We developed two-photon (TP) probes for DNA (ABI-Nu), cytoplasm (Pyr-CT) and mitochondria (BF-MT). We found that ABI-Nu binds to AT in the minor groove, while ABI-Nu and BF-MT are effective for tracking in the cytoplasm and mitochondria, respectively. These probes showed very large effective two-photon action cross section values of 2230, 1555 and 790 Göppert-Mayer units (1 GM  =  10⁻⁵⁰ cm⁴ s photon⁻¹molecule⁻¹) at 740 nm with emission maxima at 473, 561 and 560 nm, respectively, in each organelle. Using these probes, we quantitatively estimated the mean nuclear area and the ratios of nuclei to cytoplasm and mitochondria to nuclei in human colon tissues by dual-colour two-photon microscopy imaging within 2  h after biopsy. The mean nuclear area and the nuclei to cytoplasm and mitochondria to cytoplasm ratios increased in the following order: normal colon mucosa <colon adenoma <colon adenocarcinoma. Furthermore, the nuclear areas of these tissues showed significant differences that were well outside of the ranges of experimental errors, indicating the diagnostic potential of this method.