Feasibility of parathyroid gland autofluorescence imaging after indocyanine green fluorescence angiography.

Background: In thyroid surgery, autofluorescence allows the parathyroid glands (PTGs) to be located very early to protect them. Moreover, indocyanine green (ICG) fluorescence angiography (ICG-FA) allows for assessing the viability of the PTGs and identifying blood vessels to preserve them. The main limitation of using ICG-FA is that once ICG has been injected, it is no longer possible to observe PTG autofluorescence using existing devices. This study aimed to describe an approach that allows for visualization of the PTGs by autofluorescence, even after ICG injection. Methods: We redesigned the FLUOBEAM® LX system to excite fluorescence at 685 nm and detect fluorescence between 700 and 900 nm. This device had short-pass filters at 775 nm that helped to split the contributions of the PTG autofluorescence and ICG fluorescence. Tests were performed on extemporaneous PTG preparations placed next to ICG droplets to assess for rejection of the ICG signal. Results: A low-pass filter at 775 nm detected 60% of the autofluorescence signals and 10% of the ICG signals. Conclusion: These findings support the possibility of visualizing PTG autofluorescence despite multiple ICG injections and measuring the balance between ICG and autofluorescence signals.

Clinical evaluation of a device providing simultaneous white-light and fluorescence video streams as well as panoramic imaging during fluorescence assisted-transurethral resection of bladder cancer.

The current clinical study is aimed at evaluating the clinical relevance of an innovative device (called CyPaM2 device) that for the first time provides urologists with (i) a panoramic image of the bladder inner wall within the surgery time, and with (ii) a simultaneous (bimodal) display of fluorescence and white-light video streams during the fluorescence assisted-transurethral resection of bladder cancers procedure. The clinical relevance of this CyPaM2 device was evaluated on 10 patients according to three criteria (image quality, fluorescent lesions detection relevance, and ergonomics) compared with a reference medical device. Innovative features displayed by the CyPaM2 device were evaluated without any possible comparison: (i) simultaneous bimodal display of white-light and fluorescence video streams, (ii) remote light control, and (iii) time delay for the panoramic image building. The results highlight the progress to achieve in order to obtain a fully mature device ready for commercialization and the relevance of the innovative features proposed by the CyPaM2 device confirming their interest.

A palm-sized high-sensitivity near-infrared fluorescence imager for laparotomy surgery.

In laparotomy surgery guided by near-infrared fluorescence imaging, the access to the field of operation is limited by the illumination and/or the imaging field. The side of cavities or organs such as the liver or the heart cannot be examined with the systems available on the market, which are too large and too heavy. In this article, we describe and evaluate a palm sized probe, whose properties, weight, size and sensitivity are adapted for guiding laparotomy surgery. Different experiments have been performed to determine its main characteristics, both on the illumination and imaging sides. The device has been tested for fluorescent molecular probe imaging in preclinical procedures, to prove its ability to be used in cancer nodule detection during surgery. This system is now CE certified for clinical procedures and Indocyanine Green imaging has been performed during clinical investigations: lymphedema and surgical resection of liver metastases of colorectal cancers.

Targeted imaging of α(v)ß(3) expressing sarcoma tumor cells in vivo in pre-operative setting using near infrared: a potential tool to reduce incomplete surgical resection.

Tumor size and location along with efficacy of pre-operative imaging are limiting factors for optimal surgical excision for osteosarcoma. Our general hypothesis is that targeting αvß3 integrin-rich osteosarcoma neoangiogenesis should provide improved delivery of diagnostic compounds and assist surgeons intra operatively using near-infrared imaging techniques. We evaluated in an orthotopic metastatic osteosarcoma in rats the potential of AngioStamp™ targeting αvß3 integrins and detected intra operatively by near infrared (NIR) illumination (Fluobeam™) as a novel, intra operative imaging technique. To determine the potential of this association in improving tumor and metastasis detection, we compared the quality and sensitivity of tumor/metastasis margin delineation and tumor resection using intra-operative NIR imaging to the ones guided by pre-operative imaging (i.e., MRI subsequently confirmed by histopathological analysis). Chemotherapy being essential in osteosarcoma treatment, we evaluated the capacity of AngioStamp™ to specifically localize to the tumor after chemotherapy treatment. We showed a significantly lesser extent of healthy tissue resection after surgical excision when assessing tumor margin intra operatively using AngioStamp™/Fluobeam™ association compared to pre-operative MRI post-operatively confirmed by histopathological analysis (p<0.01). Importantly, intra-operative NIR illumination of lungs revealed more metastases than were detected by CT Scan or under intra-operative white light examination (p<0.01). Importantly, chemotherapy did not alter AngioStamp™ tumor specific targeting nor the sensitivity of tumor detection. Our preclinical data confirm the potential of intra-operative imaging for improved primary tumor and lung metastasis excision. Based on these promising results, we now propose to evaluate this approach as a mean to improve surgical excision while maintaining tumor control in other sarcoma or tumors overexpressing αvß3 integrins.

Intraoperative fluorescence imaging of peritoneal dissemination of ovarian carcinomas. A preclinical study.

OBJECTIVE: Improvement of the management and outcome of ovarian cancers may require intraoperative detection and therapeutic intervention to treat minimal residual disease after complete surgery. The aim of this study was to validate the importance of fluorescence in the peroperative detection of human ovarian adenocarcinoma cells and to determine its efficiency in detecting infra millimetric tumor metastases. METHODS: A fluorescent RAFT-(cRGD)4 tracer molecule (AngioStamp®) was used. The tracer is based on a biomarker, which has a very high affinity for the α(v)ß3 integrin, which is overexpressed in a large ratio of cancer cells and neovessel endothelial cells during angiogenesis. Infrared fluorescence was visualized with Fluobeam®, an open fluorescent imaging system that could potentially be used in peroperative conditions in the future. RESULTS: This novel technique allowed the specific detection of residual tumor deposits and inframillimetric metastases, smaller than 500µm, which were resected under fluorescent guidance. AngioStamp® was able to detect all types of cell lines, derived from human ovarian adenocarcinomas, before or after chemotherapy treatment in animals. The effectiveness of AngioStamp® for the detection of various human ovarian adenocarcinomas was assessed on 10 different fragments of tumor, implanted subcutaneously in nude mice. All implanted tumor fragments were visualized by AngioStamp®. CONCLUSIONS: The high rate of recurrence after apparently complete surgery and/or complete clinical response to chemotherapy implies that most patients have undetected minimal residual disease. Novel techniques such as laparoscopic or laparotomic fluorescence may prove to be crucial in reassessing the definition of primary outcome in ovarian cancer management.

Real time optical method for localization of inclusions embedded in turbid media.

A simple and fast time-domain method for localizing inclusions, fluorescent optical probes or absorbers, is presented. The method offers new possibilities for situations where complete tomographic measurements are not permitted by the examined object, for example in endoscopic examination of the human prostate or the oesophagus. Feasibility has been envisioned with a phantom study conducted on a point-like fluorochrome embedded in a diffusing medium mimicking the optical properties of biological tissues.

Fluorescence diffuse optical tomography for free-space and multifluorophore studies.

We present two major advances in preclinical fluorescence-enhanced diffuse optical tomography (fDOT) system and assess its performance. It is now possible to perform experiments without adaptation liquid or a glass plate over the animal, and our system is equipped with a filter wheel in order to discriminate two injected fluorophores. Evaluation carried out on characterization phantoms and in vivo on mice demonstrates enriched use of the system for biological studies on small animals.

Chi2 analysis for estimating the accuracy of optical properties derived from time resolved diffuse-reflectance.

Weighted residuals and the reduced chi(2) (chi(R) (2)) value are investigated with regard to their relevance for assessing optical property estimates using the diffusion equation for time-resolved measurements in turbid media. It is shown and explained, for all photon counting experiments including lifetime estimation, why chi(R) (2) increases linearly with the number of photons when there is a model bias. Only when a sufficient number of photons has been acquired, chi(R) (2) is a pertinent value for assessing the accuracy of mu(a) and mu(s)' estimates. It was concluded that chi(R) (2) is of particular interest for cases of small interfiber separation, low-level scattering, strong absorption and incorrect measurement of instrument response function. It was also found that chi(R) (2) is less pertinent for judging mu(a) in case of air boundary effects.

Optical calibration protocol for an x-ray and optical multimodality tomography system dedicated to small-animal examination.

A small-animal multimodality tomography system dedicated to the coregistration of fluorescence optical signal and x-ray measurements has been developed in our laboratory. The purpose of such a system is to offer the possibility of getting in vivo anatomical and functional information simultaneously. Moreover, anatomical measurements can be used as a regularization factor to achieve more accurate reconstructions of the biodistribution of fluorochromes and to speed up treatment. A dedicated acquisition protocol has been established, and the methodology of the reconstruction of the three-dimensional distribution of the biomarkers under cylindrical geometry consistent with classic computed tomography has been implemented. A phantom study was conducted to evaluate and to fix the parameters for the coregistration. These test experiments were reproduced by considering anesthetized mice that had thin glass tubes containing fluorochromes inserted into their esophagus. The instrument is also used for an in vivo biological study conducted on mice with lung tumors, tagged with near-infrared optical probes (targeting probes such as Transferin-AlexaFluor750).

In vivo mice lung tumor follow-up with fluorescence diffuse optical tomography.

We present in vivo experiments conducted with a new fluorescence diffuse optical tomographic (fDOT) system on cancerous mice bearing mammary murine tumors. We first briefly present this new system that has been developed and its associated reconstruction method. Its main specificity is its ability to reconstruct the fluorescence yield even in heterogeneous and highly attenuating body regions such as lungs and to enable mouse inspection without immersion in optical index matching liquid (Intralipid and ink). Some phantom experiments validate the performance of this new system for heterogeneous media inspection. Its use for a mice study is then related. It consists in the follow-up of the lungs at different stages of tumor development after injection of RAFT-(cRGD)4-Alexa700. As expected, the reconstructed fluorescence increases along with the tumor stage. These results validate the use of our system for biological studies of small animals.

From bench-top small animal diffuse optical tomography towards clinical imaging.

Fluorescence enhanced diffuse optical tomography is an emergent diagnosis tool for the localization and the quantification of fluorescent probes; this technique comes as a supplement or sometimes replaces the classical ionizing radiation imaging techniques, and in particular if a simple, inexpensive, non invasive and accurate instrumentation is sought. For 4 years now, the CEA-LETI has built a base of knowledge in markers and instrumentation within the framework of small animal imaging. More recently, an instrumentation has been developed, the purpose of which is a specific approach to the examination of underlying structures, deeply embedded within the tissues, and in fine for human being screening.

Coupling X-ray and optical tomography systems for in vivo examination of small animals.

For the purpose of the co-registration of fluorescence optical signal and X-rays measurements, a multimodality tomographer has been developed in our laboratory. Such a system brings the possibility to get, on the same bench, in vivo both anatomical and functional information. Moreover, the information on the morphology of the animal can be used as a regularization factor in order to get the reconstructions of the biodistribution of fluorochromes more accurate and to reduce the computation time. A study on homogeneous phantoms was conducted to evaluate the feasibility, to test the linearity and the reproducibility, and to fix the parameters for the co-registration. More cumbersome phantoms (sacrificed mice) have then been considered and the test experiments were reproduced. Finally, results of a study conducted in vivo on mice bearing tumors in the lungs, tagged with different types of optical probes, are presented.