High-resolution multiphoton tomography and fluorescence lifetime imaging of UVB-induced cellular damage on cultured fibroblasts producing fibres

Abstract

Background: Multiphoton tomography (MPT) is suitable to perform both ex vivo and in vivo investigations of living skin and cell cultures with submicron resolution. Fluorescence lifetime imaging (FLIM) generates image contrast between different states of tissue characterized by various fluorescence decay rates. Our purpose was to combine MPT and FLIM to evaluate fibroblasts and collagen fibres produced in vitro.

Methods: Fibroblast cultures, 2-4 days old, at a subconfluent stage, were evaluated before and after irradiation with a single UVB dose. One month old cultures stimulated with ascorbic acid were also assessed.

Results: After UVB radiation, fibroblasts appear irregular in size, lose their alignment and show a decrease in fluorescence lifetime. One month-old fibroblasts, producing collagen fibres after stimulation with ascorbic acid, appear as small roundish structures intermingled by filaments showing a granular arrangement.

Conclusion: The combination of MPT and FLIM may be useful for the in vitro study of cell modifications induced by injurious or protective agents and drugs.

Keywords: collagen; extracellular; fibroblasts; fluorescence lifetime imaging; matrix; multiphoton tomography.