Abstract
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. 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. 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. 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.
Highlights
Multiphoton tomography (MPT) is suitable to perform both ex vivo and in vivo investigations of living skin and cell cultures with submicron resolution
After 1 month, when a transparent sheet comprising cells and fibres was produced within the culture, cell cultures were investigated for the presence of fibres by multiphoton tomography and Fluorescence lifetime imaging (FLIM)
The main endogenous fluorophores observed in the skin include the reduced coenzyme NAD(P)H, which emits in the blue/ green spectral range with a maximum around 460 nm [15], oxidized flavoproteins, which emit in the green spectral region [15], lipofuscin, which emits in the yellow spectral region [16], and melanin, which emits in the green/yellow spectral region [17]
Summary
Multiphoton tomography (MPT) is suitable to perform both ex vivo and in vivo investigations of living skin and cell cultures with submicron resolution. 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 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
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