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Confocal endomicroscopes aim at providing to the clinician microscopic imaging of a living tissue. The currently available microendoscopic devices use the principle of confocal fluorescent microscopy, in which the objective is replaced by an optical fiber and a miniaturized scanhead at the distal end of the endoscope or by a retractable bundle of optical fibers. Such systems have recently been applied to the explorations of several organs, including the gastrointestinal tract, and more recently to the proximal and distal airways in vivo. Respiratory fluorescence microendoscopes use 488 nm or 660 nm excitation laser light and thin flexible miniprobes that are introduced into the working channel of the bronchoscope. The devices have a lateral resolution of 3 microm, a field of view of 600 microm, and produce real-time imaging at 9 frames per second. For in vivo imaging, the miniprobe is applied onto the bronchial wall surface or advanced into a distal bronchiole down to the acinus. In nonsmokers, the 488-nm excitation device images the autofluorescence of the elastin that is contained in the basement membrane of the proximal airways and that participates to the axial backbone of the peripheral interstitial respiratory system. In smokers, a specific tobacco tar-induced fluorescence allows in vivo macrophage and alveolar wall imaging. Using 660 nm excitation and topical methylene blue, the technique enables cellular imaging of both bronchial epithelial layer and peripheral lung nodules. This article reviews the capabilities and possible limitations of confocal microendoscopy for in vivo proximal and distal lung explorations.

The aim of the present study was to assess fibred confocal fluorescence microscopy (FCFM) as a tool for imaging the alveolar respiratory system in vivo during bronchoscopy. A 488-nm excitation wavelength FCFM device was used in 41 healthy subjects including 17 active smokers. After topical anaesthesia, the 1.4-mm miniprobe was introduced into the bronchoscope working channel and advanced distally to the alveoli. Morphometric and cellular analyses were performed on selected frames harbouring a minimal compression effect. In vivo acinar microimaging was obtained from each lung segment except for the apical and posterior segments of both upper lobes. Reproducible patterns, corresponding to the elastic framework of the axial and peripheral interstitial systems, were recorded from 192 separate acini. The mean+/-sd thickness of the acinar elastic fibres was 10+/-2.7 microm. Alveolar mouth diameters (mean+/-sd 278+/-53 microm) were normally distributed but appeared smaller in the right upper lobe and right medial basal segment. Lobular microvessels (median diameter 90 microm) were equally distributed throughout the lungs. Alveolar macrophages were not detectable in nonsmokers, whereas a specific tobacco-tar-induced fluorescence was observed in smoking subjects, providing fine details of the alveolar walls and macrophages. A strong correlation was found between the number of cigarettes smoked per day and the amount of large and mobile macrophages observed in vivo, as well as with the intensity of the macrophage alveolitis. Fibred confocal fluorescence microscopy enables accurate exploration of the peripheral lung in vivo in both smokers and nonsmokers.

Rationale: Fibered confocal fluorescence microscopy (FCFM) is a new technique that produces microscopic imaging of a living tissue through a 1-mm fiberoptic probe that can be introduced into the working channel of the bronchoscope., Objectives: To analyze the microscopic autofluorescence structure of normal and pathologic bronchial mucosae using FCFM during bronchoscopy., Methods: Bronchial FCFM and spectral analyses were performed at 488-nm excitation wavelength on two bronchial specimens ex vivo and in 29 individuals at high risk for lung cancer in vivo. Biopsies of in vivo FCFM-imaged areas were performed using autofluorescence bronchoscopy., Results: Ex vivo and in vivo microscopic and spectral analyses showed that the FCFM signal mainly originates from the elastin component of the basement membrane zone. Five distinct reproducible microscopic patterns were recognized in the normal areas from the trachea down to the more distal respiratory bronchi. In areas of the proximal airways not previously biopsied, one of these patterns was found in 30 of 30 normal epithelia, whereas alterations of the autofluorescence microstructure were observed in 19 of 22 metaplastic or dysplastic samples, five of five carcinomas in situ, and two of two invasive lesions. Disorganization of the fibered network could be found on 9 of 27 preinvasive lesions, compatible with early disruptions of the basement membrane zone. FCFM alterations were also observed in a tracheobronchomegaly syndrome and in a sarcoidosis case., Conclusions: Endoscopic FCFM represents a minimally invasive method to study specific basement membrane alterations associated with premalignant bronchial lesions in vivo. The technique may also be useful to study the bronchial wall remodeling in nonmalignant chronic bronchial diseases.