Fig. 1

Experimental setup schematic and workflow. In (a) the experimental setup, the pump and Stokes pulses are chirped by glass blocks (GB), combined at a dichroic mirror (DM), and scanned through an objective (OBJ) onto the sample (S) using galvanometer mirrors (GM). The light is then collected using a condenser (CON). Another dichroic mirror redirects the SHG light to the PMT, and the remaining light is collected in the SRS path after filtering the 1040 nm light and focusing it on the photodetector. (b) A representative annotated H&E image of a prostate tissue core with tissue regions annotated by a pathologist. HGC regions are shown in red and LGC regions in green, with the remainder being benign. Images were collected in the four regions demarcated by black squares on the core. The imaging process yields (c) 1450 cm⁻¹ SRS images and (d) 1668 cm⁻¹ SRS images after background subtraction, and (e) SHG images. (f) A co-registered image where SHG is shown in red, 1450 cm⁻¹ SRS is shown in green, and 1668 cm⁻¹ SRS is shown in blue. All the different multimodal NLO images (FOVs) are split into 3 × 3 subimages as shown in (g) to train and test an SVM (ECOC) model.