Darine Abi Haidar

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.

Nanoprobe Synthesized by Magnetotactic Bacteria, Detecting Fluorescence Variations under Dissociation of Rhodamine B from Magnetosomes following Temperature, pH Changes, or the Application of Radiation

We report a method of fabrication of fluorescent magnetosomes, designated as MCR400, in which 400 μM of rhodamine B are introduced in the growth medium of AMB-1 magnetotactic bacteria and fluorescent magnetosomes are then extracted from these bacteria. These fluorescent magnetosomes behave differently from most fluorescent nanoprobes, which often lead to fluorescence losses over time due to photobleaching. Indeed, when MCR400 are heated to 30-90 °C, brought to an acidic pH, or exposed to radiations, we observed that their fluorescence intensity increased. We attributed this behavior to the dissociation of rhodamine B from the magnetosomes. Interestingly, enhanced fluorescence was also observed in vitro when MCR400 were mixed with either primary macrophages or tumor cells (TC1-GFP or RG2-Cells) or in vivo when MCR400 were introduced in rat glioblastoma. We showed that MCR400 internalize in tumor and immune cells (macrophages) leading to enhanced fluorescence, suggesting that fluorescent magnetosomes could be used during cancer treatments such as magnetic hyperthermia to image cells of interest such as immune or tumor cells.

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.

The Impact of Compressed Femtosecond Laser Pulse Durations on Neuronal Tissue Used for Two-Photon Excitation Through an Endoscope

Accurate intraoperative tumour margin assessment is a major challenge in neurooncology, where sparse tumours beyond the bulk tumour are left undetected under conventional resection. Non-linear optical imaging can diagnose tissue at the sub-micron level and provide functional label-free histopathology in vivo. For this reason, a non-linear endomicroscope is being developed to characterize brain tissue intraoperatively based on multiple endogenous optical contrasts such as spectrally- and temporally-resolved fluorescence. To produce highly sensitive optical signatures that are specific to a given tissue type, short femtosecond pulsed lasers are required for efficient two-photon excitation. Yet, the potential of causing bio-damage has not been studied on neuronal tissue. Therefore, as a prerequisite to clinically testing the non-linear endomicroscope in vivo, the effect of short laser pulse durations (40–340 fs) on ex vivo brain tissue was investigated by monitoring the intensity, the spectral, and the lifetime properties of endogenous fluorophores under 800 and 890 nm two-photon excitation using a bi-modal non-linear endoscope. These properties were also validated by imaging samples on a benchtop multiphoton microscope. Our results show that under a constant mean laser power, excitation pulses as short as 40 fs do not negatively alter the biochemical/ biophysical properties of tissue even for prolonged irradiation.

Multimodal imaging to explore endogenous fluorescence of fresh and fixed human healthy and tumor brain tissues

To complement a project toward label-free optical biopsy and enhanced resection which the overall goal is to develop a multimodal nonlinear endomicroscope, this multimodal approach aims to enhance the accuracy in classifying brain tissue into solid tumor, infiltration and normal tissue intraoperatively. Multiple optical measurements based on one- and two-photon spectral and lifetime autofluorescence, including second harmonic generation imaging, were acquired. As a prerequisite, studying the effect of the time of measurement postexcision on tissues spectral/lifetime fluorescence properties was warranted, so spectral and lifetime fluorescences of fresh brain tissues were measured using a point-based linear endoscope. Additionally, a comparative study on tissues optical properties obtained by multimodal nonlinear optical imaging microscope from fresh and fixed tissue was necessary to test whether clinical validation of the nonlinear endomicroscope is feasible by extracting optical signatures from fixed tissue rather than from freshly excised samples. The former is generally chosen for convenience. Results of this study suggest that an hour is necessary postexcision to have consistent fluorescence intensities\lifetimes. The fresh (a,b,c) vs fixed (d,e,f) tissue study indicates that while all optical signals differ after fixation. The characteristic features extracted from one- and two-photon excitation still discriminate normal brain (a,d) cortical tissue, glioblastoma (GBM) (b,e) and metastases (c,f).

Multimodal Analysis of Central Nervous System Tumor Tissue Endogenous Fluorescence With Multiscale Excitation

The primary therapeutic approach for high-grade brain tumor is surgical resection. However, identifying tumor margins in vivo remains a major challenge. Biopsy analysis remains the standard diagnostic technique on tumor margins. This ex-vivo analysis is time consuming and delays treatment. The aim of this study is tissue discrimination using label free autofluorescence and application in intraoperative optical probe for optical biopsy. Biopsy samples from fifty-one patients who underwent brain tumor surgery (21 metastasis tumors, 17 glioblastoma tumors, GBM, and 13 control samples) were included in this study. The samples underwent a multiscale and multi-contrast optical analysis. The excitation were performed with a deep-UV synchrotron beam, at 275nm, and a near-infrared Ti:sapphire pulsed laser, from 690nm to 1040nm. The detection modalities were fluorescence imaging, spectroscopy and fluorescence lifetime. Using deep-UV excitation, and combining three molecular ratios (tyrosin-tryptophan, tryptophan-collagen, tryptophan-NADH) resulted in discrimination with a sensitivity of 90% and a specificity of 73%. Using a two-photon excitation, and combining average lifetime, NADH-FAD ratio and Porphyrin-NADH ratio, resulted in discrimination with a sensitivity of 97% and a specificity of 100%. A multiscale algorithm resulted in an overlap of only 1.8% between control and tumor samples.

Multimodal optical imaging database from tumour brain human tissue: endogenous fluorescence from glioma, metastasis and control tissues

Eliminating time-consuming process of conventional biopsy is a practical improvement, as well as increasing the accuracy of tissue diagnoses and patient comfort. We addressed these needs by developing a multimodal nonlinear endomicroscope that allows real-time optical biopsies during surgical procedure. It will provide immediate information for diagnostic use without removal of tissue and will assist the choice of the optimal surgical strategy. This instrument will combine several means of contrast: non-linear fluorescence, second harmonic generation signal, reflectance, fluorescence lifetime and spectral analysis. Multimodality is crucial for reliable and comprehensive analysis of tissue. Parallel to the instrumental development, we currently improve our understanding of the endogeneous fluorescence signal with the different modalities that will be implemented in the stated. This endeavor will allow to create a database on the optical signature of the diseased and control brain tissues. This proceeding will present the preliminary results of this database on three types of tissues: cortex, metastasis and glioblastoma.

Detection of human brain tumor infiltration with multimodal multiscale optical analysis

Brain tumor surgeries are facing major challenges to improve patients’ quality of life. The extent of resection while preserving surrounding eloquent brain areas is necessary to equilibrate the onco-functional. A tool able to increase the accuracy of tissue analysis and to deliver an immediate diagnostic on tumor, could drastically improve actual surgeries and patient survival rates. To achieve such performances a complete optical study, ranging from ultraviolet to infrared, of biopsies has been started by our group. Four different contrasts were used: 1) spectral analysis covering the DUV to IR range, 2) two photon fluorescence lifetime imaging and one photon time domain measurement, 3) second harmonic generation imaging and 4) fluorescence imaging using DUV to IR, one and two photon excitation. All these measurements were done on the endogenous fluorescence of tissues to avoid any bias and further clinical complication due to the introduction of external markers. The different modalities are then crossed to build a matrix of criteria to discriminate tumorous tissues. The results of multimodal optical analysis on human biopsies were compared to the gold standard histopathology.

Autofluorescence and second harmonic generation spectral analysis under two-photon excitation between 700 and 980 nm

The study of endogenous fluorescence appears as the future of medical imaging, coupled with a two photon excitation it could lead to numerous medical progresses, especially in early diagnosis. However due to the lack of miniaturized two photon technology it’s still a largely unknown field. As we project to build such a device, we also study the endogenous response of human brain biopsy under a two-photon excitation. The samples were excited with wavelengths between 700 and 980 nm and the spectral responses were measured. Two types of samples were study, thin and thick ones, the thick biopsy had a broader spectrum at each wavelength. Moreover three interesting wavelength were found, 750 nm that excite the most NADH molecules, 850 nm were we measure second harmonic generation (SHG) and a fluorescence were FAD molecules are predominant and 890 nm were SHG is the dominant response.