Fluorescence properties of collagen types I-V: a comprehensive study of spectral and lifetime characteristics.

Significance: Collagen autofluorescence provides valuable intrinsic contrast for assessing tissue structure, composition, and pathology. However, a comprehensive understanding of the fluorescence properties across different collagen types remains limited. This knowledge gap may limit the development of advanced label-free fluorescence spectroscopy and imaging techniques for specific tissue characterization and diagnostic applications. Aim: This study aims to comprehensively characterize the fluorescence intensity excitation-emission matrices (I-EEMs) and time-resolved excitation-emission matrices (TR-EEMs) of collagen standards from types I, II, III, IV, and V obtained from various organ sources under both dry and hydrated conditions, to identify optimal excitation-emission parameters for each collagen type discrimination, and to establish a reference dataset that supports future research in label-free tissue characterization. Approach: We employed a time-resolved fluorescence spectroscopy system equipped with an optical parametric oscillator laser (excitation: 200 to 2000 nm, pulse width: 30 ps) as an excitation source to generate I-EEMs and TR-EEMs of human and bovine collagen types I-V. The fluorescence light was obtained by a multichannel plate photomultiplier tube through a monochromator (spectral range: 200 to 1000 nm). Measurements were conducted using collagen standards, under both dry and hydrated states. In addition, photobleaching effects were assessed to ensure the reliability and reproducibility of fluorescence data. Results: Each collagen type exhibited distinct I-EEM and TR-EEM signatures, with fluorescence lifetimes ranging from 2.5 ns (type III, bovine skin) to 5.3 ns (types II and V). Fibrillar collagens (types I and V) displayed broader I-EEMs, whereas basement membrane collagen (type IV) showed the narrowest spectral distribution. Organ-source-dependent variations were evident within the same collagen type. Type I collagen from human placenta exhibited an inverse lifetime-emission wavelength relationship compared with bovine sources. Hydration consistently red-shifted emission peaks into the 395 to 420 nm range and reduced fluorescence lifetimes across all collagen types (e.g., type I bovine Achilles tendon: 3.2 to 5.0 ns dry versus 3.0 to 4.5 ns hydrated). Despite excitation wavelength- and fluence-dependent photobleaching of fluorescence intensity, fluorescence lifetimes remained relatively stable, confirming the robustness of lifetime-based measurements. Conclusions: This study establishes a comprehensive reference dataset for the fluorescence properties of collagen types I-V and demonstrates the potential of combined I-EEMs and TR-EEMs analysis for tissue characterization. The results highlight species-, organ-, type-, and environment-specific optical fingerprints of similar collagens, which must be considered before implementing more in-depth studies on how the optical properties of collagen change in different medical applications.