On the Nature of Stationary and Time-Resolved Fluorescence Spectroscopy of Collagen Powder from Bovine Achilles Tendon.

This paper presents a more systematic study of steady-state and time-resolved autofluorescence spectroscopy of collagen isolated from bovine Achilles tendon. In steady-state fluorescence measurements, the excitation and emission spectra of collagen powder, recorded at different fluorescence excitation and detection wavelengths, were compared with the fluorescence excitation and emission spectra of the amino acids phenylalanine, tyrosine, and tryptophan, as well as with similar spectra for 13 autofluorescent collagen cross-links, which have been identified and described in the literature so far. In time-resolved studies, fluorescence was excited by the pulsed light of different wavelengths, and for each excitation wavelength, fluorescence decay was recorded for several detection wavelengths. Data analysis allowed recovery of the fluorescence decay times for each experimental excitation detection event. The obtained information on the decay times of the measured fluorescent signals was discussed, taking into account the available literature data from similar studies of isolated collagen and collagen-rich tissues. Based on the obtained results, it was found that the shape and position of the measured fluorescence excitation and emission spectra of collagen strongly depend on the emission and excitation wavelengths selected in the measurements. From the recorded excitation and emission bands of collagen, it can be concluded with high probability that there are additional, so far unidentified, collagen cross-links, which can be excited at longer excitation wavelengths. In addition, the collagen excitation spectra were measured at longer emission wavelengths at which the collagen cross-links emit fluorescent light. In addition to the emission spectra obtained for excitation in the deep-UV region, the results of time-resolved fluorescence studies with excitation in the deep-UV region and detection at longer wavelengths suggest that fluorescence excitation energy transfer processes occur from the amino acids to the collagen cross-links, and also between the cross-links themselves.