(A) Confocal microscopic imaging of three cell types: normal keratinocytes NHEK, SCC13 cells, or HEK1 cells that received either no preconditioning (panels a–c), or preconditioning with 0.01 mg/L MTX for 72 hr (panels d–f), followed by incubation with ALA (4 hr) and visualization with confocal microscopy. Phase-contrast images (panels g–i) correspond to PpIX images directly above. White-boxed insets are from dishes that did not receive ALA (negative controls, -ALA). Note that PpIX levels are preferentially elevated in the carcinoma cells pretreated with MTX (e, f) relative to NHEK (d). Scale bar, 50 μm. (B) Confocal images at higher magnification to illustrate localization of PpIX in normal NHEK (j) versus carcinoma SCC13 (k) cells; both had received MTX preconditioning. PpIX signal is observable in plasma membranes (arrows) and cytoplasm (Cy), but not in the nucleus (Nu). Scale bar, 50 μm.