Ultra-fast confocal fluorescence microscopy for neck lymph node imaging in head and neck cancer.

OBJECTIVES: We evaluated ultra-fast confocal fluorescence microscopy (UFCM) as a new modality for pathology practice in head and neck cancer (HNC). This was assessed through an ex vivo study to estimate the accuracy, specificity, and sensitivity of interpretation of UFCM images by pathologists for the detection of metastatic lymph nodes in HNC patients undergoing sentinel lymph node biopsy or selective or complete neck dissection. MATERIALS AND METHODS: 44 patients with 32 cN0 and 12 cN+ HNC were included. The macroscopically non-invaded fresh bisected lymph nodes were stained with acridine orange and imaged with Histolog® Scanner (UFCM). Two pathologists interpreted independently the UFCM images postoperatively and gave a consensus diagnosis in case of disagreement. The gold standard was the diagnosis based on hematoxylin-eosin-saffron (HES) sections. RESULTS: 201 lymph nodes were imaged by UFCM. Thirty nodes (15 %) were invaded on final histology: 3 with micrometastases and 27 with macrometastases. The concordance rate between the pathologists on the UFCM images was 192/201 = 95.5 % and the Cohen kappa coefficient was 0.80. The accuracy of UFCM was 95.5 % (95 %CI: 91.7 %-97.9 %) with a high specificity at 98.8 % (95 %CI: 95.8 %-99.9 %) but an insufficient sensitivity at 76.7 % (95 %CI: 57.7 %-90.1 %). The three micrometastases and four of the 27 macrometastases were missed on UFCM images. CONCLUSION: The UFCM is providing promising detection values with a very good specificity and moderate sensitivity carrying room for improvement.

Breast tissue imaging atlas using ultra-fast confocal microscopy to identify cancer lesions.

New generation ultra-fast fluorescence confocal microscopy (UFCM) allows to image histological architecture of fresh breast tissue and may be used for ex vivo intraoperative analysis for margin status. The criteria to identify breast tumoral and non-tumoral tissues in UFCM images are still objects of investigation. The objective of the study was to create an atlas of ex vivo UFCM images of breast tissues and breast carcinomas based on the first extensive collection of large field-of-view UFCM breast images. One hundred sixty patients who underwent conserving surgery for breast cancer were included. Their fresh surgical specimens were sliced, stained with acridine orange, and imaged at high resolution with large-field-of-view UFCM. The resulting images were digitally false colored to resemble frozen sections. Each UFCM image was correlated with the corresponding definitive histology. Representative images of normal tissue, inflammation, benign lesions, invasive carcinoma (IC), and ductal carcinoma in situ (DCIS) were collected. A total of 320 large-field images were recorded from 58 IC of no special type, 44 invasive lobular carcinomas, 1 invasive mucinous carcinoma, 47 DCIS, 2 lobular carcinomas in situ, and 8 specimens without cancer. Representative images of the main components of the normal breast and the main types of ICs and DCIS were annotated to establish an UFCM atlas. UFCM enables the imaging of the fresh breast tissue sections. Main morphological criteria defined in traditional histopathology such as tissue architecture and cell features can be applied to describe UFCM images content. The generated atlas of the main normal or tumoral tissue features will support the adoption of this optical technology for the intraoperative examination of breast specimens in clinical practice as it can be used to train physicians on UFCM images and develop artificial intelligence algorithms. Further studies are needed to document rare breast lesions.

Breast carcinoma detection in ex vivo fresh human breast surgical specimens using a fast slide-free confocal microscopy scanner: HIBISCUSS project.

BACKGROUND: New generation ultra-fast fluorescence confocal microscopy allows the ex vivo intraoperative analysis of fresh tissue. The High resolution Imaging for Breast carcInoma detection in ex vivo Specimens after breast Conserving sUrgery by hiStolog Scanner (HIBISCUSS) project aimed to develop an online learning program to recognize the main breast tissue features on ultra-fast fluorescence confocal microscopy images and to evaluate the performance of surgeons and pathologists in diagnosing cancerous and non-cancerous breast tissue in ultra-fast fluorescence confocal microscopy images. METHODS: Patients who underwent conservative surgery or mastectomy for breast carcinoma (invasive or in situ lesions) were included. The fresh specimens were stained with a fluorescent dye and imaged using a large field-of-view (20 cm2) ultra-fast fluorescence confocal microscope. RESULTS: One hundred and eighty-one patients were included. The images from 55 patients were annotated to generate learning sheets and images from 126 patients were blindly interpreted by seven surgeons and two pathologists. The time for tissue processing and ultra-fast fluorescence confocal microscopy imaging was between 8 and 10 min. The training program was composed of 110 images divided into nine learning sessions. The final database for blind performance assessment comprised 300 images. The mean duration for one training session and one performance round was 17 and 27 min respectively. The performance of pathologists was almost perfect with 99.6 per cent (standard deviation (s.d.) 5.4 per cent) accuracy. Surgeons' accuracy significantly increased (P = 0.001) from 83 per cent (s.d. 8.4 per cent) in round 1 to 98 per cent (s.d. 4.1 per cent) in round 7 as well as the sensitivity (P = 0.004). Specificity increased without significance from 84 per cent (s.d. 16.7 per cent) in round 1 to 87 per cent (s.d. 16.4 per cent) in round 7 (P = 0.060). CONCLUSION: Pathologists and surgeons showed a short learning curve in differentiating breast cancer from non-cancerous tissue in ultra-fast fluorescence confocal microscopy images. Performance assessment for both specialties supports ultra-fast fluorescence confocal microscopy evaluation for intraoperative management. REGISTRATION NUMBER: NCT04976556 (http://www.clinicaltrials.gov).

Intraoperative Autofluorescence Imaging for Parathyroid Gland Identification during Total Laryngectomy with Thyroidectomy.

OBJECTIVE: Hypoparathyroidism is a known complication of total laryngectomy, although parathyroid preservation and/or reimplantation are not routine. Autofluorescence is a new technique for identifying parathyroid glands intraoperatively. The aim of this study was to evaluate the feasibility of autofluorescence in this context. MATERIALS AND METHODS: A retrospective study of patients undergoing total laryngectomy/pharyngectomy with concomitant thyroidectomy using the Fluobeam® (Fluoptics, Grenoble, France) and frozen section of a parathyroid fragment in case of reimplantation. The rates of identification using autofluorescence, reimplantation, and hypoparathyroidism were evaluated. RESULTS: Eighteen patients (16 males, median age 67) underwent total laryngectomy/pharyngectomy with total thyroidectomy (n = 12) or hemithyroidectomy (n = 6). A median of 2 parathyroid glands were identified per patient. Ninety-two percent were identified by autofluorescence before visualisation. All parathyroids were reimplanted due to devascularization. Temporary hypoparathyroidism occurred in nine patients, and was permanent in one patient. After 34 months of median follow-up (range 1-49), no tumor recurrence was observed in the reimplantation sites. CONCLUSIONS: To our knowledge, this is the largest study to evaluate autofluorescence during total laryngectomy with thyroidectomy. No tumor recurrence occurred in the sites of parathyroid reimplantation.

Intraoperative Parathyroid Gland Identification Using Autofluorescence Imaging in Thyroid Cancer Surgery with Central Neck Dissection: Impact on Post-Operative Hypocalcemia.

Hypoparathyroidism is the most frequent complication in thyroid surgery. The aim of this study was to evaluate the impact of intraoperative parathyroid gland identification, using autofluorescence imaging, on the rate of post-operative (PO) hypoparathyroidism in thyroid cancer surgery. Patients undergoing total thyroidectomy with central neck dissection from 2018 to 2022 were included. A prospective cohort of 77 patients operated on using near-infrared autofluorescence (NIRAF+) with the Fluobeam® (Fluoptics, Grenoble, France) system was compared to a retrospective cohort of 94 patients (NIR-). The main outcomes were the rate of PO hypocalcemia, with three cutoffs: corrected calcium (Cac) < 2.10 mmol/L, <2.00 mmol/L and <1.875 mmol/L, and the rate of permanent hypoparathyroidism, at 12 months. The rate of PO Cac < 2.10 mmol/L was statistically lower in the NIRAF+ group, compared to the control group (36% and 60%, p = 0.003, respectively). No statistically significant difference was observed for the other two thresholds. There was a lower rate of permanent hypoparathyroidism in the NIRAF+ group (5% vs. 14% in the control group), although not statistically significant (p = 0.07). NIRAF is a surgically non-invasive adjunct, and can improve patients' outcomes for thyroid cancer surgery by reducing post-operative temporary hypoparathyroidism. Larger prospective studies are warranted to validate our findings.

Axillary reverse mapping using near-infrared fluorescence imaging in invasive breast cancer (ARMONIC study).

BACKGROUND: Axillary lymph node dissection (ALND) in patients with breast cancer has potential side effects, including upper-limb lymphedema. Axillary reverse mapping (ARM) is a technique that enables discrimination of the lymphatic drainage of the upper limb in the axillary lymph node basin from that of the breast. We aimed to evaluate ARM node identification by near-infrared (NIR) fluorescence imaging during total mastectomy with ALND and then to analyze potential predictive factors of ARM node involvement. METHODS: The study enrolled 119 patients diagnosed with invasive breast cancer with an indication for ALND. NIR imaging using indocyanine green dye was performed in 109 patients during standard ALND to identify ARM nodes and their corresponding lymphatic ducts. RESULTS: 94.5% of patients had ARM nodes identified (95%CI = [88.4-98.0]). The ARM nodes were localized in zone D in 63.4% of cases. Metastatic axillary lymph nodes were found in 55% in the whole cohort, and 19.4% also had metastasis in ARM nodes. Two patients had metastatic ARM nodes but not in the remaining axillary lymph nodes. No serious adverse events were observed. Only the amount of mitosis was significantly associated with ARM node metastasis. CONCLUSIONS: ARM by NIR fluorescence imaging could be a reliable technique to identify ARM nodes in real-time when ALND is performed. The clinical data compared with ARM node histological diagnosis showed only the amount of mitosis in the diagnostic biopsy is a potential predictive factor of ARM node involvement. CLINICAL TRIAL REGISTRATION: NCT02994225.

Near-Infrared Fluorescence Axillary Reverse Mapping (ARM) Procedure in Invasive Breast Cancer: Relationship between Fluorescence Signal in ARM Lymph Nodes and Clinical Outcomes.

The near-infrared (NIR) fluorescence axillary reverse mapping (ARM) procedure is a promising tool to identify and preserve arm lymphatic drainage during axillary lymph node dissection (ALND). The ARMONIC clinical trial was conducted to validate the technique on a large cohort of patients and to analyze the predictive clinical factors for ARM lymph node metastasis. For the first time, the fluorescence signal intensity from the ARM lymph nodes was measured and correlated with clinical findings. A total of 109 patients with invasive breast cancer and indications of mastectomy and ALND underwent the NIR fluorescence ARM procedure. Indocyanine green was administered by intradermal injection followed by intraoperative identification and resection of the ARM lymph nodes with NIR fluorescence camera guidance. The fluorescence signal intensity and signal distribution were then measured ex vivo and compared with clinical outcomes. ARM lymph nodes were successfully identified by fluorescence in 94.5% of cases. The mean normalized fluorescence signal intensity value was 0.47 with no significant signal difference between metastatic and non-metastatic ARM lymph nodes (p = 0.3728). At the microscopic level, the fluorescence signal distribution was focally intense in lymphoid tissue areas. Only the preoperative diagnosis of metastasis in the axillary nodes of patients was significantly associated with a higher ARM node fluorescence signal intensity (p = 0.0253), though it was not significantly associated with the pathological nodal (pN) status (p = 0.8081). Based on an optimal cut-off fluorescence value, the final sensitivity and specificity of the NIR fluorescence ARM procedure for ARM lymph node metastatic involvement were 64.7% and 47.3%, respectively. Although our preliminary results did not show that fluorescence signal intensity is a reliable diagnostic tool, the NIR fluorescence ARM procedure may be useful for ARM lymph node identification. Clinical trial registration: NCT02994225.

Confocal laser endomicroscopy and confocal microscopy for head and neck cancer imaging: Recent updates and future perspectives.

Confocal laser endomicroscopy (CLE) is a promising technology that provides real-time histological visualization of living tissue, described as "optical biopsy". It has been used successfully in dermatology, gastroenterology and ophthalmology for clinical diagnosis and tumor margin assessment. In addition to in vivo tissue assessment, confocal microscopy can also be used ex vivo on freshly excised or fixed tissue for intraoperative tumor margin assessment. While CLE and ex vivo confocal microscopy (EVCM) have shown promising results for the identification of head and neck cancers at cell level, several methodological issues need to be addressed before they can be widely used in routine clinical use. The present review will focus on recent advances in CLE and EVCM in the diagnosis and management of head and neck cancer and discuss remaining challenges for their clinical application.

Diagnostic accuracy of in vivo early tumor imaging from probe-based confocal laser endomicroscopy versus histologic examination in head and neck squamous cell carcinoma.

OBJECTIVES: Probe-based confocal laser endomicroscopy (pCLE) is a noninvasive and real-time imaging technique allowing acquisition of in situ images of the tissue microarchitecture during oral surgery. We aimed to assess the diagnostic performance of pCLE combined with patent blue V (PB) in improving the management of early oral cavity, oro/hypopharyngeal, and laryngeal cancer by imaging squamous cell carcinoma in vivo. MATERIALS AND METHODS: The prospective study enrolled 44 patients with early head and neck lesions. All patients underwent white-light inspection or panendoscopy depending on the lesion's location, followed by pCLE imaging of the tumor core and its margins after topical application of PB. Each zone imaged by pCLE was interpreted at distance of the exam by three pathologists blinded to final histology. RESULTS: Most imaged zones could be presented to pathologists; the final sensitivity and specificity of pCLE imaging in head and neck cancers was 73.2-75% and 30-57.4%, respectively. During imaging, head and neck surgeons encountered some challenges that required resolving, such as accessing lesions with the flexible optical probe, achieving sufficiently precise imaging on the targeted tissues, and heterogeneous tissue staining by fluorescent dye. CONCLUSION: Final sensitivity scores were reasonable but final specificity scores were low. pCLE zones used to calculate specificity were acquired in areas of tumor margins, and the poor quality of the images acquired in these areas explains the final low specificity scores. CLINICAL RELEVANCE: Practical adjustments and technical training are needed to analyze head and neck lesions in various anatomical sites in real-time by pCLE.

Value of Full-Field Optical Coherence Tomography Imaging for the Histological Assessment of Head and Neck Cancer.

BACKGROUND AND OBJECTIVES: In head and neck surgery, intraoperative and postoperative evaluation of tumor margins is achieved by histopathological assessment, which is a multistep process. Intraoperative analysis of tumor margins to obtain a preliminary diagnosis is usually carried out on frozen sections. Analysis of frozen sections is challenging due to technical difficulties in processing. Full-field optical coherence tomography (FFOCT) provides ex vivo images of fresh tissue samples at a microscopic scale without tissue processing. The objectives of our study were to define the diagnostic criteria required to interpret head and neck FFOCT images and to evaluate the reliability of a histological diagnosis made on an "optical biopsy" produced by head and neck FFOCT imaging compared with conventional histology. STUDY DESIGN/MATERIALS AND METHODS: First, we established an atlas of comparative images (FFOCT/standard histology) and defined the diagnostic criteria based on FFOCT images. Two pathologists subsequently performed a blinded review on 57 FFOCT images (32 patients). Specificity and sensitivity were measured by comparison with the standard histological diagnosis. The primary endpoint was major concordance, defined as two classifications leading to the same therapeutic decision (treatment/no treatment). RESULTS: Pathologists identified four main criteria for tissue diagnosis on FFOCT images: heterogeneous cell distribution, stromal reaction, coiling, and keratinization abnormalities. The correlation study showed good results, with sensitivity from 88% to 90% and specificity from 81% to 87%, regardless of whether the FFOCT image review was performed by a pathologist with or without previous experience in optical imaging. CONCLUSIONS: Our results demonstrate that FFOCT images can be used by pathologists for differential diagnosis, and that high-resolution FFOCT imaging can provide an assessment of microscopic architecture in head and neck tissues without tissue processing requirements. Lasers Surg. Med. © 2020 Wiley Periodicals, Inc.

Near-infrared fluorescence imaging for the prevention and management of breast cancer-related lymphedema: A systematic review.

Sentinel lymph node identification by near infrared (NIR) fluorescence with indocyanine green (ICG) is recognized in the literature as a useful technique. NIR fluorescence technology could become key in the prevention and management of lymphedema after axillary dissection for breast cancer. Here, we conducted a systematic review focusing on ICG imaging to improve lymphedema prevention and treatment after axillary surgery. A systematic literature review was performed using MEDLINE and Embase to identify articles focused on ICG imaging for breast-cancer-related lymphedema (BCRL). Qualitative analysis was performed to summarize the characteristics of reported ICG procedures. In situ tissue identification and functionality assessment based on fluorescence signal were evaluated. Clinical outcomes were appraised when reported. Studies relating to axillary reverse mapping, lymphography and upper limb supermicrosurgery combined with ICG imaging were identified. We included a total of 33 relevant articles with a total of 2016 patients enrolled. ICG imaging for axillary reverse mapping was safe for all 951 included patients, with identification of arm nodes in 80%-88% of patients with axillary lymph nodes dissection. However, the papers discuss the oncologic safety of the approach and how - regardless of the contrast agent - concerns limit its adoption. ICG lymphography is openly supported in BCRL management, with 1065 patients undergoing this procedure in 26 articles. The technique is reported for lymphedema diagnosis, with high sensitivity and specificity, staging, intraoperative mapping and patency control in lymphaticovenular anastomosis. The substantial advantages/disadvantages of ICG imaging procedures are finally described.

Parathyroid gland management using optical technologies during thyroidectomy or parathyroidectomy: A systematic review.

New optical technologies enhancing localization or assessing viability of parathyroid glands (PG) during endocrine surgery have been reported in clinical studies. These technologies could become complementary to the surgeon's eyes and may improve surgical outcomes in thyroidectomy and parathyroidectomy. Here, we conducted a systematic review focusing on PG identification and functional assessment using optical methods to enhance surgery. A systematic literature review was performed using MEDLINE and Embase database. Two authors selected studies and extracted data; qualitative analysis was performed to summarize the characteristics of reported optical tools for thyroidectomy or parathyroidectomy. Identification and vascularisation of PG during surgery were evaluated. Clinical and biochemical outcomes were appraised when reported. Studies relating to parathyroidectomy or thyroidectomy combined with autofluorescence, fluorescent methylene blue, 5-aminolevulinic acid, indocyanine green (ICG), optical coherence tomography, laser speckle contrast imaging, dynamic optical contrast imaging and Raman spectroscopy were identified with MEDLINE and Embase. We included a total of 47 relevant articles with a total of 1615 patients enrolled. Each optical technique is described and appreciated related to its surgical purpose. Autofluorescence and ICG imaging of PG are the most widely reported optical technologies for identification and assessment of vascularisation of PG. Results are mainly based on observational studies and argue for the feasibility of both techniques in endocrine surgery but prospective randomized studies have not been performed. In vivo applications are still limited for the other methods and further investigations correlating these techniques with post-operative parathormone measurements are still needed before considering these technologies in clinical practice.

Prospective evaluation of the limitations of near-infrared imaging in detecting axillary sentinel lymph nodes in primary breast cancer.

We compared the performance of near-infrared imaging using indocyanine green (ICG) with the radioisotope (ISO) method to detect sentinel lymph nodes (SLNs) in breast cancer, to analyze predictive factors for negative ICG identification. The study included 122 patients who underwent sentinel lymph node biopsy (SLNB) using the combined ISO and ICG technique for primary breast cancer. We assessed the putative association between pathologic/clinical variables and ICG failure to detect SLNs. The ISO identification rate was 96.7% and ICG identification 81.9%. Overweight patients or presence of macrometastasis in SLNB were associated with the risk of ICG failing to detect SLNs (P = 0.02).

Intraoperative Near-infrared Imaging for Parathyroid Gland Identification by Auto-fluorescence: A Feasibility Study.

BACKGROUND: Parathyroid glands (PGs) can be particularly hard to distinguish from surrounding tissue and thus can be damaged or removed during thyroidectomy. Postoperative hypoparathyroidism is the most common complication after thyroidectomy. Very recently, it has been found that the parathyroid tissue shows near-infrared (NIR) auto-fluorescence which could be used for intraoperative detection, without any use of contrast agents. The work described here presents a histological validation ex vivo of the NIR imaging procedure and evaluates intraoperative PG detection by NIR auto-fluorescence using for the first time to our knowledge a commercially available clinical NIR imaging device. METHODS: Ex vivo study on resected operative specimens combined with a prospective in vivo study of consecutive patients who underwent total or partial thyroid, or parathyroid surgery at a comprehensive cancer center. During surgery, any tissue suspected to be a potential PG by the surgeon was imaged with the Fluobeam 800 (®) system. NIR imaging was compared to conventional histology (ex vivo) and/or visual identification by the surgeon (in vivo). RESULTS: We have validated NIR auto-fluorescence with an ex vivo study including 28 specimens. Sensitivity and specificity were 94.1 and 80 %, respectively. Intraoperative NIR imaging was performed in 35 patients and 81 parathyroids were identified. In 80/81 cases, the fluorescence signal was subjectively obvious on real-time visualization. We determined that PG fluorescence is 2.93 ± 1.59 times greater than thyroid fluorescence in vivo. CONCLUSIONS: Real-time NIR imaging based on parathyroid auto-fluorescence is fast, safe, and non-invasive and shows very encouraging results, for intraoperative parathyroid identification.

Patent blue V and indocyanine green for fluorescence microimaging of human peritoneal carcinomatosis using probe-based confocal laser endomicroscopy.

BACKGROUND: Peritoneal carcinomatosis is a metastatic stage aggravating abdominal and pelvic cancer dissemination. The preoperative evaluation of lesions remains difficult today. Probe-based confocal laser endomicroscopy (pCLE) provides dynamic images of tissue architecture and cellular details. This technology allows in vivo histological interpretation of tissue. The main limitation of pCLE for adoption in the clinic is the unavailability of fluorescent contrast agents. The aim of our study was to evaluate the staining performance of indocyanine green and patent blue V for histological diagnosis of pCLE images of pathological and non-pathological peritoneal tissue. METHODS: We performed a correlative study with the histological gold standard on ex vivo human specimens from 25 patients operated for peritoneal carcinomatosis; 70 specimens were stained by topical application with ICG or patent blue V and then imaged with a probe-based confocal laser endomicroscope. A total of 350 pCLE images and 70 corresponding histological sections were randomly and blindly interpreted by two pathologists (PT1 and PT2). The images were first classified into two categories, tumoral versus non-tumoral, and a refined histological diagnosis was then given. RESULTS: All presented images were interpreted by PT1 (who received prior training on PCLE image reading) and PT2 (no training). 100 % sensitivity for PT1 and PT2 was noticed with tissues stained with ICG to differentiate tumoral and non-tumoral tissue. Global scores were always better for PT1 (major concordance between 86 and 94 %) than for PT2 (major concordance between 77 and 89 %) independently of the fluorescent dye when histological diagnosis was done on pCLE images. CONCLUSION: In conclusion, the pair ICG-pCLE offers the best combination for a non-trained pathologist for the interpretation of pCLE images from peritoneum.

Red and far-red fluorescent dyes for the characterization of head and neck cancer at the cellular level.

BACKGROUND: Primary upper aerodigestive tract malignancy remains a cancer having a poor prognosis, despite current progress in treatment, due to a generally late diagnosis. OBJECTIVES: We conducted a preliminary assessment of five dyes approved for human use for the imaging of head and neck tissues at the cellular level, which could be considered for clinical examination. METHODS: We investigated fluorescence endomicroscopic images on fresh samples obtained from head and neck surgeries after staining with hypericin, methylene blue, toluidine blue, patent blue or indocyanine green to provide a preliminary consideration as to whether these images contain enough information for identification of non-pathologic and pathologic tissues. The distribution pattern of dye has been examined using probe-based confocal laser endomicroscopy (pCLE) in ex vivo specimens and compared with corresponding histology. RESULTS: In most samples, the image quality provided by pCLE with both dyes allowed pathologists to recognize histological characteristics to identify the tissues. CONCLUSION: The combination of pCLE imaging with these dyes provides interpretable images close to conventional histology; a promising clinical tool to assist physicians in examination of upper aerodigestive tract, as long as depth imaging issues can be overcome.

Confocal laser endomicroscopy for non-invasive head and neck cancer imaging: a comprehensive review.

Histological assessment is an essential tool in the diagnosis and guidance of the treatment of various diseases, in particular cancer, of the head and neck. Recent major advances in optical imaging techniques have made it possible to acquire high-resolution in vivo images at the cellular scale. Confocal endomicroscopy is a non-invasive technique, which can be highly useful whenever meaningful in situ histological information is required. The technical aspects of confocal endomicroscopy are introduced, followed by an overview of major clinical studies in the field of head and neck cancer. Ongoing technical developments, contributing to improvements in imaging of the upper aero-digestive tract, are also discussed. Finally, the potential complementarities of functional and molecular imaging, as compared to morphological endomicroscopy, are highlighted.

Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication.

The role of gap junctional intercellular communication (GJIC) in regulation of normal growth and differentiation is becoming increasingly recognized as a major cellular function. GJIC consists of intercellular exchange of low molecular weight molecules, and is the only means for direct contact between cytoplasms of adjacent animal cells. Disturbances of GJIC have been associated with many pathological conditions, such as carcinogenesis or hereditary illness. Reliable and accurate methods for the determination of GJIC are therefore important in cell biology studies. There are several methods used successfully in numerous laboratories to measure GJIC both in vitro and in vivo. This review comments on techniques currently used to study cell-to-cell communication, either by measuring dye transfer, as in methods like microinjection, scrape loading, gap-fluorescence recovery after photobleaching (gap-FRAP), the preloading assay, and local activation of a molecular fluorescent probe (LAMP), or by measuring electrical conductance and metabolic cooperation. As we will discuss in this review, these techniques are not equivalent but instead provide complementary information. We will focus on their main advantages and limitations. Although biological applications guide the choice of techniques we describe, we also review points that must be taken into consideration before using a methodology, such as the number of cells to analyze.

Gap junctional intercellular communication capacity by gap-FRAP technique: a comparative study.

Gap junctions play an important role in vital functions, including the regulation of cell growth and cell differentiation. Connexins 43 (Cx43) are the most widely expressed gap junction proteins. Cellular localization of phosphorylated Cx43 has been implicated in the capacity of gap junctional intercellular communication (GJIC). To follow the functionality of GJIC of different cell types, in monolayer cultures, characterized by different patterns of phosphorylated Cx43, we used a fluorescence recovery after photobleaching (FRAP) technique, and compared two tracers, 5(6)-carboxyfluorescein diacetate (CFDA) and calcein acetoxymethylester (AM). The GJIC capacity was quantified by estimating fluorescence redistribution parameters. The functionality of GJIC was in relation with the staining localization of phosphorylated Cx43 to the cell-cell contact areas, corresponding to gap junctions between contacting cells. GJIC involvement in fluorescence restitution after photobleaching was checked by a gap junction channel inhibition assay. We demonstrated that the choice of the dye did not significantly influence the fluorescence recovery percentages despite a cell line-dependent CFDA release, whereas it had an important impact on fluorescence kinetic profiles. This study reinforces the interest of the gap-FRAP approach to quantify modifications in the functionality of gap junctions and, above all, argues about the limits of CFDA for 3-D future approaches.