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1 Rouen University Hospital, Rouen, France; 2 Faculté de Médecine-Pharmacie, Rouen, France; LITIS EA 4108 (groupe Quant-IF), Rouen, France; 3 Université Pierre et Marie Curie, Paris, France; and 4 UMR CNRS 7033, BioMoCeTi, Paris, France
Correspondence and requests for reprints should be addressed to Luc Thiberville, M.D., Clinique Pneumologique, Hôpital Charles Nicolle, CHU de Rouen, 1 rue de Germont, 76031 Rouen Cedex, France. E-mail: Luc.Thiberville{at}univ-rouen.fr
ABSTRACT
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 µm, a field of view of 600 µm, 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.
Key Words: bronchoscopy • diagnostic imaging • laser scanning • confocal microscopy • pulmonary alveoli
Over the last decade, in vivo intravital microscopy has proven to be an important tool in physiology and pathophysiology research. Within this field, confocal microscopy offers unique capabilities, allowing in vivo optical sectioning of cells and tissue, with enhanced lateral and axial resolutions (1, 2). The principle by which confocal microscopy images a thin slide of a sample relies on both the use of a narrow point source on the illumination path and of a small aperture or pinhole on the light detection path. According to this principle, a laser source (the point source) focuses on a single spot in the sample, and the light emitted from this focal point is imaged through the pinhole onto a detector. This results in the rejection of the light coming from depths adjacent to the focal plane region, and therefore of out-of-focus information from the material above and below a very thin plane of focus. The illumination and detection systems being conjugated on the same focal plane are termed "confocal." To obtain a two-dimensional image within the tissue, confocal microscopes use various systems that scan the sample in both lateral dimensions.
Translating the confocal microscopy principles into the clinic for endomicroscopic explorations is currently the subject of significant scientific efforts (1, 2), which recently ended in the availability of two commercial systems for both animal (3–5) and human in vivo explorations (6–8).
CONFOCAL ENDOMICROSCOPES FOR HUMAN EXPLORATION
Confocal endomicroscopes aim at providing to the clinician "optical biopsies,"—that is, in vivo microscopic imaging—of a living tissue (9, 10). Such systems have been successfully applied to the in vivo explorations of the human skin (11, 12), cervix (13), and oral cavity (14, 15), as well as to the endomicroscopic exploration of the gastric and colonic mucosae, biliary tract (16–19), and more recently to the microscopic imaging of the proximal (7) and distal respiratory systems (8).
Both commercially available confocal endomicroscopes use fluorescence imaging instead of reflectance imaging. The consequences of this choice will be discussed later in this article. These two systems can be distinguished by the technical approach used to conduct the light to the tissue. The distal scanning principle is used in the Optisan/Pentax endomicroscopic system (20). In distal scanning, the light is conducted by a single fiber back and forth from the distal tip of the system, and the scanning function is accomplished by a very small scanhead (4.5 cm long x 3.5 mm diameter) that is included in the distal end of the endoscope. Optiscan endomicroscopic images from the gastro intestinal tract appear very close to conventional histology, with a lateral resolution below 1 µm and optical slices of 7 µm for a field of view of 475 x 475 µm. The system offers the possibility to adjust the Z-depth range from 0 to 250 µm below the contact surface, so that three-dimensional structures in the specimen and successive layers of the mucosae can be imaged. Two potential drawbacks explain why this system is not yet available for the respiratory tract imaging. First, because of the added sizes of the distal scanhead, working channel, conventionnal light guide, and CCD camera, the diameter of the distal tip of the endoscope is currently larger than 12 mm, a size barely compatible with the exploration of the human trachea and large main bronchi. Second, the miniaturization of the distal scanhead results in scanning rates of 1 frame/second, which needs a very efficient stabilization system of the distal tip of the endoscope onto the mucosae, to produce crisp microscopic images of the epithelium.
The second commercially available confocal endomicroscopy system (Cellvizio; Mauna Kea Technologies, Paris, France) uses the principle of proximal scanning, in which the illumination light scans the proximal part of a coherent fiber bundle or miniprobe. This bundle conducts the light back and forth from the imaged area at the tip of the miniprobe (21). The light delivery, scanning, spectral filtering, and imaging systems are located at the proximal part of the device, the distal part being a separate miniprobe, including both the fiber bundle and its connector to the Laser Scanning Unit (Figure 1).
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The main advantages of this design are the very small size and the flexibility of the probe that can reach the more distal part of the lungs (8), as well as the fast image collection speed that helps to avoid artifacts due to tissue movement.
Specific miniprobes for bronchial and alveolar imaging have a diameter of 1 mm that can enter the working channel of any adult bronchoscope. These miniprobes are devoid of distal optics and have a depth of focus of 0 to 50 µm and a lateral resolution of 3 µm, for a field of view of 600 x 600 µm. The system produces endomicroscopic imaging in real time at 9 to 12 frames/second.
Two different wavelengths are available. The Cellvizio 488 nm is used for autofluorescence imaging of the respiratory tract as well as for fluorescein-induced imaging of the GI tract (7, 8, 22). Another device at 660 nm excitation can be used for epithelial cell imaging after topical application of exogenous fluorophores such as methylene blue (23–25).
The main limitations of the system are related to its maximal imaging capabilities (30,000 pixels), which restrict the lateral resolution to the fiber intercore distance (3 µm), and the fact that the focus point of the system cannot be adjusted. As discussed later, interpretation of the data also relies on the fluorescence properties of the imaged tissue.
HUMAN IN VIVO CONFOCAL MICROIMAGING OF THE NORMAL LUNG USING FCFM
FCFM Imaging of the Proximal Bronchi
FCFM can easily be performed during a fiberoptic bronchoscopy under local anesthesia (7, 8). The technique of in vivo bronchial FCFM 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, FCFM produces very precise microscopic fluorescent images of the bronchial basement membrane zone. As seen in Figure 2, FCFM bronchial microimaging reveals a mat of large fibers mainly oriented along the longitudinal axis of the airways with crosslinked smaller fibers, as well as larger openings—100 µm 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).
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As a result, 488 nm excitation FCFM 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 FCFM device using several wavelengths (30), or devices based on a multiphoton approach (2), may enable imaging of collagen, elastin, and flavins simultaneously.
Distal Lung FCFM Imaging In Vivo: From the Distal Bronchioles Down to the Lung Acini
Earlier work has demonstrated that elastin represents up to 50% of the peripheral lung connective tissue fibers (31). 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 (32, 33). After our previous observation in the proximal bronchi, we demonstrated that FCFM could also image the elastic framework of the distal lung (8, 28).
For distal lung microimaging, we use a 4.4-mm bronchoscope (MP60 model; Olympus, Tokyo, Japan) that is inserted into the airways down to the smallest reachable bronchi. The FCFM miniprobe is then gently advanced into the distal bronchiole until the alveolar system is observed. During the procedure, several acinar areas can be successively explored by selecting different bronchioles, with real-time imaging. Once the alveoli are reached, the probe is slightly pulled back until the contact is lost, to ensure that the probe compression effect onto the alveolar system is minimal.
Experience of FCFM alveolar imaging in more than 150 healthy volunteers and patients has demonstrated that the technique is very well tolerated under topical anesthesia, in spontaneously breathing awake subjects. Due to the lack of pain receptors in the bronchial tree down to the subpleural level, the penetration of the miniprobe into the pulmonary lobule through the distal bronchiolar wall is painless (8). In addition, acinar imaging is not associated with significant bleeding in the proximal airways, in contrast to what is usually observed with transbronchial biopsy sampling. This can be explained by the low pressure in the alveolar capillaries that could be altered during the progression of the probe, as well as by the smooth design of the probe tip that can displace the extra-alveolar microvessels without damage. Finally, no pleural complication occurred in our experience when the system is used in awake, spontaneously breathing subjects, despite multiple lung segment imaging during the endoscopy.
Acinar FCFM Imaging in Nonsmoking Subjects
Acinar imaging is easily obtained by pushing forward the probe a few centimeters after the endoscope is distally blocked into a subsegmental bronchi. Due to the respective sizes of the probes and of the distal bronchiolar structures, the presence of alveolar buds in the respiratory bronchioles is rarely identified during the bronchoalveoscopy procedure (Figure 2D). When progressing toward the more distal parts of the lungs, the entry into the alveolar space is therefore obtained by penetration through the bronchiolar wall.
In nonsmokers, FCFM mainly produces images of the elastic fibers that encircle the alveolar openings, reinforce the virtual wall of the alveolar ducts, and surround the extra-alveolar microvessels (Figure 3).
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The reproducibility of the technique has been demonstrated in a series of healthy volunteers, where alveolar opening sizes measured from in vivo imaging were found normally distributed with mean values (around 275 µm) close to what is observed using complex stereological methods in vitro (32, 33), with thickness of the elastic fibers 10 ± 2.7 µm. In the published series, smaller alveolar mouths were observed in the right upper lobe and paracardiac segments, presumably in relation to the lower ventilation of these segments in supine position (8). The technique also enables precise measurements of the extra alveolar lobular microvessels. A significant variation in the intensity of the autofluorescence signal could be observed between the subjects in relation with their age, the oldest individuals presenting the strongest signal.
Acinar and Alveolar Imaging in Active Smokers
Alveolar fluorescence imaging in active smokers dramatically differs from imaging in nonsmokers (Figures 3C and 3D). 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. Using FCFM, morphologic markers of alveolar macrophage activation such as size, number, and mobility can be assessed that appear highly correlated with the amount of cigarettes smoked per day (8).
The alveolar autofluorescence intensity appears significantly higher in active smokers compared with non-smokers, in relation to the intensity of the macrophage alveolitis. In situ alveolar microspectrometric measurements have been performed in active smokers, which evidenced that the main fluorophore contributing to the FCFM alveolar signal corresponds to the tobacco tar by itself, explaining this difference (8, 28). Due to this specific contrast imaging in smokers, details of the alveolar and ductal surface could often be obtained. (Figure 3C).
POTENTIAL CLINICAL APPLICATIONS OF BRONCHOALVEOLAR CONFOCAL IMAGING
Proximal Bronchial Exploration
Preliminary studies have shown that per endoscopic FCFM could be used to study specific basement membrane remodeling alterations in benign or malignant/premalignant bronchial alterations (7). The FCFM microstructure of the bronchial walls underlying premalignant epithelia is significantly altered. In these precancerous conditions, the elastic fibered pattern of the lamina reticularis is absent or disorganized in almost every preinvasive lesion, supporting the hypothesis of an early degradation of the basement membrane components in preinvasive bronchial lesions. However, although this observation shed some light on the origin of the autofluorescence defect in precancerous bronchial lesions, the absence of epithelial cell visualization does not allow the technique to differentiate between the different grades of progression of the precancerous bronchial lesions such as metaplasia/dysplasia/carcinoma in situ.
To be successfully applied to the exploration of precancerous/cancerous bronchial epithelial layer, the FCFM 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). Exogenous fluorophores that could be activated at 488 nm (such as Acriflavin—a putative mutagen agent—or fluorescein solution, which does not stain the nuclei) (34) are not approved for intrabronchial use. Recently, Lane and coworkers have used a confocal microendoscope prototype at 488 nm excitation and topical physiologic PH cresyl violet to provide cellular contrast in the bronchial epithelium both in vitro and in vivo (35).
Methylene blue is a nontoxic agent that is commonly used during bronchoscopy for the diagnosis of bronchopleural fistulae. MB is also used in gastroenterology for chromo-endoscopic detection of precancerous lesions (36–38), as well as for in vivo microscopic examination of the GI tract and bronchus using a novel endocytoscopic system (39, 40). MB is a potent fluorophore that enters the nuclei and reversibly binds to the DNA, before being reabsorbed by the lymphatics. 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.
Preliminary study has demonstrated that Cellvizio 660/topical methylene blue makes it possible to reproducibly image the epithelial layer of the main bronchi as well as cellular patterns of peripheral solide lung nodules (24, 25) (Figure 4). Future studies using this technique could make it possible to differentiate normal, premalignant, and malignant alterations at the microscopic level. If this strategy is successful, FCFM may become a very powerful technique for in vivo diagnosis of early malignant and premalignant conditions of the bronchial tree, allowing the analysis of both the epithelial and subepithelial layers during the same procedure.
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Whereas the technique appears to have a great potential for in vivo distal lung explorations, both experimentally and clinically, some technical aspects and potential limitations for its clinical use should be discussed.
First, because of the orthogonal branching and the small caliber of the terminal and respiratory bronchioles in humans compared with the probe size, alveolar imaging regularly bypasses the transitional respiratory bronchioles. This could represent a limitation for the study of the distal membranous and respiratory bronchioles, unless thinner probes, currently devoted to experimental animal imaging, become clinically available in the future (5).
Second, the probe progression into the lobule supposes the disruption of alveolar walls, followed by a compression effect on the more resistant ductal structures. Minimal imaging distortion is observed when the probe is applied on the axis of the duct, resulting in the visualization of more details in the background planes than theoritically allowed by the 50-µm depth of focus of the system (5). This compression effect may be difficult to control in vivo. In our hands, the more reproducible results were obtained in gently pulling back the probe once the alveolar imaging is obtained and analyzing the last images before the contact is lost. Therefore, future studies should include a standardization of the endoscopic technique.
Third, fluorescence microimaging by itself has some advantages and limitations. In contrast to reflectance imaging, fluorescence microimaging is devoid of interference with the reflected and refracted light at the air–liquid interface, because the backscattered excitation light is filtered out by the detection system. This merely produces a small decrease in the signal intensity at the air–liquid interface, without any other optical artifact. This property allows the visualization of bubble-like structures as well as the presence of fluorescent cells within this liquid phase. These observations indicate that a small quantity of liquid interposes at the probe tip that may contribute to the imaging signal. The nature of this liquid interface as well as its consequences on alveolar imaging should be further analyzed.
On the other hand, FCFM exclusively records the signal coming from fluorescent structures in response to appropriate excitation wavelengths. In this regards, in vivo FCFM in nonsmokers only images the elastin of the peripheral and axial connective tissues. Data from the literature indicate that such information might be helpful for the exploration of several peripheral lung diseases (41, 42). However, as the confocal fluorescence imaging of the distal lung is likely to appear very different from the corresponding histopathology, the semeiology of the FCFM elastin lung network imaging will have to be characterized in pathological conditions. In this regard, in vivo comparative studies on confocal alveolar imaging in patients with peripheral lung diseases and healthy volunteers appear mandatory, before the place of FCFM in the routine exploration of the peripheral lung could be appreciated.
Until now, confocal microendoscopy of the airways has only used endogenous autofluorescence or simple fluorescent contrast agents to visualize the in vivo cellular and interstitial organization of the airways and distal lung parenchyma. In the future, using molecular contrast coumponds, it will be possible to extend the range of biomarkers that may be imaged. Pilot studies exploring this strategy have recently been published, that provided specific confocal imaging of molecular probes in precancerous conditions of the oral cavity ex vivo (43) and of colonic dysplasia in vivo (44). Coupled to FCFM, molecular imaging may help in the future to enable early diagnosis, rapid typing of molecular markers, and assessment of therapeutic outcome in many lung diseases.
FOOTNOTES
Funded by Program Hospitalier de Recherche Clinique 2007, French ministry of Health; Canceropole Nord Ouest, Lille, France; Institut National du Cancer (Grant INCa-Lilly # 0612-3D1317-31), Boulogne Billancourt, France; and Association pour le traitement à Domicile de l'Insuffisance Respiratoire (ADIR), Rouen, France.
Conflict of Interest Statement: L.T. received grant support from the French National Institute of Cancer, Lilly Institute, and is listed as a coinventor in a patent concerning the use of a system for imaging confocal fluorescence in vivo in situ. M.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.D. received lecture fees from Actelion up to $1,000 for nonpromotional CME activity. S.M.-S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.V.-B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.B.-H. is listed as a coinventor in a patent concerning the use of a system for imaging confocal fluorescence in vivo in situ.
(Received in original form February 23, 2009; accepted in final form April 28, 2009)
REFERENCES
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