Fig. 1.

The mesoscopic photoacoustic image analysis pipeline. 1) Images are acquired and reconstructed at a resolution of 20 × 20×4 µm³ (PDX tumour example shown with axial and lateral maximum intensity projections – MIPs). 2) Image volumes are pre-processed to remove noise and homogenise the background signal (high-pass and Wiener filtering followed by slice-wise background correction). Vesselness image filtering (VF) is an optional and additional feature enhancement method. 3) Regions of interest (ROIs) are extracted and segmentation is performed on standard and VF images using auto-thresholding (AT or AT + VF, respectively) or random forest-based segmentation with ilastik (RF or RF + VF, respectively). 4) Each segmented image volume is skeletonised (skeletons with diameter and length distributions shown for RF and RF + VF, respectively). 5) Statistical and topological analyses are performed on each skeleton to quantify vascular structures for a set of vascular descriptors. All images in steps 2–4 are shown as x-y MIPs.