Photo-induced processes in collagen-hypericin system revealed by fluorescence spectroscopy and multiphoton microscopy

Abstract

Collagen is the main structural protein and the key determinant of mechanical and functional properties of tissues and organs. Proper balance between synthesis and degradation of collagen molecules is critical for maintaining normal physiological functions. In addition, collagen influences tumor development and drug delivery, which makes it a potential cancer therapy target. Using second harmonic generation, two-photon excited fluorescence microscopy, and spectrofluorimetry, we show that the natural pigment hypericin induces photosensitized destruction of collagen-based tissues. We demonstrate that hypericin-mediated processes in collagen fibers are irreversible and may be used for the treatment of cancer and collagen-related disorders.

Keywords: (170.3880) Medical and biological imaging; (180.4315) Nonlinear microscopy; (190.1900) Diagnostic applications of nonlinear optics.

Fig. 1

Experimental setup for spectrofluorimetric measurement of laser-induced photoprocesses.

Fig. 2

Fluorescence spectra of BAT (a) and gelatin (b) before and after Hyp sensitization and UV (337 nm) or green (532 nm) laser irradiation. Samples were illuminated for 15 min at the intensity of 15 mW/cm². Excitation of the samples was carried out at 337 nm and intensity of 0.5 mW/cm².

Fig. 3

Fluorescence spectra of chicken tendon, before and after 8μM Hyp sensitization and UV (337 nm) illumination. The sample was illuminated at the intensity of 15 mW/cm² for 30 minutes. Excitation was carried out at 337 nm at the intensity of 0.5 mW/cm².

Fig. 4

(a) Multiphoton images of Hyp-sensitized BAT acquired with a 20 × /NA 0.5 objective at different time points of halogen lamp illumination. Red pseudocolor is the SHG from collagen fibers, green pseudocolor represents the TPEF signal from Hyp and collagen autofluorescence (spectral range of 435-700 nm). (b) Dynamics of TPEF (series 2) and SHG (series 4) intensities in ROI 2 localized within collagen fibers during the irradiation of Hyp-sensitized BAT with a halogen lamp. Series 1 and 3 show the dynamics of the fluorescence in the range of 435-700 nm measured from outside of the collagen fibers (ROIs 1 and 3, respectively). Arrow indicates the starting point of the light illumination.

Fig. 5

TPEF (green) and SHG (red) imaging of Hyp-induced destruction of CLT acquired with 20x/NA 0.5 objective. (a), (b): Native collagen fibers after 40 min treatment with EtOH(75%)–PBS. Same samples after 60 min treatment with 10 μM (c) and 20 μM (d) of Hyp in EtOH(75%)–PBS solutions. Dynamics of SHG and TPEF intensities during collagen treatment with 20 μM Hyp-EtOH(75%)–PBS solution, and EtOH–PBS solvent is shown in (e).