American Chemical Society, Analytical Chemistry, 15(87), p. 7575-7582, 2015
DOI: 10.1021/acs.analchem.5b01903
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Based on an infrared femtosecond Cr:forsterite laser, we developed a semi-quantitative method to analyze the microscopic distribution of bilirubins. Using 1230 nm femtosecond pulses, we selectively excited the two-photon red fluorescence of bilirubin dimers around 660 nm. Autofluorescences from other endogenous fluorophores were greatly suppressed. Using this distinct fluorescence measure, we found that poorly-differentiated hepatocellular carcinoma (HCC) tissues on-average showed 3.7-times lower concentration of bilirubins than the corresponding non-tumor parts. The corresponding fluorescence lifetime measurements indicated that HCC tissues exhibited a longer lifetime (500 ps) than those of non-tumor parts (300 ps). Similarly, oral cancer cell lines had longer lifetimes (>330 ps) than those of non-tumor ones (250 ps). We anticipate the developed methods of bilirubin molecular imaging to be useful in diagnosing cancers or studying the dynamics of bilirubin metabolisms in live cells.