The PHDFM dataset is composed of fluorescence microscopy images of root tissue samples from A. thaliana, using the ratiometric fluorescent indicator 8‐hydroxypyrene‐1,3,6‐trisulfonic acid trisodium salt (HPTS). This semantic segmentation training dataset consists of 2D microscopy images (the brightfield channel for excitation at 405 nm), each containing a segmentation mask as an additional image channel (manually annotated by plant biologists). The segmentation masks classify pixels into the following 5 labels with the corresponding IDs: background (0), root tissue (1), early elongation zone (2), late elongation zone (3), and meristematic zone (4).