Comparative Study Between a Customized Bimodal Endoscope and a Benchtop Microscope for Quantitative Tissue Diagnosis.

Nowadays, surgical removal remains the standard method to treat brain tumors. During surgery, the neurosurgeon may encounter difficulties to delimitate tumor boundaries and the infiltrating areas as they have a similar visual appearance to adjacent healthy zones. These infiltrating residuals increase the tumor recurrence risk, which decreases the patient's post-operation survival time. To help neurosurgeons improve the surgical act by accurately delimitating healthy from cancerous areas, our team is developing an intraoperative multimodal imaging tool. It consists of a two-photon fluorescence fibered endomicroscope that is intended to provide a fast, real-time, and reliable diagnosis information. In parallel to the instrumental development, a large optical database is currently under construction in order to characterize healthy and tumor brain tissues with their specific optical signature using multimodal analysis of the endogenous fluorescence. Our previous works show that this multimodal analysis could provide a reliable discrimination response between different tissue types based on several optical indicators. Here, our goal is to show that the two-photon fibered endomicroscope is able to provide, based on the same approved indicators in the tissue database, the same reliable response that could be used intraoperatively. We compared the spectrally resolved and time-resolved fluorescence signal, generated by our two-photon bimodal endoscope from 46 fresh brain tissue samples, with a similar signal provided by a standard reference benchtop multiphoton microscope that has been validated for tissue diagnosis. The higher excitation efficiency and collection ability of an endogenous fluorescence signal were shown for the endoscope setup. Similar molecular ratios and fluorescence lifetime distributions were extracted from the two compared setups. Spectral discrimination ability of the bimodal endoscope was validated. As a preliminary step before tackling multimodality, the ability of the developed bimodal fibered endoscope to excite and to collect efficiently as well as to provide a fast exploitable high-quality signal that is reliable to discriminate different types of human brain tissues was validated.

Molecular changes tracking through multiscale fluorescence microscopy differentiate Meningioma grades and non-tumoral brain tissues.

Meningioma is the most common primary intracranial extra-axial tumor. Total surgical removal is the standard therapeutic method to treat this type of brain tumors. However, the risk of recurrence depends on the tumor grade and the extent of the resection including the infiltrated dura mater and, if necessary, the infiltrated bone. Therefore, proper resection of all invasive tumor borders without touching eloquent areas is of primordial in order to decrease the risk of recurrence. Nowadays, none of the intraoperative used tools is able to provide a precise real-time histopathological information on the tumor surrounding areas to help the surgeon to achieve a gross total removal. To respond to this problem, our team is developing a multimodal two-photon fluorescence endomicroscope, compatible with the surgeon tool, to better delimitate tumor boundaries, relying on the endogenous fluorescence of brain tissues. In this context, we are building a tissue database in order to specify each brain tissue, whether healthy or tumoral, with its specific optical signature. In this study, we present a multimodal and multiscale optical measurements on non-tumoral control brain tissue obtained in epilepsy surgery patients and several meningioma grades. We investigated tissue auto-fluorescence to track the molecular changes associated with the tumor grade from deep ultra-violet (DUV) to near infrared (NIR) excitation. Micro-spectroscopy, fluorescence lifetime imaging, two-photon fluorescence imaging and Second Harmonic Generation (SHG) imaging were performed. Several optically derived parameters such as collagen crosslinks fluorescence in DUV, SHG emission in NIR and long lifetime intensity fraction of Nicotinamide Adenine Dinucleotide and Flavins were correlated to discriminate cancerous tissue from control one. While collagen response managed to discriminate meningioma grades from control samples with a 100% sensitivity and 90% specificity through a 3D discriminative algorithm.

A Customized Two Photon Fluorescence Imaging Probe Based on 2D scanning MEMS Mirror Including Electrothermal Two-Level-Ladder Dual S-Shaped Actuators.

We report the design and characterization of a two-photon fluorescence imaging miniature probe. This customized two-axis scanning probe is dedicated for intraoperative two-photon fluorescence imaging endomicroscopic use and is based on a micro-electro-mechanical system (MEMS) mirror with a high reflectivity plate and two-level-ladder double S-shaped electrothermal bimorph actuators. The fully assembled probe has a total outer diameter of 4 mm including all elements. With a two-lens configuration and a small aperture MEMS mirror, this probe can generate a large optical scan angle of 24° with 4 V drive voltage and can achieve a 450 µm FOV with a 2-fps frame rate. A uniform Pixel Dwell Time and a stable scanning speed along a raster pattern were demonstrated while a 57-fs pulse duration of the excitation beam was measured at the exit of the probe head. This miniature imaging probe will be coupled to a two-photon fluorescence endomicroscope oriented towards clinical use.

Comparison of optically-derived biomarkers in healthy and brain tumor tissue under one- and two-photon excitation.

The surgical outcome of brain tumor resection and needle biopsy is significantly correlated to the patient's prognosis. Brain tumor surgery is limited to resecting the solid portion of the tumor as current intraoperative imaging modalities are incapable of delineating infiltrative regions. For accurate delineation, in situ tissue interrogation at the submicron scale is warranted. Additionally, multimodal detection is required to remediate the genetically and molecularly heterogeneous nature of brain tumors, notably, that of gliomas, meningioma and brain metastasis. Multimodal detection, such as spectrally- and temporally-resolved fluorescence under one- and two-photon excitation, enables characterizing tissue based on several endogenous optical contrasts. In order to assign the optically-derived parameters to different tissue types, construction of an optical database obtained from biopsied tissue is warranted. This report showcases the different quantitative and semi-quantitative optical markers that may comprise the tissue discrimination database. These include: the optical index ratio, the optical redox ratio, the relative collagen density, spectrally-resolved fluorescence lifetime parameters, two-photon fluorescence imaging and second harmonic generation imaging.

Optical Signatures Derived From Deep UV to NIR Excitation Discriminates Healthy Samples From Low and High Grades Glioma.

Among all the tumors of the central nervous system (CNS), glioma are the most deadly and the most malignant. Surgical resection is the standard therapeutic method to treat this type of brain cancer. But the diffusive character of these tumors create many problems for surgeons during the operation. In fact, these tumors migrate outside the tumor solid zone and invade the surrounding healthy tissues. These infiltrative tissues have the same visual appearance as healthy tissues, making it very difficult for surgeons to distinguish the healthy ones from the diffused ones. The surgeon, therefore, cannot properly remove the tumor margins increasing the recurrence risk of the tumor. To resolve this problem, our team has developed a multimodal two-photon fibered endomicroscope, compatible with the surgeon trocar, to better delimitate tumor boundaries by relying on the endogenous fluorescence of brain tissues. In this context, and in order to characterize the optical signature of glioma tumors, this study offers multimodal and multi-scaled optical measurements from healthy tissues to high grade glioma. We can interrogate tissue from deep ultra-violet to near infrared excitation by working with spectroscopy, fluorescence lifetime imaging, two-photon fluorescene imaging and Second Harmonic Generation (SHG) imaging. Optically derived ratios such as the Tryptophan/Collagen ratio, the optical redox ratio and the long lifetime intensity fraction, discriminated diseased tissue from its normal counterparts when fitted by Gaussian ellipsoids and choosing a threshold for each. Additionally two-photon fluorescence and SHG images were shown to display similar histological features as Hematoxylin-Eosin stained images.

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 tissue's 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 tissue's 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).

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.