The bichromophore nature of bilirubin explains the presence of at least two partially overlaying bands in both absorption and fluorescence emission spectra, and accounts for interchromophore exciton transfer events responsible for the emission sensitivity to the molecular environment and excitation wavelength. These concepts were likely responsible for the previously reported good yield of the unexpected remarkable bilirubin fluorescence emission under excitation at 366 nm, at which bilirubin absorption is very low. In this connection, aim of this work is to further investigate bilirubin spectral excitation properties and their diagnostic potential, until now poorly considered. Fluorescence excitation spectra of pure bilirubin in solution with solubilizing agents observed at 520 and 570 nm showed a wide region in the 430–510 nm range, similar to the absorption profile. In addition, an excitation band centered at about 400 nm was detected. Comparable excitation features were detected in rat serum. The 430–510 nm excitation region was well separated from a main band at shorter wavelength, ascribable to other endogenous fluorophores, with a shoulder at about 400 nm which was also easily discriminated by spectral fitting analysis. The bands ascribable to bilirubin showed changes of their relative contribution to the overall spectral region after liver ischemia/reperfusion, comparable to bilirubin biochemical data. Excitation spectra proved to discriminate the fluorescence of serum bilirubin at levels much lower than emission spectra, opening promising perspectives to improve the real time fluorescence analysis of crude serum in the absence of any exogenous labelling agent, and advance the diagnostic application of optical-biopsy in experimental hepatology and biomedicine.

胆红素的双色团性质解释了在吸收和荧光发射光谱中至少存在两个部分重叠的谱带，并解释了发色团间的激子转移事件，这对分子环境和激发波长具有发射敏感性。这些概念可能是先前报道的在366 nm激发下出乎意料的显着胆红素荧光发射的良好产率的原因，在该波长下胆红素的吸收非常低。在这方面，这项工作的目的是进一步研究胆红素的光谱激发特性及其诊断潜力，直到现在仍未充分考虑。在520和570 nm处观察到的纯胆红素与增溶剂在溶液中的荧光激发光谱显示在430–510 nm范围内有一个宽广的区域，类似于吸收曲线。另外，检测到以约400nm为中心的激发带。在大鼠血清中检测到可比的激发特征。430-510 nm的激发区域与较短波长的主谱带良好隔离，归因于其他内源荧光团，其肩部约为400 nm，这也很容易通过光谱拟合分析来区分。与胆红素生化数据相当，归因于胆红素的条带在肝脏缺血/再灌注后显示出其对总光谱区域的相对贡献的变化。激发光谱被证明能够以比发射光谱低得多的水平区分血清胆红素的荧光，为在没有任何外源标记剂的情况下改善粗血清的实时荧光分析提供了广阔的前景，