Methods

Tissue collection and study population

11 placental biopsies were collected from Maastricht University Medical Centre, the Netherlands, between November 2020 and May 2021. 3 placental biopsies from uncomplicated pregnancies and 1 PE sample were collected directly after birth. 6 Placental samples (n=2 PE) were collected from the pathology department and received on formalin. Collection and usage of the placentas was approved by the Medical Ethics Committee Academic Hospital Maastricht and Maastricht University (METC, 16-4-047). Clinical characteristics of all placental samples are summarized in Table S1.

Tissue dissection and fixation

Placental tissue (~20 cm3) was sectioned from a central region of the chorion, stored for 24h at 4°C and frequently washed with phosphate buffered saline (PBS) to remove blood. Subsequently it was fixed (1h) in 4% paraformaldehyde (PFA) and stored in PBS at 4°C.

Nuclear and vascular fluorescent dyes

Placental tissue slabs (~10mm3) were fluorescently labelled to visualize nuclei and vascular structures. Tissue was permeabilized in 0,1% triton, 1% BSA (20 min), and incubated with 10µg/ml Ulex Europaeus Agglutinin I (UEA I), DyLight® 594 (Vector laboratories, CA, USA, cat#DL-1067-1, emmax=618nm) for 1-3h, in the dark, at room temperature. After washing with TBS, nuclei were labelled with 5µM SYTO 13 green (Invitrogen, cat#S7575, emmax= 509nm) for 30 minutes in the dark at room temperature.

Mounting and MPM imaging

For imaging, samples were mounted on a 50 mm glass bottom petri dish (MatTek, Ashland, MA, USA) filled with PBS. MPM was performed with a Leica TCS SP 5 (Leica Microsystems, Wetzlar, Germany) multiphoton microscope. Excitation was at 780 nm. Fluorescence was collected with a 20x Leica HCX APO L, NA 1.0 water immersion objective or a 25x NA1.05 Olympus objective. Alternatively, the Leica Stellaris 8 Dive (Leica Microsystems, Wetzlar, Germany) employing similar settings was used.
Autofluorescence and second harmonic generation (SHG) from unstained samples was detected by an external detector with bandpass filter 460-525 nm, and a forward detector with 380-420 nm bandpass filter, correspondingly.
Signal of stained samples was detected using 4 channels. SHG was collected as described before. Internal detectors with detection bandwidth 444-496 nm, 508-559 nm, and 586-650 nm recorded autofluorescence, nuclear stain, and vascular stain respectively.
All images were acquired in 8-bit, with bidirectional scanning mode and at least 1024x1024 pixels. Z-step size ranged from 1.5 µm to 5µm. Image stitching was performed with the Leica Stellaris Dive microscope equipped with motorized stage.