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 mm. Alveolar mouth diameters (mean¡SD 278¡53 mm) were normally distributed but appeared smaller in the right upper lobe and right medial basal segment. Lobular microvessels (median diameter 90 mm) 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.

We present a two-photon microendoscope capable of in vivo label-free deep-tissue high-resolution fast imaging through a very long optical fiber. First, an advanced light-pulse spectro-temporal shaping device optimally precompensates for linear and nonlinear distortions occurring during propagation within the endoscopic fiber. This enables the delivery of sub-40-fs duration infrared excitation pulses at the output of 5 meters of fiber. Second, the endoscopic fiber is a custom-made double-clad polarization-maintaining photonic crystal fiber specifically designed to optimize the imaging resolution and the intrinsic luminescence backward collection. Third, a miniaturized fiber-scanner of 2.2 mm outer diameter allows simultaneous second harmonic generation (SHG) and two-photon excited autofluorescence (TPEF) imaging at 8 frames per second. This microendoscope’s transverse and axial resolutions amount respectively to 0.8 μm and 12 μm, with a field-of-view as large as 450 μm. This microendoscope’s unprecedented capabilities are validated during label-free imaging, ex vivo on various fixed human tissue samples, and in vivo on an anesthetized mouse kidney demonstrating an imaging penetration depth greater than 300 μm below the surface of the organ. The results reported in this manuscript confirm that nonlinear microendoscopy can become a valuable clinical tool for real-time in situ assessment of pathological states.

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.