Combined non-linear laser imaging (two-photon excitation fluorescence microscopy, fluorescence lifetime imaging microscopy, multispectral multiphoton microscopy) in cutaneous tumours: first experiences

Year: 2009

Authors: De Giorgi V., Massi D., Sestini S., Cicchi R., Pavone F.S., Lotti T.

Autors Affiliation: Department of Dermatology, University of Florence, Florence, Italy; Department of Human Pathology and Oncology, University of Florence, Florence, Italy; LENS, Department of Physics, University of Florence, Florence, Italy

Abstract: Background
Two-photon excitation (TPE) fluorescence microscopy is a high-resolution laser-scanning imaging technique enabling deep imaging inside biological tissues. TPE microscopy has the triple advantage of offering high spatial resolution (250 nm radially, 800 nm axially), high penetration depth inside skin (200mm ), and low photodamage effects. Further, cells and extracellular matrix intrinsically contain a variety of fluorescent molecules (NADH, tryptophan, keratins, melanin, elastin, cholecalciferol and others), so that biological tissues can be imaged by TPE microscopy without any exogenous probe. The time-resolved analysis of the fluorescence signal, known as fluorescence lifetime imaging microscopy (FLIM), is an additional non-invasive microscopy technique useful to characterize endogenous fluorescence species and their surrounding medium by measuring the mean lifetime of fluorescent emission. Finally, multispectral (MTPE) tissue imaging can also be used to identify different endogenous fluorescent species by measuring their two photon emission spectra. Those techniques offer functional information about the relative quantities of fluorescent molecules, which are correlated
with tissue structure in physiological and pathological states.
Objective
We have decided to apply these three methods at the same time for cutaneous tumors in order to evaluate their possible future use.
Method
We have analyzed a melanoma and a basal cell carcinoma, with their surrounding healthy skin, to evaluate any
difference in healthy skin and neoplasia. The samples were excised during dermatological surgery, then cut, saving some healthy skin in both, to obtain a regular shape, allowing its positioning either with the skin surface parallel to the optical axis (horizontal optical sectioning), or perpendicular (vertical optical sectioning).
Conclusion
This first result demonstrates that FLIM is effective in discriminating healthy skin from MM, while MTPE is effective in discriminating healthy skin from BCC.

Journal/Review: JOURNAL OF THE EUROPEAN ACADEMY OF DERMATOLOGY AND VENEREOLOGY

Volume: 23 (3)      Pages from: 314  to: 316

KeyWords: analytic method; article; basal cell carcinoma; fluorescence microscopy; human; melanoma; multiphoton microscopy; priority journal; sample; skin surface; skin tumor; tissue structure, Carcinoma, Basal Cell; Humans; Melanoma; Microscopy, Fluorescence; Skin Neoplasms
DOI: 10.1111/j.1468-3083.2008.03045.x

Citations: 43
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