Mathieu Salaün

Human in vivo fluorescence microimaging of the alveolar ducts and sacs during bronchoscopy

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

Bronchoscopic Advances: On the Way to the Cells

In the past 15 years, new endoscopic methods have been developed in order to improve the detection of early bronchial cancers, with autofluorescence bronchoscopy being the leading technique. However, autofluorescence bronchoscopy is hampered by the low specificity of the fluorescence defect which ranges from 25 to 50%, and its limitation to the proximal bronchial tree from which arise only half of the lung cancers that are currently diagnosed. To overcome these limitations, other techniques emerge including video/autofluorescence bronchoscopy, narrow band imaging, optical coherence tomography, and ‘endomicroscopy’ using confocal fluorescent laser microscopy. These emerging techniques provide new insight into bronchology, extending the field of exploration from the proximal bronchus down to the most distal part of the lungs, and from macroscopy to in vivo cellular imaging. In the near future, they may enable in vivo, minimally invasive, ‘pathological grade’ evaluation of abnormal bronchial or parenchymal lung tissue. Whereas promising pioneer work has recently been published, careful assessment is required before these methods find a place in the evaluation strategy of early lung cancer and other lung diseases.

In vivo imaging of pulmonary alveolar proteinosis using confocal endomicroscopy

To the Editors: Fibred confocal fluorescence microscopy (FCFM) is a new technology that can provide microscopic imaging of a living tissue through a 1-mm flexible fibreoptic miniprobe 1, 2. To image the alveolar structures in vivo , the miniprobe is advanced through the working channel of the bronchoscope down to the alveolar ducts and sacs. In nonsmoking subjects, the FCFM is able to image the peripheral elastin framework of the alveolar ducts 3. In active smokers, the technique also images fluorescent alveolar macrophages which contain tobacco tar 3. The technique has the potential to improve the in vivo diagnosis of peripheral lung diseases during bronchoscopy. Herein we report the use of FCFM during bronchoscopy on a smoking patient presenting with diffuse alveolar and interstitial opacities on chest radiograph. The in vivo confocal fluorescence endomicroscopy in real-time showed the globular lipoproteinaceous material which is typical of pulmonary alveolar proteinosis (PAP). This diagnosis was confirmed by cytological analysis of the bronchoalveolar lavage (BAL) fluid, and by the presence of high levels of anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) serum antibodies. This is the first report of the in vivo confocal microscopic appearance of lipoproteinaceous material during bronchoscopy, which appears to be typical of PAP. A 42-yr-old, 25 pack-yr, active smoking male presented with a 4-month history of increasing breathlessness on exertion and dry cough. He reported a history of heavy and short household plaster dust exposure preceding the onset of symptoms. The physical examination was unremarkable. The chest radiograph showed bilateral alveolar and interstitial opacities. A high-resolution computed …

In vivo probe-based confocal laser endomicroscopy in amiodarone-related pneumonia

Probe-based confocal laser endomicroscopy (pCLE) allows microscopic imaging of the alveoli during bronchoscopy. The objective of the study was to assess the diagnostic accuracy of pCLE for amiodarone-related pneumonia (AMR-IP). Alveolar pCLE was performed in 36 nonsmoking patients, including 33 consecutive patients with acute or subacute interstitial lung disease (ILD), of which 17 were undergoing treatment with amiodarone, and three were amiodarone-treated patients without ILD. Nine out of 17 patients were diagnosed with high-probability AMR-IP (HP-AMR-IP) by four experts, and three separate observers. Bronchoalveolar lavage findings did not differ between HP-AMR-IP and low-probability AMR-IP (LP-AMR-IP) patients. In HP-AMR-IP patients, pCLE showed large (>20 &mgr;m) and strongly fluorescent cells in 32 out of 38 alveolar areas. In contrast, these cells were observed in only two out of 39 areas from LP-AMR-IP patients, in one out of 59 areas from ILD patients not receiving amiodarone and in none of the 10 areas from amiodarone-treated patients without ILD (p<0.001; HP-AMR-IP versus other groups). The presence of at least one alveolar area with large and fluorescent cells had a sensitivity, specificity, negative predictive value and positive predictive value for the diagnosis of AMR-IP of 100%, 88%, 100% and 90%, respectively. In conclusion, pCLE appears to be a valuable tool for the in vivo diagnosis of AMR-IP in subacute ILD patients. Probe-based confocal laser endomicroscopy appears useful for diagnosis of amiodarone-related pneumonia in subacute ILD http://ow.ly/ongMj

In vivo molecular microimaging of pulmonary aspergillosis

The early diagnosis of invasive pulmonary aspergillosis (IPA) is challenging. Fibered confocal fluorescence microscopy (FCFM) is a new technique that allows in vivo imaging of the lung microstructure during bronchoscopy. In this study, we investigated the ability of FCFM to detect a fluorescent peptide-tracer bound to Aspergillus fumigatus in experimental IPA in 13 immunosuppressed, non-neutropenic rats. Subpleural IPA microabscesses were imaged through a transthoracic window using FCFM in vivo after i.v. injection of the c(CGGRLGPFC)-NH2([FITC]) peptide (n = 7) or saline. Results were compared to 10 immunosuppressed, non-infected rats and to six immunosuppressed Geosmithia argillacea-infected rats with and without i.v. injection of the peptide. The peptide in vitro specifically labeled A. fumigatus grown under biofilm growth conditions but not G. argillacea. In vivo, FCFM showed a local infiltration of fluorescent host cells in both Aspergillus and Geosmithia infections. Lung/inner thoracic wall fluorescence intensity ratio (FI) did not differ before and after peptide administration on healthy lung areas, on non-specific inflammatory areas, or on Geosmithia micro-abscesses. In contrast, FI increased from 1.05 without peptide to 1.83 after peptide injection on Aspergillus micro-abscesses (p < 0.0001). In peptide-injected rats, FI from IPA foci was higher than from non-specific inflammation or from Geosmithia abscesses (p ≤ 0.002). Using c(CGGRLFPC)-NH2([FITC]) peptide, FCFM allows the in vivo specific imaging of pulmonary aspergillosis. These data provide the basis for the in vivo diagnosis of human pulmonary aspergillosis using alveolar confocal endomicroscopy.

In vivo and in situ imaging of experimental invasive pulmonary aspergillosis using fibered confocal fluorescence microscopy

Invasive pulmonary aspergillosis (IPA) is a highly fatal disease in immunosuppressed patients. In this study, we assessed fibered confocal fluorescence microscopy (FCFM), a new endoscopic technique that enables in vivo microscopic imaging of the distal lung, as a tool for in vivo imaging of IPA. IPA was induced in immunosuppressed rats using a wild strain of Aspergillus fumigatus (n = 6) or a fluorescent transformed TAG-RFP A. fumigatus strain (n = 10). Subpleural areas of pulmonary infection were imaged in vivo using FCFM employing a transthoracic approach. Results were compared to three immunosuppressed control groups, i.e., non-inoculated rats (n = 4), rats inoculated with sterile Phospate-buffer saline (PBS; n = 5), and rats inoculated with Geosmithia argillacea (n = 6). Only hyphae of TAG-RFP A. fumigatus were detectable both in vitro and in vivo by FCFM. In vivo, a local infiltration of fluorescent alveolar macrophages was observed with FCFM in IPA areas in all fungal infections groups, but also in focal inflammatory areas in the immunosuppressed PBS group. A specific fibrillar fluorescence was observed in IPA areas with the TAG-RFP A. fumigatus group, with a 83% sensitivity, a 100% specificity, a 100% positive predictive value and 94% negative predictive value. FCFM provides a new tool to study host-aspergillus interactions in vivo.

Endomicroscopie confocale en pneumologie : de la bronche à l’alvéole

INTRODUCTION Fibred confocal fluorescence microscopy, also named probe based confocal laser endomicroscopy (pCLE), is a new endoscopic technique that can be applied for in-vivo microscopic imaging of both upper airways and distal lung structures during bronchoscopy. BACKGROUND Two recent in-vivo human studies using pCLE at 488nm light excitation have described the normal fluorescence endomicroscopic features of the bronchial wall and the elastic framework of the alveolus. These studies have demonstrated that elastin, a major component of the bronchial basement membrane and of the acinar elastic framework, is the main endogenous fluorophore in the non-smoking population. In smoking subjects, the tobacco tar itself is highly fluorescent and, thereby, acts as an additional fluorophore, allowing study of the macrophage alveolitis associated with smoking. These studies have also confirmed the safety of this endoscopic procedure. VIEWPOINT In the near future, confocal endomicroscopy of the airways should make it possible to investigate the semiology of focal and diffuse distal lung diseases, to characterize cancerous and precancerous lesions of both upper and distal airways and to study the lung microcirculation. These studies may also use exogenous molecular fluorescent probes, which will enable functional imaging of the lung structures in-vivo. CONCLUSION Confocal endomicroscopy has the potential to explore accurately the peripheral lung in-vivo and may become a useful tool to improve endoscopic diagnosis of many lung diseases.

Long-term follow-up of severe dysplasia and carcinoma in situ of the bronchus.

To the Editor: In a previous issue of the journal, Banerjee1 brilliantly reviewed the available data from the literature about the natural history of bronchial preinvasive lesions. We congratulate the author for this very important and difficult work. In his review, the author cited our work published in the American Journal of Respiratory and Critical Care Medicine in 20012 that described the 24 months follow-up of 416 preinvasive lesions in 104 patients. Indeed, we agree with the author’s conclusions that limited individual lesion follow-up in most studies, as well as interferences with local or even systemic treatments, complicate the assessment of the potential aggressiveness of lesions known to be characterized by a long carcinogenesis process. In a recent published work, not cited in Banerjee’s review, we provided some answers to these limitations3 in studying the long-term evolution of 37 patients and 54 high-grade preinvasive lesions—31 carcinoma in situ (CIS) and 23 severe dysplasia (SD)—during a 12year period. Our study was performed using auto-fluorescence bronchoscopy with repeated bronchial biopsy sampling over time, with a mean of seven biopsies per individual lesion during the follow-up and molecular analysis of the baseline lesions. We believe that this very long monitoring authorizes to draw reliable conclusions on the aggressiveness of high-grade lesions, despite the fact that conservative endobronchial treatment was applied to persisting or relapsing lesions. To reliably identify the factors that are linked to the lesions’ evolution over time, we used a very restrictive definition of “progression,” including local progression to invasive cancer. We also defined a locally treated lesion as “treatment resistant” that recurred at any time during the monitoring period and “treatment sensitive” those that did not reoccur after local treatment, whereas regressing lesions were those that spontaneously disappeared without treatment. This allowed us to classify accurately the more aggressive lesions in the progression or treatment resistant groups, while ascertaining that the spontaneously regressing lesions were truly benign ones (Table 1). In this study, we observed that all the lesions that progressed to invasive cancer were initially classified as CIS (7 of 7) according to World Health Organization 1999 standards,4 and 10 of 31 CIS were resistant to several courses of endoscopic treatments, whereas 10 of 31 CIS were treatment sensitive and only 4 of 31 CIS spontaneously regressed without recurrence during follow-up. Our study also provided valuable information on long-term SD and CIS patients outcome, with, at the study end point, nine deaths attributable to lung cancer including four deaths directly related to the progression of the initial high-grade bronchial lesion. In addition, using careful laser microdissection and robust molecular analysis on the baseline lesions,3,5 we could demonstrate that losses of heterozygosity (LOH) of chromosome 3p and 9p are significantly more frequent in CIS compared with SD, whereas 3p LOH seems to be a strong predictor of progression in the whole group of lesions and in the CIS group (Table 1). Altogether, our results lend strong support to the World Health Organization classification for the premalignant bronchial lesions, which clearly differentiates CIS from SD,4 not only in confirming their different outcomes, as we had previously observed,2 but also in providing evidence of significant differences at the molecular level. Finally, our contribution shows that CIS histology as defined by 1999 WHO classification and the molecular analysis of 3p LOH are useful indicators of the evolution of high-grade preinvasive bronchial lesions. The molecular analysis could easily be integrated in the management decision tree of CIS and SD of the bronchial epithelium.

Scattering features for lung cancer detection in fibered confocal fluorescence microscopy images

OBJECTIVE To assess the feasibility of lung cancer diagnosis using fibered confocal fluorescence microscopy (FCFM) imaging technique and scattering features for pattern recognition. METHODS FCFM imaging technique is a new medical imaging technique for which interest has yet to be established for diagnosis. This paper addresses the problem of lung cancer detection using FCFM images and, as a first contribution, assesses the feasibility of computer-aided diagnosis through these images. Towards this aim, we have built a pattern recognition scheme which involves a feature extraction stage and a classification stage. The second contribution relies on the features used for discrimination. Indeed, we have employed the so-called scattering transform for extracting discriminative features, which are robust to small deformations in the images. We have also compared and combined these features with classical yet powerful features like local binary patterns (LBP) and their variants denoted as local quinary patterns (LQP). RESULTS We show that scattering features yielded to better recognition performances than classical features like LBP and their LQP variants for the FCFM image classification problems. Another finding is that LBP-based and scattering-based features provide complementary discriminative information and, in some situations, we empirically establish that performance can be improved when jointly using LBP, LQP and scattering features. CONCLUSIONS In this work we analyze the joint capability of FCFM images and scattering features for lung cancer diagnosis. The proposed method achieves a good recognition rate for such a diagnosis problem. It also performs well when used in conjunction with other features for other classical medical imaging classification problems.

An SVM-based distal lung image classification using texture descriptors

A novel imaging technique can now provide microscopic images of the distal lung in vivo, for which quantitative analysis tools need to be developed. In this paper, we present an image classification system that is able to discriminate between normal and pathological images. Different feature spaces for discrimination are investigated and evaluated using a support vector machine. Best classification rates reach up to 90% and 95% on non-smoker and smoker groups, respectively. A feature selection process is also implemented, that allows us to gain some insight about these images. Whereas further tests on extended databases are needed, these first results indicate that efficient computer based automated classification of normal vs. pathological images of the distal lung is feasible.