Intraoperative confocal laser endomicroscopy for real-time in vivo tissue characterization during surgical procedures.

INTRODUCTION: Probe-based confocal laser endomicroscopy (pCLE) is an innovative technique providing real-time, in vivo optical biopsies. A previous ex vivo phase of the study (PERSEE) allowed identifying accurate pCLE criteria for the diagnosis of hepatic and peritoneal surgical specimens. This study aimed at evaluating the pCLE role for in vivo intra-abdominal tissue characterization during digestive cancer surgical procedures. METHODS: Between October 2014 and July 2015, consecutive patients diagnosed with digestive cancers and scheduled for a surgical resection or an exploratory laparoscopy were prospectively enrolled. Endomicroscopic images were acquired using a motorized Confocal Miniprobe™ with a bending distal tip providing easy access to abdominal organs. It was connected to an endomicroscopy system that allowed near-infrared illumination (at a wavelength of 785 nm) in conjunction with indocyanine green for contrast agent. A live audiovisual transmission was established between the surgeon and the pathologist for real-time interpretation of optical biopsies. Intraoperative pCLE performance for the diagnosis of suspicious nodules was assessed using corresponding surgical histopathology as reference standard. RESULTS: 21 consecutive patients were successfully enrolled. Live audiovisual transmission between the surgeon and the pathologist was successfully established in all cases. 62 pCLE sequences were acquired from different tissues [peritoneum (n = 27), liver (n = 21), lymph node (n = 4), diaphragm (n = 3), colon (n = 3), stomach (n = 2), and adrenal gland (n = 2)]. Malignant tissues were identified by fluorescently enhanced irregular cancerous tubes contrasting with dark glandular lumen and extracellular matrix. pCLE sensitivities and specificities were 67% and 100%, and 38% and 100% for peritoneal and hepatic carcinogenesis, respectively. One benign incident was reported during the trial with no patient consequence. CONCLUSIONS: Real-time intraoperative pCLE with near-infrared illumination is feasible and safe, provides additional information in terms of tissue characterization, and, in combination with telepathology, allows interactive collaboration between the surgeon and the pathologist during surgical procedures. Trial registration clinicaltrials.gov Identifier: NCT02312167.

Diagnostic accuracy of confocal laser endomicroscopy for the ex vivo characterization of peritoneal nodules during laparoscopic surgery.

BACKGROUND: Intraoperative characterization of peritoneal nodules can be challenging. Probe-based confocal laser endomicroscopy (pCLE) is an innovative technique enabling real-time microscopic analysis. This study aimed to assess the role of pCLE in the discrimination of benign versus malignant peritoneal nodules during laparoscopic staging. MATERIALS AND METHODS: During this prospective trial, pCLE was performed ex vivo on fresh samples of peritoneal nodules in 30 consecutive patients, after topical application of indocyanine green. The final diagnosis was obtained histologically, as per standard of care. pCLE image criteria for normal versus inflammatory versus malignant nodules were established (phase I); these criteria were tested retrospectively on selected videos by two examiners (phase II). The primary endpoints were values of accuracy in diagnosing malignant nodules. RESULTS: pCLE criteria for malignant nodules defined in phase I were: strongly fluorescent irregular clusters of cancerous cells, nonfluorescent nuclei of cancerous cells, and substantially lower fluorescence of the extracellular matrix fluorescence compared with cancerous clusters. In phase II, the detection rate of these criteria was significantly higher in malignant compared with benign nodules. Overall sensitivity, specificity, positive and negative predictive values to detect malignant nodules were 75, 100, 100 and 89 %, respectively. Interobserver agreement was substantial (kappa 0.69). CONCLUSION: These preliminary results suggest that pCLE is a valuable tool to discriminate between benign and malignant peritoneal nodules, with a high positive predictive value.

Optical fiber-based full Mueller polarimeter for endoscopic imaging using a two-wavelength simultaneous measurement method.

This paper reports a technique based on spectrally differential measurement for determining the full Mueller matrix of a biological sample through an optical fiber. In this technique, two close wavelengths were used simultaneously, one for characterizing the fiber and the other for characterizing the assembly of fiber and sample. The characteristics of the fiber measured at one wavelength were used to decouple its contribution from the measurement on the assembly of fiber and sample and then to extract sample Mueller matrix at the second wavelength. The proof of concept was experimentally validated by measuring polarimetric parameters of various calibrated optical components through the optical fiber. Then, polarimetric images of histological cuts of human colon tissues were measured, and retardance, diattenuation, and orientation of the main axes of fibrillar regions were displayed. Finally, these images were successfully compared with images obtained by a free space Mueller microscope. As the reported method does not use any moving component, it offers attractive integration possibilities with an endoscopic probe.

Polarimetric imaging of uterine cervix: a case study.

We present a preliminary investigation of macroscopic polarimetric imaging of uterine cervix. Orthogonal state contrast (OSC) images of healthy and anomalous cervices have been taken in vivo at 550 nm. Four ex vivo cervix samples have been studied in full Muller polarimetry, at 550 nm and 700 nm, and characterized in detail by standard pathology. One sample was totally healthy, another one carried CIN lesions at very early stage (CIN1) in its visible exocervical region, while for the other two samples more advanced (CIN3) lesions were present, together with visible glandular epithelium (ectropion). Significant birefringence has been observed in the healthy regions of all six samples, both in vivo and ex vivo. Standard treatments of the Mueller images of the ex vivo samples allowed to quantify both retardation and depolarization. Retardation reached 60° in healthy regions, and disappeared in the anomalous regions of the other three ex vivo samples. The depolarization power was largest in healthy regions, and lower in CINs and ectropion. Possible origins of the observed effects are briefly discussed.

Multispectral Mueller polarimetric imaging detecting residual cancer and cancer regression after neoadjuvant treatment for colorectal carcinomas.

This work is devoted to a first exploration of Mueller polarimetric imaging for the detection of residual cancer after neoadjuvant treatment for the rectum. Three samples of colorectal carcinomas treated by radiochemotherapy together with one untreated sample are analyzed ex vivo before fixation in formalin by using a multispectral Mueller polarimetric imaging system operated from 500 to 700 nm. The Mueller images, analyzed using the Lu-Chipmann decomposition, show negligible diattenuation and retardation. The nonirradiated rectum exhibits a variation of depolarization with cancer evolution stage. At all wavelengths on irradiated samples, the contrast between the footprint of the initial tumor and surrounding healthy tissue is found to be much smaller for complete tumor regression than when a residual tumor is present, even at volume fractions of the order of 5%. This high sensitivity is attributed to the modification of stromal collagen induced by the cancer. The depolarization contrast between treated cancer and healthy tissue is found to increase monotonously with the volume fraction of residual cancer in the red part of the spectrum. Polarimetric imaging is a promising technique for detecting short-time small residual cancers, which is valuable information for pathological diagnosis and patient management by clinicians.

Ex vivo photometric and polarimetric multilayer characterization of human healthy colon by multispectral Mueller imaging.

Healthy human colon samples were analyzed ex vivo with a multispectral imaging Mueller polarimeter operating from 500 to 700 nm in a backscattering configuration with diffuse light illumination impinging on the innermost tissue layer, the mucosa. The intensity and polarimetric responses were taken on whole tissues first and after progressive exfoliation of the outer layers afterwards. Moreover, these measurements were carried out with two different substrates (one bright and the other dark) successively placed beneath each sample, allowing a reasonably accurate evaluation of the contributions to the overall backscattered light by the various layers. For the shorter investigated wavelengths (500 to 550 nm) the major contribution comes from mucosa and submucosa, while for the longer wavelengths (650 to 700 nm) muscular tissue and fat also contribute significantly. The depolarization has also been studied and is found to be stronger in the red part of the spectrum, mainly due to the highly depolarizing power of the muscular and fat layers.

Impact of model parameters on Monte Carlo simulations of backscattering Mueller matrix images of colon tissue.

Polarimetric imaging is emerging as a viable technique for tumor detection and staging. As a preliminary step towards a thorough understanding of the observed contrasts, we present a set of numerical Monte Carlo simulations of the polarimetric response of multilayer structures representing colon samples in the backscattering geometry. In a first instance, a typical colon sample was modeled as one or two scattering "slabs" with monodisperse non absorbing scatterers representing the most superficial tissue layers (the mucosa and submucosa), above a totally depolarizing Lambertian lumping the contributions of the deeper layers (muscularis and pericolic tissue). The model parameters were the number of layers, their thicknesses and morphology, the sizes and concentrations of the scatterers, the optical index contrast between the scatterers and the surrounding medium, and the Lambertian albedo. With quite similar results for single and double layer structures, this model does not reproduce the experimentally observed stability of the relative magnitudes of the depolarizing powers for incident linear and circular polarizations. This issue was solved by considering bimodal populations including large and small scatterers in a single layer above the Lambertian, a result which shows the importance of taking into account the various types of scatterers (nuclei, collagen fibers and organelles) in the same model.

Ex-vivo characterization of human colon cancer by Mueller polarimetric imaging.

Cancerous and healthy human colon samples have been analyzed ex-vivo using a multispectral imaging Mueller polarimeter operated in the visible (from 500 to 700 nm) in a backscattering configuration with diffuse light illumination. Three samples of Liberkühn colon adenocarcinomas have been studied: common, mucinous and treated by radiochemotherapy. For each sample, several specific zones have been chosen, based on their visual staging and polarimetric responses, which have been correlated to the histology of the corresponding cuts. The most relevant polarimetric images are those quantifying the depolarization for incident linearly polarized light. The measured depolarization depends on several factors, namely the presence or absence of tumor, its exophytic (budding) or endophytic (penetrating) nature, its thickness (its degree of ulceration) and its level of penetration in deeper layers (submucosa, muscularis externa and serosa). The cellular density, the concentration of stroma, the presence or absence of mucus and the light penetration depth, which increases with wavelength, are also relevant parameters. Our data indicate that the tissues with the lowest and highest depolarizing powers are respectively mucus-free tumoral tissue with high cellular density and healthy serosa, while healthy submucosa, muscularis externa as well as mucinous tumor probably feature intermediate values. Moreover, the specimen coming from a patient treated successfully with radiochemotherapy exhibited a uniform polarimetric response typical of healthy tissue even in the initially pathological zone. These results demonstrate that multi-spectral Mueller imaging can provide useful contrasts to quickly stage human colon cancer ex-vivo and to distinguish between different histological variants of tumor.

Mueller matrix imaging of human colon tissue for cancer diagnostics: how Monte Carlo modeling can help in the interpretation of experimental data.

Colon samples with both healthy and cancerous regions have been imaged in diffuse light and backscattering geometry by using a Mueller imaging polarimeter. The tumoral parts at the early stage of cancer are found to be less depolarizing than the healthy ones. This trend clearly shows that polarimetric imaging may provide useful contrasts for optical biopsy. Moreover, both types of tissues are less depolarizing when the incident polarization is linear rather than circular. However, to really optimize an optical biopsy technique based on polarimetric imaging a realistic model is needed for polarized light scattering by tissues. Our approach to this goal is based on numerical Monte-Carlo simulations of polarized light propagation in biological tissues modeled as suspensions of monodisperse spherical scatterers representing the cell nuclei. The numerical simulations were validated by comparison with measurements on aqueous polystyrene sphere suspensions, which are commonly used as tissue phantoms. Such systems exhibit lower depolarization for incident linear polarization in the Rayleigh scattering regime, i.e. when the sphere diameters are smaller than the wavelength, which is obviously not the case for cell nuclei. In contrast, our results show that this behaviour can also be seen for "large" scatterers provided the optical index contrast between the spheres and the surrounding medium is small enough, as it is likely to be the case in biological tissues.