readout. The difference of both datasets was normalized to the signal contrast of a Rabi oscillation to yield a quantitative estimate of ${∣⟨0∣\psi ⟩∣}^2$ .

Image acquisition of Annexin V-FITC stained cells was performed with an inverted confocal microscope (Fluoview FV1000, Olympus) using a UPLSAPO 60 oil-immersion objective (numerical aperture: 1.35) and 2x zoom. Annexin V–FITC was excited at 488 nm and emission was recorded at 520 nm. Images were acquired with the same laser and detection settings for each experimental setup. Phase contrast images for quantification of blebbing cells were taken with a Canon DS126291 camera and a Hund Wetzlar Wllovert30 microscope. Calculation was performed by counting blebbing cells in 3 random microscope fields of six cover slips from independent experiments, respectively, and expressing these counts as a proportion of total cells in identical fields.

Confocal measurements were performed using a MicroTime200 (PicoQuant, Berlin, Germany). The fluorophores were excited using LDH-D-C 485B and LDH-D-C 640B lasers with 485 nm and 640 nm emission (PicoQuant, Berlin, Germany) and a power of 21 μW and 18 μW, respectively. For smFRET and Brightness-gated two-color coincidence detection (BTCCD) measurements, lasers were operated in a pulsed-interleaved excitation (PIE) scheme, in which blue and red excitation was alternated in order to directly excite both channels [22 (link)]. The excitation light was focused and collected by a high numerical aperture water immersion objective (UPLSAPO 60×; Olympus, Hamburg, Germany) and directed through a 75 μm pinhole. The emission signal was separated by a dichroic mirror (T600lpxr, Chroma Technology, Olching, Germany) and filtered by band pass filters of 535 nm (FF01-535/55-25, Semrock, Rochester, NY, USA) and 685 nm (ET685/80m, Chroma Technology, Olching, Germany) for the blue and red channels, respectively. Photons were detected by single-photon avalanche diodes (τ-SPAD, PicoQuant, Berlin, Germany; COUNT-T, Laser Components, Olching, Germany).

Images were acquired with an EVOS-FL microscope (Life Technologies, Bleiswijk, The Netherlands), equipped with three fluorescent LEDs combined with dichroic cubes (Cy5, RFP, GFP), an Olympus UPLSAPO 60 × oil-immersion objective, and a sensitive 1360 × 1024-pixel CCD camera. Images were quantified for surface-area-coverage and scored for characteristic thrombus parameters, utilizing semi-automated ImageJ scripts [29 (link)]. Parameters 1–5 were generated from brightfield images, and parameters 6–8 from single color fluorescence images (Suppl. Table 1).