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- Guldbrand, Stina, 1970, et al.
(författare)
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Two-photon fluorescence correlation spectroscopy as a tool for measuring molecular diffusion within human skin
- 2013
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Ingår i: European Journal of Pharmaceutics and Biopharmaceutics. - : Elsevier BV. - 0939-6411. ; 84:2, s. 430-436
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Tidskriftsartikel (refereegranskat)abstract
- There is a need for tools enabling quantitative imaging of biological tissue for pharmaceutical applications. In this study, two-photon fluorescence microscopy (TPM) has been combined with fluorescence correlation spectroscopy (FCS), demonstrating proof-of-principle providing quantitative data of fluorophore concentration and diffusion in human skin. Measurements were performed on excised skin exposed to either rhodamine B (RB) or rhodamine B isothiocyanate (RBITC), chosen based on their similarity in fluorescence yield and molecular weight, but difference in chemical reactivity. The measurements were performed at tissue depths in the range 0 and 20 pm, and the diffusion coefficients at skin depths 5 and 10 mu m were found to be significantly different (P < 0.05). Overall median values for the diffusion coefficients were found to be 4.0 x 10(-13) m(2)/s and 2.0 x 10(-13) m(2)/s for RB and RBITC, respectively. These values correspond to the diffusion of a hard sphere with a volume eight times larger for RBITC compared to RB. This indicates that the RBITC have bound to biomolecules in the skin, and the measured signal is obtained from the RBITC-biomolecule complexes, demonstrating the potential of the TPM-FCS method to track molecular interactions in an intricate biological matrix such as human skin.
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| 2. |
- Kantere, Despoina, et al.
(författare)
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Anti-Stokes fluorescence from endogenously formed protoporphyrin IX - Implications for clinical multiphoton diagnostics.
- 2013
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Ingår i: Journal of biophotonics. - : Wiley. - 1864-0648 .- 1864-063X. ; 6:5, s. 409-415
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Tidskriftsartikel (refereegranskat)abstract
- Multiphoton imaging based on two-photon excitation is making its way into the clinics, particularly for skin cancer diagnostics. It has been suggested that endogenously formed protoporphyrin IX (PpIX) induced by aminolevulinic acid or methylaminolevulinate can be applied to improve tumor contrast, in connection to imaging of tissue autofluorescence. However, previous reports are limited to cell studies and data from tissue are scarce. No report shows conclusive evidence that endogenously formed PpIX increases tumor contrast when performing multiphoton imaging in the clinical situation. We here demonstrate by spectral analysis that two-photon excitation of endogenously formed PpIX does not provide additional contrast in superficial basal cell carcinomas. In fact, the PpIX signal is overshadowed by the autofluorescent background. The results show that PpIX should be excited at a wavelength giving rise to one-photon anti-Stokes fluorescence, to overcome the autofluorescent background. Thus, this study reports on a plausible method, which can be implemented for clinical investigations on endogenously formed PpIX using multiphoton microscopy (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
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