Assembly, characterization, and validation of a fluorescence lifetime rigid endoscope for clinical imaging of skin lesions

• Autor(a) principal: Rosa, Ramon Gabriel Teixeira
• Data de Publicação: 2018
• Tipo de documento: Tese
• Idioma: eng
• Título da fonte: Biblioteca Digital de Teses e Dissertações da USP
• Texto Completo: http://www.teses.usp.br/teses/disponiveis/76/76132/tde-04062018-104844/
• topic: Biofotônica; Biophotonics; FLIM; FLIM; Microscopia; Microscopy

Fluorescence based microscopy techniques have been extensively used in biological sciences. The most common approach is the steady-state fluorescence microscopy. Although the said approach is powerful, it often lacks sensitivity to detect several biochemical processes that may indicate relevant conditions of biological tissues. The fluorescence dynamics analysis not only brings intrinsic information about the tissue, but is also less sensitive to the medium scattering and absorption, and sometimes capable of distinguishing between fluorescent structures with indistinguishable spectra. The intrinsic fluorescence lifetime of biological tissues is usually affected by some clinical conditions, especially when those conditions cause or are correlated with metabolic modifications. Time-resolved spectroscopy techniques can be used to detect those modifications and may be used as a tool to improve the detection and diagnosis rate of such conditions. Fluorescence Lifetime Imaging Microscopy (FLIM) combines the temporal resolution and the microscopy concept, so fluorescence lifetime images can be generated. This technique has a great potential for clinical applications since it may be able to detected lesions and delineate its borders. However, FLIM usually demands a more sophisticated instrumentation than most techniques based on the steady-state approach, what creates a difficulty for moving such a system to a clinical setting. We report the assembly, characterization, validation, and clinical application of a multispectral FLIM system featuring a handheld probe composed of a laser scanning rigid endoscope. The assembled system uses a 355 nm short pulsed laser as excitation and has three spectral channels, targeting the emission of collagen, NADH, and FAD, which are important endogenous fluorophores. The system acquires images of 8.65 x 8.65 mm2 areas in ~ 2.4 s. MATLAB codes were written to process the images using a biexponential model and a modified phasor approach. In vivo validation measurements of tumors induced in mice were performed. The system was also validated with in vivo imaging of skin of healthy volunteers. The assembled FLIM system was moved to Hospital Amaral Carvalho, where we performed a pilot clinical study, in which different types of skin lesions were imaged in vivo in a clinical setting. A significant contrast was achieved on seborrheic keratosis, Bowen´s disease, and sclerodermiform basal cell carcinoma tumors. These results indicate the potential of this technique for clinical imaging of skin lesions.