pCLE can easily be performed during a fiber-optic bronchoscopy under local anesthesia [7, 8]. The technique of in vivo bronchial pCLE imaging is simple: the miniprobe is introduced into the 2 mm working channel of the bronchoscope and the probe tip applied onto the bronchial mucosae under sight control. The depth of focus being 50 μm below the contact surface, the system can image the first layers of the bronchial subepithelial connective tissue, presumably the lamina densa and the lamina reticularis [7].

At 488 nm excitation, pCLE produces very precise microscopic fluorescent images of the bronchial basement membrane zone (Fig. 15.3). pCLE bronchial microimaging reveals a mat of large fibers mainly oriented along the longitudinal axis of the airways with cross-linked smaller fibers, as well as larger openings—100 to 200 μm—corresponding to the bronchial glands origins. In vivo, the technique also makes it possible to record high-resolution images of small airways such as terminal bronchioles, which are recognizable by the presence of the helicoidal imprint of the smooth muscle on the inner part of the bronchiole. [7]

Fluorescence properties of the bronchial mucosae at 488 nm excitation are determined by the concentration of various cellular and extracellular fluorophores, including the intracellular flavins, which could originate from the epithelial cells, and specific cross-links of collagens and elastin present in the subepithelial areas [2, 16, 17]. Microspectrometer experiments coupled with pCLE imaging have clearly demonstrated that the main fluorescence signal emitted after 488 nm excitation from both bronchial and alveolar human system originates from the elastin component of the tissue [7, 8, 18]. Indeed, flavin cellular autofluorescence appears too weak to allow imaging of the epithelial layer using 488 nm pCLE without exogenous fluorophore [19]. Similarly, the collagen fluorescence does not significantly affect the pCLE image produced at 488 nm, the fluorescence yield of collagen at this wavelength being at least one order of magnitude smaller than that of elastin.

As a result, 488 nm excitation pCLE specifically images the elastin respiratory network that is contained in the basement membrane of the proximal airways and participates to the axial backbone of the peripheral interstitial respiratory system. In the future, it is possible that a modified pCLE device using several wavelengths [20] or devices based on a multiphoton approach [21–23] may enable imaging of collagen, elastin, and flavins simultaneously.

In the acinus , elastin is present in the axial backbone of the alveolar ducts and alveolar entrances, as well as in the external sheath of the extra-alveolar microvessels [24, 25]. pCLE acinar imaging is easily obtained by pushing forward the probe a few centimeters after the endoscope is distally blocked into a subsegmental bronchi. When progressing toward the more distal parts of the lungs, the entry into the alveolar space is obtained by penetration through the bronchiolar wall. Alveolar fluorescence imaging in active smokers dramatically differs from imaging in nonsmokers. The alveolar areas of smokers are usually filled with highly fluorescent cells corresponding to alveolar fluorescent macrophages, the presence of which appears very specific of active smoking [8]. In situ alveolar microspectrometric measurements have been performed in active smokers, which evidenced that the main fluorophore contributing to the pCLE alveolar signal corresponds to the tobacco tar by itself, explaining this difference [8, 18].

Preliminary studies have shown that per endoscopic pCLE could be used to study specific basement membrane remodeling alterations in benign or malignant/premalignant bronchial alterations [7, 26]. In the first human study using pCLE in the respiratory tract in vivo, the structure of the bronchial wall was analyzed in twenty-nine patients at high risk for lung cancer that also underwent an autofluorescence bronchoscopy [7]. In this study, the fibered network of the basement membrane zone underlying premalignant epithelia was found significantly altered. This was observed in one invasive cancer, three CIS, two mild and one moderate dysplastic, and three metaplastic lesions. In these precancerous conditions, the elastic fibered pattern of the lamina reticularis was found absent or disorganized (Fig. 15.4). This supported the hypothesis of an early degradation of the basement membrane components in preinvasive bronchial lesions. However, while this observation shed some light on the origin of the autofluorescence defect in precancerous bronchial lesions, the absence of epithelial cell visualization did not allow the technique to differentiate between the different grades of progression of the precancerous bronchial lesions such as metaplasia/dysplasia/carcinoma in situ.

In order to be successfully applied to the exploration of precancerous/cancerous bronchial epithelial layer, the pCLE technique would need to be coupled with the use of an exogenous nontoxic fluorophore. Ex vivo studies have shown that the resolution of the system is not a limitation for nuclear or cellular imaging [7, 8].

A few exogenous fluorophores could be activated at 488 nm.

Acriflavine hydrochloride is an acridine-derived dye containing both proflavine and euflavine, which binds to DNA by intercalating between base pairs. Acriflavine produces a strong nuclear fluorescence with 488 nm pCLE when topically applied on the top of the bronchial epithelium ex vivo [7]. Acriflavine has been used in a couple of in vivo studies using CLE in the GI tract [27], without demonstrated side effect. However, comet assay of cells exposed in vitro to acriflavine solution shows significant DNA damage after 2 nm illumination with 488 nm Cellvizio (personal data). This observation needs further studies before acriflavine use for bronchial explorations, especially in patients at risk for cancer. Acriflavine is not currently approved for bronchial use.

Fluorescein has been used in Musani study with some success [11]. However, fluorescein, which does not enter the cells and therefore does not stain the nuclei [28], does not provide cellular imaging using pCLE. This is probably linked to the lower lateral resolution of pCLE compared to CLE and the impossibility to distinguish intercellular space with pCLE. Recently, Lane et al. have used a confocal microendoscope prototype at 488 nm excitation and topical physiological pH cresyl violet to provide cellular contrast in the bronchial epithelium both in vitro and in vivo [29].

Methylene blue is a nontoxic agent which is commonly used during bronchoscopy for the diagnostic of bronchopleural fistulae. MB is also used in gastroenterology for chromoendoscopic detection of precancerous lesions [30–32], as well as for in vivo microscopic examination of the GI tract and bronchus using a novel endocytoscopic system [33, 34]. MB is a potent fluorophore which enters the nuclei and reversibly binds to the DNA, before being reabsorbed by the lymphatics. In order to give a fluorescent signal, MB needs to be excited around 660 nm and is therefore accessible to FCFM intravital imaging using this excitation wavelength. In our hands, no DNA damage could be observed using comet assay from lymphocytes exposed to methylene blue in vitro and 660 nm Cellvizio for 2 min.