Fanny Poulon

Spectral and fluorescence lifetime endoscopic system using a double-clad photonic crystal fiber

We present a customized small-core double-clad photonic crystal fiber for spectral and fluorescence lifetime measurements of human samples. In this Letter, the new fiber has been characterized on different fluorophores and samples of human brain tumor; a comparison to a bi-fiber homemade system and a commercial fiber probe was made.

Multimodal optical analysis discriminates freshly extracted human sample of gliomas, metastases and meningiomas from their appropriate controls

Delineating tumor margins as accurately as possible is of primordial importance in surgical oncology: extent of resection is associated with survival but respect of healthy surrounding tissue is necessary for preserved quality of life. The real-time analysis of the endogeneous fluorescence signal of brain tissues is a promising tool for defining margins of brain tumors. The present study aims to demonstrate the feasibility of multimodal optical analysis to discriminate fresh samples of gliomas, metastases and meningiomas from their appropriate controls. Tumor samples were studied on an optical fibered endoscope using spectral and fluorescence lifetime analysis and then on a multimodal set-up for acquiring spectral, one and two-photon fluorescence images, second harmonic generation signals and two-photon fluorescence lifetime datasets. The obtained data allowed us to differentiate healthy samples from tumor samples. These results confirmed the possible clinical relevance of this real-time multimodal optical analysis. This technique can be easily applied to neurosurgical procedures for a better delineation of surgical margins.

Multimodal optical analysis of meningioma and comparison with histopathology.

Meningioma is the most frequent primary central nervous system tumor. The risk of recurrence and the prognosis are correlated with the extent of the resection that ideally encompasses the infiltrated dura mater and, if required, the infiltrated bone. No device can deliver real-time intraoperative histopathological information on the tumor environment to help the neurosurgeon to achieve a gross total removal. This study assessed the abilities of nonlinear microscopy to provide relevant and real-time data to help resection of meningiomas. Nine human meningioma samples (four World Health Organization Grade I, five Grade II) were analyzed using different optical modalities: spectral analysis and imaging, lifetime measurements, fluorescence lifetime imaging microscopy, fluorescence emitted under one- and two-photon excitation and the second-harmonic generation signal imaging using a multimodal setup. Nonlinear microscopy produced images close to histopathology as a gold standard. The second-harmonic generation signal delineated the collagen background and two-photon fluorescence underlined cell cytoplasm. The matching between fluorescence images and Hematoxylin and Eosin staining was possible in all cases. Grade I meningioma emitted less autofluorescence than Grade II meningioma and Grade II meningioma exhibited a distinct lifetime value. Autofluorescence was correlated with the proliferation rates and seemed to explain the observed differences between Grade I and II meningiomas. This preliminary multimodal study focused on human meningioma samples confirms the potential of tissue autofluorescence analysis and nonlinear microscopy in helping intraoperatively neurosurgeons to reach the actual boundaries of the tumor infiltration. Correspondence between H&E staining (top pictures) and the two-photon fluorescence imaging (bottom pictures).

Characterization of fiber ultrashort pulse delivery for nonlinear endomicroscopy

In this work, we present a detailed characterization of a small-core double-clad photonic crystal fiber, dedicated and approved for in vivo nonlinear imaging endomicroscopy. A numerical and experimental study has been performed to characterize the excitation and collection efficiencies through a 5 m-long optical fiber, including the pulse duration and spectral shape. This was first done without any distal optics, and then the performances of the system were studied by using two kinds of GRIN lenses at the fiber output. These results are compared to published data using commercial double clad fibers and GRIN lenses.

Optical properties, spectral, and lifetime measurements of central nervous system tumors in humans

A key challenge of central nervous system tumor surgery is to discriminate between brain regions infiltrated by tumor cells and surrounding healthy tissue. Although monitoring of autofluorescence could potentially be an efficient way to provide reliable information for these regions, we found little information on this subject, and thus we conducted studies of brain tissue optical properties. This particular study focuses on the different optical quantitative responses of human central nervous system tumors and their corresponding controls. Measurements were performed on different fixed human tumoral and healthy brain samples. Four groups of central nervous system tumors (glioblastoma, diffuse glioma, meningioma and metastasis) were discriminated from healthy brain and meninx control tissues. A threshold value was found for the scattering and absorption coefficient between tumoral and healthy groups. Emission Spectra of healthy tissue had a significant higher intensity than tumoral groups. The redox and optical index ratio were thenn calculated and these also showed significant discrimination. Two fluorescent molecules, NADH and porphyrins, showed distinct lifetim values among the different groups of samples. This study defines several optical indexes that can act as combinated indicators to discriminate healthy from tumoral tissues.

Two-photon optical imaging, spectral and fluorescence lifetime analysis to discriminate urothelial carcinoma grades

In the framework of urologic oncology, mini-invasive procedures have increased in the last few decades particularly for urothelial carcinoma. One of the essential elements in the management of this disease is still the diagnosis, which strongly influences the choice of treatment. The histopathologic evaluation of the tumor grade is a keystone of diagnosis, and tumor characterization is not possible with just a macroscopic evaluation. Even today intraoperative evaluation remains difficult despite the emergence of new technologies which use exogenous fluorophore. This study assessed an optical multimodal technique based on endogenous fluorescence, combining qualitative and quantitative analysis, for the diagnostic of urothelial carcinoma. It was found that the combination of two-photon fluorescence, second harmonic generation microscopy, spectral analysis and fluorescence lifetime imaging were all able to discriminate tumor from healthy tissue, and to determine the grade of tumors. Spectral analysis of fluorescence intensity and the redox ratio used as quantitative evaluations showed statistical differences between low-grade and high-grade tumors. These results showed that multimodal optical analysis is a promising technology for the development of an optical fiber setup designed for an intraoperative diagnosis of urothelial carcinoma in the area of endo-urology.

Comparison between fresh and fixed human biopsies using spectral and lifetime measurements: Fluorescence analysis using one and two photon excitations

The purpose of this study is to make a comparison between the fluorescence emissions of fresh extracted human biopsies and fixed human biopsies, in order to evaluate the impact of fixation on autofluoresence signal. Our group is developing an endomicroscope to image brain tissues in-vivo, however to date, in order to validate our technology the easiest type of samples we can access are fixed samples. However, the fixation is still challenging. For that, we aim through this study to determine whether we should pursue to work on fixed samples or we should shift to work on fresh biopsies. Data were collected on spectroscopic, lifetime measurement and fluorescence imaging set-ups with visible and two-photon excitations wavelengths. Five fresh and five fixed samples are involved in the experiment. Endogenous fluorescence of fixed biopsies were calculated. Experimental results reveal that at 405 nm and 810 nm, the fresh samples have an intensity of fluorescence two times higher than that of fixed samples. However, for each fluorophore and each excitation wavelength, the lifetime for fresh samples is shorter than that for fixed samples. Still, further studies and investigations involving the comparison between different samples are required to strengthen our findings.

Identifying Isolated Glioblastoma Tissues in Human Patients through Their Optical and Spectral Properties

Survival rates and health-related quality of life of adult patients suffering from glioblastoma depend significantly on the extent (no residual tumor tissue) and precision (no collateral damage) of the surgical resection. Assistance in defining the borders of the infiltrating component of the glioblastoma would be valuable to improve outcomes. A tissue can be defined by its optical properties : absorption, scattering, intensity of fluorescence, that will give a unique signature. In this work we look at the absorption and scattering coefficients of glioblastoma and control tissues from adult patients using an integrating sphere, spectral measurements were also took on the samples using a fiber endoscope. The preliminary results show the potential of using endogenous fluorescence for intraoperative identification of residual glioblastoma tissue in the wall of the surgical cavity of resection.

MP54-18 QUANTITATIVE AND QUALITATIVE MULTIMODAL OPTICAL ANALYSIS TO DISCRIMINATE UROTHELIAL CARCINOMA GRADES

INTRODUCTION AND OBJECTIVES: In the framework of urologic oncology, mini-invasive procedures have increased the last decades particularly for urothelial carcinoma. One of the essential step in the management of this disease stay the diagnostic, which strongly impacts the treatment choice. The histopathologic evaluation of the grade of the tumor is a keystone of the diagnosis, and its recognition is not possible with a macroscopic evaluation. Nowadays, this specific intraoperative characteristic evaluation remains difficult despite the emergence of new technologies which use exogenous fluorophore. In our study, we assessed the use of an optical multimodal technique based on endogenous fluorescence combining qualitative and quantitative analysis for the diagnostic of urothelial carcinoma grades. METHODS: Urothelial samples from bladder and upper urinary tract were prospectively included (IRB-00003835) and analyzed on a specific optical multimodal setup based on endogenous fluorescence.The analysis included qualitative analyses with two-photons fluorescence imaging (TPF) and quantitative analyses with spectral analyses and fluorescence lifetime imaging (FLIM). Qualitative analyses were compared with pathological examination. Quantitative analyses was performed with a 870nm excitation wavelength, in the spectral analysis we evaluated the spectra, the redox ratio (NADH and FAD) and the fluorescence lifetime for each sample. RESULTS: We identified 3 major urothelial aspects by TPF. All this samples were correlated with pathological examinations, and permitted to differentiate healthy tissue from low and high grade urothelial carcinoma. In the quantitative analysis, the spectral results shown that the intensity level of the emitted spectra was correlated with the histopathological characteristics: the higher the grade was, the lower was the fluorescence signal. The redox ratio analysis was significantly higher in the healthy urothelium compared with tumors samples (p<0.001). Moreover, we were able to discriminate low grade from high grade tumors, the low grade had a significantly higher redox ratio (p1⁄40.002). We were able to identify the cells structure in the FLIM images and to compare the different grade. This analysis also permitted to discriminate the different origins. The average lifetime in low grade tumor appeared shorter on our images and in the histogram shown significant differences between healthy, low grade and high grade urothelial carcinoma: healthy vs. low grade (p 1⁄4 0.002), healthy vs. high grade (p < 0.001) and high grade vs. low grade (p < 0.001). CONCLUSIONS: Those results show that multimodal optical analysis was able to discriminate low grade from high grade urothelial carcinoma without using exogenous fluorophore. This is a promising technology for the development of an optical fiber setup designed for an intraoperative diagnosis of urothelial carcinoma in the area of endourology.

Real-time Brain Tumor imaging with endogenous fluorophores: a diagnosis proof-of-concept study on fresh human samples

The primary line of therapy for high-grade brain tumor is surgical resection, however, identifying tumor margins in vivo remains a major challenge. Despite the progress in computer-assisted imaging techniques, biopsy analysis remains the standard diagnostic tool when it comes to delineating tumor margins. Our group aims to answer this challenge by exploiting optical imaging of endogenous fluorescence in order to provide a reliable and reproducible diagnosis close to neuropathology. In this study, we first establish the ability of two-photon microscopy (TPM) to discriminate normal brain tissue from glioblastomas and brain metastasis using the endogenous fluorescence response of fresh human brain sample. Two-photon fluorescence images were compared to gold standard neuropathology. “Blind” diagnosis realized by a neuropathologist on a group of TPM images show a good sensitivity, 100%, and specificity, 50% to discriminate non tumoral brain tissue versus glioblastoma or brain metastasis. Quantitative analysis on spectral and fluorescence lifetime measurements resulted in building a scoring system to discriminate brain tissue samples.