Laser-induced fluorescence (LIF) and intrinsic fluorescence spectroscopy (IFS) have been used experimentally for diagnosing coronary atherosclerosis. In this study, we demonstrated the diagnostic superiority of IFS at 342-nm excitation (IFS<sub>342</sub>) versus LIF (LIF<sub>342</sub>) and described a protocol for head-to-head comparison of old (LIF) versus new (IFS) generations of similar diagnostic methods, labeled as “generational comparison model”. IFS<sub>342</sub> and LIF<sub>342</sub> were modeled with basis spectra of media, fibrous caps, and superficial foam cells and of their correspondent chemicals (elastin, collagen, and lipoproteins). The average accuracy and receiver operating characteristic area under the curve of IFS<sub>342</sub> in single-, double-, and triple-parameter diagnostic algorithm iterations, geared toward identifying 84 atherosclerotic specimens from a group of 117 coronary segments, was 90% ± 1% and 0.87 ± 0.025, superior to LIF<sub>342</sub> (84% ± 3% and 0.84 ± 0.016; <i>P</i> = 0.0002 and 0.02, respectively) in a generational comparison model.

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