KEYWORDS: Microelectromechanical systems, Mirrors, Luminescence, Micromirrors, Photodynamic therapy, In vivo imaging, Prototyping, Tumors, Tissues, Surgery
Nowadays, the ability to diagnose brain tumors intraoperatively and to delineate their margins, as accurately as possible, is of primordial importance during surgery. However, the exact tumor boundaries and the infiltrated tumor cells outside its solid volume are difficult to be found by the surgeon due to the similar visual appearances especially at the margins, leading in many cases to poor surgical outcomes and a high risk of recurrences. To solve this issue, our group develop a two-photon multimodal endomicroscope to analyze the endogenous fluorescence of cerebral tissues in order to provide reliable informations intraoperatively on the tissues nature at cellular level. For that, and in order to implement the imaging modality in our endomicroscope, we launched a study based on an original miniaturized scanning system to be coupled with our endomicroscope. In this work, we characterize a scanning system prototype based on an electrothermally-actuated scanning micro-mirror. This type of scanning devices can provide a wide linear scan range at a low driving voltage and a high stable scanning speed along a straight scan line which help to acquire high-quality images.
Among Central Nervous System (CNS) tumors, diffuse glioma are the most infiltrating and malignant tumors. According to the World Health Organization (WHO), they are classified into different grades, referring to their pathological class and histological properties. To treat glioma tumors, many methods have been proposed, still the standard one remains the maximal safe total resection. During the operation, many difficulties obstruct the surgeon to identify the infiltrated areas, which contains diffuse tumors cells, around the solid area of the tumor, overlapping the healthy areas, and presenting the same visual appearances. If not totally removed, these infiltrating zones can increase the risk of recurrence and affects the survival rate of patient. To overcome this problem, we develop a multimodal two-photon endomicroscope, based on the endogenous fluorescence of brain tissues, to assist the surgeon during the surgery. The tool will provide him information on the infiltrated areas and their histological nature. In this paper, we tried to discriminate between metastasis, low grade and high grade glioma from healthy fresh tissues, presenting a multimodal study using deep ultraviolet, visible and near infrared excitation to acquire spectral measurements, Fluorescence Lifetime Imaging (FLIM) and Two-Photon Emission Fluorescence (TPEF) imaging. We compared also our TPEF and FLIM images to the histological images.
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.
Current surgical biopsy needs several days for the analysis process to be finished. Anatomopathologists provide analysis reports to the surgeon a few days after the surgical intervention, which makes it a lengthy decision making practice. In addition, the lack of precise guidance often leads to inaccuracies in the selection of tissue regions for biopsy and so necessitates repeating the operation sometimes. Our project aims at reducing this time as well as patient discomfort. In this context, we propose to develop a multimodal nonlinear endomicroscope providing several means of contrast. Among these contrast that are useful in the detection of tumor regions, we note imaging by linear and non-linear fluorescence, by second and third harmonic generation and by reflectance. In addition, this technique allows fluorescence lifetime and spectral measurements. Our endomicroscopic system is based on a new homemade customized double-clad photonic crystal fiber (DC-PCF). Finally, this double-clad micro structured optical fiber insures visible and near infrared excitation. This system was tested by measuring fluorescence lifetime and the spectral shape of a fixed tumoral brain sample in one and two photon excitations.
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.
Several endomicroscope prototypes for nonlinear optical imaging were developed in the last decade for in situ analysis of tissue with cellular resolution by using short infrared light pulses. Fourier-transform-limited pulses at the tissue site are necessary for optimal excitation of faint endogenous signals. However, obtaining these transform-limited short pulses remains a challenge, and previously proposed devices did not achieve an optimal pulse delivery. We present a study of fibered endomicroscope architecture with an efficient femtosecond pulse delivery and a high excitation level at the output of commercially available double-clad fibers (DCFs). The endomicroscope incorporates a module based on a grism line to compensate for linear and nonlinear effects inside the system. Simulations and experimental results are presented and compared to the literature. Experimentally, we obtained short pulses down to 24 fs at the fiber output, what represents to the best of our knowledge the shortest pulse duration ever obtained at the output of a nonlinear endoscopic system without postcompression. The choice of the optimal DCF among four possible commercial components is discussed and evaluated in regard to multiphoton excitation and fluorescence emission.
Optical properties of fresh and frozen tissues of rat heart, kidney, brain, liver, and muscle were measured in the 450- to 700-nm range. The total reflectance and transmittance were measured using a well-calibrated integral sphere set-up. Absorption coefficient μ a and reduced scattering coefficient μ ′ s were derived from the experimental measurements using the inverse adding doubling technique. The influence of cryogenic processing on optical properties was studied. Interindividual and intraindividual variations were assessed. These new data aim at filling the lack of validated optical properties in the visible range especially in the blue-green region of particular interest for fluorescence and optogenetics preclinical studies. Furthermore, we provide a unique comparison of the optical properties of different organs obtained using the same measurement set-up for fresh and frozen tissues as well as an estimate of the intraindividual and interindividual variability.
Growing interest in optical instruments for biomedical applications has increased the use of optically calibrated phantoms. Often associated with tissue modeling, phantoms allow the characterization of optical devices for clinical purposes. Fluorescent gel phantoms have been developed, mimicking optical properties of healthy and tumorous brain tissues. Specific geometries of dedicated molds offer multiple-layer phantoms with variable thicknesses and monolayer phantoms with cylindrical inclusions at various depths and diameters. Organic chromophores are added to allow fluorescence spectroscopy. These phantoms are designed to be used with 405 nm as the excitation wavelength. This wavelength is then adapted to excite large endogenous molecules. The benefits of these phantoms in understanding fluorescence tissue analysis are then demonstrated. In particular, detectability aspects as a function of geometrical and optical parameters are presented and discussed.
During glioblastoma surgery, delineation of the brain tumour margins remains difficult especially since
infiltrated and normal tissues have the same visual appearance. This problematic constitutes our research
interest. We developed a fibre-optical fluorescence probe for spectroscopic and time domain measurements.
First measurements of endogenous tissue fluorescence were performed on fresh and fixed rat tumour brain
slices. Spectral characteristics, fluorescence redox ratios and fluorescence lifetime measurements were analysed.
Fluorescence information collected from both, lifetime and spectroscopic experiments, appeared promising for
tumour tissue discrimination. Two photon measurements were performed on the same fixed tissue. Different
wavelengths are used to acquire two-photon excitation-fluorescence of tumorous and healthy sites.
We are developing a non-linear fibered endomicroscope for imaging the extracellular
matrix collagen and elastin fibrillar networks during bronchoscopy. As a proof of concept, laser pulses at
the output of a standard 2 meter long single-mode fibre have been obtained with pulse duration of about
50 fs and pulse energy up to 50 uJ, using a specially designed grism line for the dispersion compensation.
With these pulses, we performed a spectroscopic characterization of the non-linear endogenous signal,
consisting of two-photon fluorescence and second harmonic generation, and originated from human
pulmonary tissue of various thickness, both in forward and backward geometry of signal collection, with
excitation at 830 nm.
KEYWORDS: Skull, Scattering, Reflectivity, Laser scattering, Confocal microscopy, Luminescence, Bone, Two photon imaging, Two photon excitation microscopy, In vivo imaging
We present experiments and analyses of confocal reflectance and two-photon microscopy studies of zebra finch skull samples. The thin and hollow structure of these birds' skulls is quite translucent, which can allow in vivo transcranial two-photon imaging for brain activation monitoring. However, the skull structure is also quite complex, with high refractive index changes on a macroscopic scale. These studies aim at exploring the geometrical and scattering properties of these skull samples with the use of several confocal microscopy contrasts. Moreover, the study of the axial reflectance exponential decay is used to estimate the scattering coefficients of the bone. Finally, two-photon imaging experiments of a fluorescent object located beneath the skull are carried out. It reveals that two-photon fluorescence can be collected through the skull with a strong signal. It also reveals that the spatial resolution loss is quite high and cannot be fully explained by the bulk scattering properties of the bone, but also by the presence of the high refractive index inhomogeneity of this pneumatic skull structure. Even if the optical properties of the skull are different during in vivo experiments, these preliminary studies are aimed at preparing and optimizing transcranial brain activation monitoring experiments on songbirds.
Zebra Finches are songbirds which constitute a model for neuro-ethologists to study the neuro-mechanisms of
vocal recognition. For this purpose, in vivo and non invasive monitoring of brain activity is required during
acoustical stimulation. MRI (Magnetic Resonance Imaging) or NIRS (Near InfraRed Spectroscopy) are suitable
methods for these measurements, even though MRI is difficult to link quantitatively with neural activity and
NIRS suffers from a poor resolution. In the particular case of songbirds (whose skin is thin and quite transparent
and whose skull structure is hollow), two-photon microscopy enables a quite deep penetration in tissues and
could be an alternative. We present here preliminary studies on the feasability of two-photon microscopy in
these conditions. To do so, we chose to image hollow fibers, filled with Rhodamine B, through the skin of Zebra
finches in order to evaluate the spatial resolution we may expect in future in vivo experiments. Moreover, we used
the reflectance-mode confocal configuration to evaluate the exponential decrease of backreflected light in skin
and in skull samples. Following this procedure recently proposed by S.L. Jacques and co-workers, we planned
to determine the scattering coefficient μs and the anisotropy g of these tissues and make a comparison between
fixed and fresh skin and skull samples for future Monte Carlo simulations of the scattering in our particular
multi-layered structure.
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