RESUMO
BACKGROUND: Fluorescence lifetime imaging (FLIM) is a novel imaging technique that generates image contrast between different states of tissue due to differences in fluorescence decay rates. OBJECTIVES: To establish whether FLIM of skin autofluorescence can provide useful contrast between basal cell carcinomas (BCCs) and surrounding uninvolved skin. METHODS: Unstained excision biopsies of 25 BCCs were imaged en face with FLIM following excitation of autofluorescence with a 355 nm pulsed ultraviolet laser. RESULTS: Using FLIM we were able to distinguish areas of BCC from surrounding skin in an ex vivo study. Significant reductions in mean fluorescence lifetimes between areas of BCC and areas of surrounding uninvolved skin were demonstrated (P < 0.0001). These differences were apparent irrespective of the decay model used to calculate the fluorescence lifetimes (single vs. stretched exponential) or the long-pass filter through which the emitted autofluorescence was collected (375 vs. 455 nm). Conversely, there was no significant difference between the BCC and uninvolved areas of each sample when mean autofluorescence intensities were examined. Moreover, wide-field false-colour images of fluorescence lifetimes clearly discriminated areas of BCC from the surrounding uninvolved skin. CONCLUSIONS: We therefore believe that FLIM has a potential future clinical role in imaging BCCs for rapid and noninvasive tumour delineation and as an aid to determine adequate excision margins with best preservation of normal tissue.
Assuntos
Carcinoma Basocelular/diagnóstico , Diagnóstico por Imagem/métodos , Neoplasias Cutâneas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Meios de Contraste , Feminino , Fluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias/métodos , Sensibilidade e EspecificidadeRESUMO
The autofluorescence of biological tissue can be exploited for the detection and diagnosis of disease but, to date, its complex nature and relatively weak signal levels have impeded its widespread application in biology and medicine. We present here a portable instrument designed for the in situ simultaneous measurement of autofluorescence emission spectra and temporal decay profiles, permitting the analysis of complex fluorescence signals. This hyperspectral fluorescence lifetime probe utilizes two ultrafast lasers operating at 355 and 440 nm that can excite autofluorescence from many different biomolecules present in skin tissue including keratin, collagen, nicotinamide adenine dinucleotide (phosphate), and flavins. The instrument incorporates an optical fiber probe to provide sample illumination and fluorescence collection over a millimeter-sized area. We present a description of the system, including spectral and temporal characterizations, and report the preliminary application of this instrument to a study of recently resected (<2 h) ex vivo skin lesions, illustrating its potential for skin cancer detection and diagnosis.
Assuntos
Biomarcadores Tumorais/análise , Medições Luminescentes/instrumentação , Neoplasias Cutâneas/diagnóstico , Espectrometria de Fluorescência/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Técnicas de Sonda Molecular , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Fluorescência/métodosRESUMO
High-speed (video-rate) fluorescence lifetime imaging (FLIM) through a flexible endoscope is reported based on gated optical image intensifier technology. The optimization and potential application of FLIM to tissue autofluorescence for clinical applications are discussed.
Assuntos
Endoscópios , Tecnologia de Fibra Óptica/instrumentação , Aumento da Imagem/instrumentação , Microscopia de Fluorescência/instrumentação , Microscopia de Vídeo/instrumentação , Animais , Sistemas Computacionais , Desenho de Equipamento , Análise de Falha de Equipamento , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/instrumentação , Interpretação de Imagem Assistida por Computador/métodos , Camundongos , Microscopia de Fluorescência/métodos , Microscopia de Vídeo/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We report the development of a high-speed wide-field fluorescence-lifetime imaging (FLIM) system that provides fluorescence-lifetime images at rates of as many as 29 frames/s. A FLIM multiwell plate reader and a potentially portable FLIM endoscopic system operating at 355-nm excitation have been demonstrated.
Assuntos
Algoritmos , Endoscópios , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Sistemas On-Line/instrumentação , Espectrometria de Fluorescência/métodos , Gravação em Vídeo/instrumentação , Endoscopia/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Estudos de Viabilidade , Gravação em Vídeo/métodosRESUMO
Target cells are thought to regulate the survival of afferent neurons during development by supplying limiting amounts of neurotrophic factors, but the degree to which afferent neurons remain dependent on target-derived support in the adult is uncertain. In this study, uninjured basal forebrain cholinergic neurons did not die after excitotoxic ablation of their target neurons in young adult rats, indicating that they are either not dependent on neurotrophic factors for survival or can obtain trophic support from other sources after target neurons are lost. This finding suggests that cholinergic cell death in neurodegenerative conditions such as Alzheimer's disease is not due solely to a loss of target neurons or factors provided by them.
