Fig. 4: 3D Stimulated Raman Histology (SRH) versus 2D standard eosin and hematoxylin safran (HES).

a A fresh biopsy from surgery (here an adenocarcinoma (ADK)) is mounted between two coverslips and placed for examination on the BondXplorer SRH microscope to perform 3D histology, b SRH images at different z depths allow for 3D virtual histology reconstruction. Tissue investigation at both low and high magnifications is necessary to perform accurate diagnosis. c SRH images stack taken at a high magnification at different depths (z step = 2 µm), allow to investigate nuclei content in 3D and to follow the collagen distribution around the gland (scale bar = 20 µm), d SRH image from the biopsy at low magnification over a FoV of 2.1 mm × 2.1 mm (scale bar = 750 µm) enabling the identification of the tumorous gland deformations and collagen surrounding, e SRH image from the biopsy at medium magnification (FoV of 420 µm × 420 µm) allows to see the tumorous gland deformations more accurately (scale bar = 150 µm), f SRH image from the biopsy at high magnification (FoV 210 µm × 210 µm) (scale bar = 40 µm), g HES image of the ADK biopsy from the same patient at low magnification (scale bar = 750 µm), h HES image of the ADK biopsy from the same patient at medium magnification (scale bar = 150 µm, and i HES image of the ADK biopsy from the same patient at high magnification (scale bar = 40 µm). e and f are the magnified square ROIs delineated in long dash and dense dash in d, respectively. Total power at the sample plane: 90 mW.