Fig. 1

Flowchart of the experimental protocol for BC detection in the placenta. a Five biopsies are taken in total, of which four on the fetal side of the placenta at distinct positions oriented according to the main blood vessel (black arrow), and one (biopsy 5) at the maternal side of biopsy 1. After sample collection, the biopsies are embedded in paraffin and sections of 4 µm are prepared. b The placental sections are illuminated using a two-photon femtosecond pulsed laser tuned to a central wavelength of 810 nm (10 mW radiant power at the sample). c The WL produced by the BC naturally present in the tissue (white dots) is detected along with the simultaneous generation and detection of two-photon excited autofluorescence (TPAF) of the cells (green) and second harmonic generation (SHG) from collagen (red) (see materials and methods for detailed protocol). A tile scan of 10 × 10 images is acquired resulting in a field of view of 9000 × 9000 µm² with a 12960 × 12960 pixel resolution (0.694 × 0.694 µm² pixel size) and a pixel dwell time of 2.51 µs. Three different locations per placental section are imaged. d The number of BC particles in the obtained images is determined using a peak-find algorithm, which counts connected pixels above a certain threshold value, i.e., 0.5% and 45% lower than the highest intensity value of the TPAF and SHG pictures, respectively. e BC particles (white dots) in the output figure are defined as the saturated pixels found in both channels, i.e., TPAF and SHG. f Finally, based on the image volume estimated from the point spread function of the optical system, the result is expressed as the total relative number, i.e., the number of detected BC particles per cubic millimeter placenta