Accurate diagnosis of metastatic lymph nodes (LNs) is essential in choosing appropriate treatment for gastrointestinal carcinoma. Our aim was to evaluate the diagnostic power of 5-aminolevulinic acid (5-ALA) for LN metastasis in mouse rectal cancer. Colorectal cancer cell lines, isolated cells from normal LNs, and orthotopic mouse model incorporating enhanced green fluorescent protein-tagged and untagged human rectal cancer cells were studied after 5-ALA administration by using confocal microscopy, fluorescence stereomicroscopy, fluorescence lifetime imaging microscopy (FLIM), multichannel spectrophotometry and macroconfocal imaging system to precisely detect LN metastases. In vitro confocal microscopic analyses showed that all colorectal cancer cell lines tested were positive for 5-ALA-induced fluorescence, whereas isolated normal LN cells were negative. 5-ALA-induced protoporphyrin IX (PPIX) fluorescence, verified by FLIM and multichannel spectrophotometry, revealed LN metastases in mice-bearing human rectal cancer cells. Occult LN metastases, unrecognized on white-light imaging and simplified hematoxylin-eosin analyses, were readily detectable on 5-ALA-induced PPIX fluorescence imaging. In vivo macroconfocal images clearly revealed PPIX-fluorescence-positive cancer cells in draining lymph vessels and nodes. Together with specific speckled patterns of PPIX-fluorescence in metastatic lesions, the PPIX-fluorescence intensity ratio of metastatic and nonmetastatic lesions discriminated metastasis with 100% sensitivity and 100% specificity in excised whole LN samples. These results show that fluorescence diagnosis with 5-ALA is very accurate in the detection of LN micrometastases of mouse rectal cancer, suggesting that this feasible diagnostic approach is applicable to target sectioning of metastases of resected fresh whole node samples in pathology laboratories. (c) 2009 UICC.