Multiple exposure speckle imaging (MESI) allows to map relative blood flows at the surface of biological tissues. MESI is an extension of laser speckle contrast imaging (LSCI). It relies on the computation of speckle contrast K for several exposure times T, allowing to discriminate the contribution of static scatters (bulk tissues) and moving scatterers (red blood cells). The MESI model describes K(T) as a function of tc, rho, beta, and v. These variables are respectively the decorrelation time of the moving scatterers, the relative contribution of static scatterers to the speckle pattern, a normalization factor for the imaging parameters and the contribution of noises to the speckle contrast. In LSCI theory, tc is commonly assumed to be inversely proportional to the flow. The acquisition of the speckle data at multiple exposures and the subsequent non-linear fit on a pixel-wise basis are instrumentally complex and time-intensive tasks that prevent real-time computation of the flow maps. In the study, we evaluated the feasibility of machine learning analysis of MESI data to bypass the non-linear fitting procedure based on the synthetic exposure acquisition. Synthetic exposures limit acquisition bias due to imperfect illumination normalization and are less sensitive to camera noises except for low illumination conditions or imaging of fast flows. A residual convolutional neural network was adopted to predict the blood flow map based on a database of representative speckle images of channels in a microfluidic chip with calibrated flows. The MESI database contains images with different exposure times for different flow and different channel diameters. The database was spitted into a training and testing data set with a 50:50 ratio. Preliminary results showed that blood flow mapping using deep learning can achieve moderate accuracy and yield a more stable prediction with high noise-resistant ability, compared to pixel-wise non-linear fit.
Multiple exposure speckle imaging (MESI) allows to map relative blood flows at the surface of biological tissues. MESI is an extension of laser speckle contrast imaging (LSCI). It relies on the computation of speckle contrast K for several exposure times T, allowing to discriminate the contribution of static scatters (bulk tissues) from that of moving scatterers (red blood cells). First, we have evaluated how a synthetic exposure acquisition scheme could strongly simplify the instrument for MESI, while remaining quantitative over a range of relevant flows. A microfluidic chip with controlled flows in channels with dimension representative of mice brain cerebral vasculature has been imaged using the classical modulated intensities approach and the synthetic exposure mode. This study allowed to propose guidelines in terms of readout dark noise and spatial response uniformity for the choice of a camera for MESI in the synthetic exposure mode. Second, we have evaluated how unwanted movements introduce bias in the speckle contrast calculation for a representative range of movement speeds. Mixed solutions of intralipid and glycerin in Brownian motion have been characterized to provide calibrated samples in terms of scatterers de-correlation times. High concentration of glycerin led to decorrelation times of several ms corresponding to actual values in small capillaries while low concentration of glycerin led to decorrelation times of 1ms or less corresponding to arterioles and arteries. The effects of the unwanted movement speed and direction have been measured for both lateral (x-y) and axial (z) movements. The bias introduced by unwanted movement in the (x-y) plane depends on the relative values of the time between frames and the scatterers decorrelation. In addition, for axial movements, parameters such as the numerical aperture (NA) and the magnification level (M) need to be considered due to their role in defining the depth of field.
Line-field confocal optical coherence tomography (LC-OCT) is an imaging technique that combines the principles of time-domain OCT and reflectance confocal microscopy (RCM). LC-OCT was designed to generate threedimensional (3D) morphological images of the skin, in vivo, with a spatial resolution of ∼ 1 μm. As in OCT and RCM, LC-OCT image contrast originates from the backscattering of incident light by the sample microstructures, which is determined by the optical scattering properties of the sample, namely the scattering coefficient μs and the scattering anisotropy parameter g. When imaging biological tissues, these properties can provide insight into tissue organization and structure, and could be used for quantitative tissue characterization in vivo. We present a method for obtaining spatially-resolved measurements of optical scattering parameters from LC-OCT images. Our approach is based on a calibration using a test sample with known optical scattering properties and on the application of a theoretical model previously developed for focus-tracking mode OCT and RCM. Assuming a single-scattering regime, this model allows to derive the optical scattering parameters μs and g from the intensity depth profiles acquired by LC-OCT. Spatially-resolved measurements are achieved by dividing the 3D LC-OCT image into “macro-voxels” and analyzing the different sample layers separately, leading to 3D distributions of μs and g. This method was experimentally tested against integrating spheres and collimated transmission measurements and validated on a set of mono- and bi-layered scattering phantoms.
We have compared multiple exposure speckle imaging using two approaches: (i) duration modulation of laser diode pulses with fixed exposure time and (ii) synthetic exposure created from the sum of frames obtained at 1ms exposure time. Both methods have been applied to evaluate controlled flows in micro-channels. The results demonstrate that the synthetic exposure method provides accurate speckle contrast data over a wide range of exposures, channel diameters and flows.
We have evaluated in vitro and in vivo the efficiency and practicability of reversible optical clearing of the skull for minimally invasive, longitudinal imaging of the rodent brain. Firstly, in vitro experiments have been conducted on resected mice skulls to evaluate the efficiency of the optical clarification process using different optical clearing agents. On the basis of recent literature, we have evaluated in vitro different optical clearing processes: (i) the direct application of PEG400; (ii) sequential EDTA and glycerol application and (iii) application of a solution of urea dissolved in ethanol. First, the time course of the clarification of the skull has been monitored quantitatively. We have carried out photometry experiments at 633 nm using a two integrating spheres system to characterize the total transmittance and reflectance of the mice skull samples. The evaluation of the optical transmission coefficient at 1300 nm was also obtained at different time points of the clearing process using sequential optical coherent tomography (OCT) imaging of the skull samples during the clearing process. Second, in vivo evaluation was carried out for repeated transcranial mapping of brain blood flow after optical clarification of the skull. Relative blood flow maps were obtained from multiple exposure laser speckle imaging. Third, post-mortem analysis of the toxicity of the chemicalstopical application was evaluated using immunohistochemistry to asses eventual cells death and inflammation. Overall our results show that in vivo brain imaging in mice could benefit from in vivo optical clearing. Yet, the use of optical clearing agents in vivo requires a proper evaluation of their efficiency, practicability of their use and potential toxicity to the tissues, especially for long term longitudinal studies.
Speckle contrast imaging allows in vivo imaging of relative blood flow changes. Multiple exposure speckle imaging (MESI) is more accurate than the standard single-exposure method since it allows separating the contribution of the static and moving scatters of the recorded speckle patterns. MESI requires experimental validation on phantoms prior to in vivo experiments to ensure the proper calibration of the system and the robustness of the model. The data analysis relies on the calculation of the speckle contrast for each exposure and a subsequent nonlinear fit to the MESI model to extract the scatterers correlation time and the relative contribution of moving scatters. We have designed two multichannel polydimethylsiloxane chips to study the influence of multiple and static scattering on the accuracy of MESI quantitation. We also propose a method based on standard C++ libraries to implement a computationally efficient analysis of the MESI data. Finally, the system was used to obtain in vivo hemodynamic data on two distinct sensory areas of the mice brain: the barrel cortex and the olfactory bulb.
The morphological and functional changes in cerebral blood vessels network is not well characterized in mice models of obesity. In order to study the hemodynamics of these models at rest and during sensory stimulation, we have developed a multi exposure speckle imaging system. It allows wide field superficial imaging of blood flow of the mice cortex. We have characterized the performances of the system using microfluidic phantoms. The capacity of the technique to retrieve accurate relative flow values was studied as a function of the diameter of vessels and the scatterers concentration. New biological data have been obtained in mice models of obesity (high fat diet mice) at rest and under sensory activation.
Vascular activity is necessary to provide suitable energy supply for cellular activity in the brain. Obesity, has become an important health and social issue worldwide. Yet, very little is known regarding morphological and functional vascular changes in the brain in obese patients. The purpose of our study is to evaluate the influence of this pathology on blood flow, vasodilation and vasoconstriction at rest and during sensory stimulation, in normal and obese mice. In order to obtain dynamic and quantitative maps of vascular activity over wide field of cortical tissues in anesthetized mice brain, we have developed a multi-exposure speckle imaging (MESI) system. MESI relies on the sequential recording of speckle images of the brain tissues illuminated with coherent light for increasing durations. For each of these multi exposure images the local speckle contrast is derived. This contrast is assumed to be related to the velocity of scatterers (red blood cells). The acquisition of speckle contrast for different expositions time allows discriminating the contribution of static and moving scatterers to the speckle pattern. Therefore, it allows mapping the blood flow changes over large cortical areas. Blood flow response to sensory activation was studied by imaging the olfactory bulb during olfactory stimulation trials. Data obtained in wild -type and high fat diet obesity model mice are presented showing a different hemodynamic response to olfactory stimulation.
In the last decade, Laser Speckle Contrast Imaging (LSCI) has been proposed and validated for imaging cerebral blood
flow at the rodent brain surface in vivo. The technique relies on the calculation of the spatial speckle contrast, which is
related to the velocity of scatterers (red blood cells). The implementation of the technique requires a partial craniotomy
so that the brain tissues of interest can be illuminated with a laser diode. However, the studies of changes in the
microcirculation during disease progression or treatment require longitudinal studies (i.e. imaging is done repeatedly
over weeks or even months). Practically, the less invasive way to obtain such data is to image through the thinned skull
without a craniotomy. However the presence of static scatterers (skull) will affect the speckle calculation and produce a
bias in the estimation of the microcirculation changes. An extension to LSCI, termed Multi-Exposure Speckle Imaging
(MESI) was proposed and validated a few years ago that address these limitations. It relies on a model of the speckle
contrast as a function of the exposure time and the proportion of static scatterers. Here, we used MESI with the aim of
repeatedly imaging the olfactory bulb of mice models of obesity. First, we have developed a MESI set up which was
characterized on microfluidic flow phantoms with different flow-rates and channel diameters to simulate blood flow in
animal model characteristics. Second, we show that MESI can discriminate flows in the presence of static scatterers and
it can measure flow changes consistently. Finally we provide an in vivo validation of the technique in mice with and
without a craniotomy.
Rodent brain is studied to understand the basics of brain function. The activity of cell populations and networks is commonly recorded in vivo with wide-field optical imaging techniques such as intrinsic optical imaging, fluorescence imaging, or laser speckle imaging. These techniques were recently adapted to unrestrained mice carrying transcranial windows. Furthermore, optogenetics studies would benefit from optical stimulation through the skull without implanting an optical fiber, especially for longitudinal studies. In this context, the knowledge of bone optical properties is requested to improve the quantitation of the depth and volume of imaged or stimulated tissues. Here, we provide experimental measurements of absorption and reduced scattering coefficients of freshly excised mice skull for wavelengths between 455 and 705 nm. Absorption coefficients from 6 to 8 months mice skull samples range between 1.67±0.28 mm−1 at 455 nm and 0.47±0.07 mm−1 at 705 nm, whereas reduced scattering coefficients were in the range of 2.79±0.26 mm−1 at 455 nm up to 2.29±0.12 mm−1 at 705 nm. In comparison, measurements carried out on 4 to 5 weeks mice showed similar spectral profiles but smaller absorption and reduced scattering coefficients by a factor of about 2 and 1.5, respectively.
2D surface maps of light distribution and temperature increase were recorded in wild type anesthetized rats brains during 90s light stimulation at 478nm (blue) and 638nm (red) with continuous or pulsed optical stimulations with corresponding power ranging from 100 up to 1200 mW/mm² at the output of an optical fiber. Post mortem maps were recorded in the same animals to assess the cooling effect of blood flow. Post mortem histological analysis were carried out to assess whether high power light stimulations had phototoxic effects or could trigger non physiological functional activation. Temperature increase remains below physiological changes (0,5 -1°) for stimulations up to 400mW/mm² at 40Hz. . Histology did not show significant irreversible modifications or damage to the tissues. The spatial profile of light distribution and heat were correlated and demonstrate as expected a rapid attenuation with diatnce to the fiber.
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.
The Geant4 Application for Emission Tomography (GATE) is an advanced open-source software dedicated to Monte-Carlo (MC) simulations in medical imaging involving photon transportation (Positron emission tomography, single photon emission computed tomography, computed tomography) and in particle therapy. In this work, we extend the GATE to support simulations of optical imaging, such as bioluminescence or fluorescence imaging, and validate it against the MC for multilayered media standard simulation tool for biomedical optics in simple geometries. A full simulation set-up for molecular optical imaging (bioluminescence and fluorescence) is implemented in GATE, and images of the light distribution emitted from a phantom demonstrate the relevance of using GATE for optical imaging simulations.
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.
Wide field multispectral imaging of light backscattered by brain tissues provides maps of hemodynamics changes (total
blood volume and oxygenation) following activation. This technique relies on the fit of the reflectance images obtain at
two or more wavelengths using a modified Beer-Lambert law1,2. It has been successfully applied to study the activation
of several sensory cortices in the anesthetized rodent using visible light1-5. We have carried out recently the first
multispectral imaging in the olfactory bulb6 (OB) of anesthetized rats. However, the optimization of wavelengths choice
has not been discussed in terms of cross talk and uniqueness of the estimated parameters (blood volume and saturation
maps) although this point was shown to be crucial for similar studies in Diffuse Optical Imaging in humans7-10. We have
studied theoretically and experimentally the optimal sets of wavelength for multispectral imaging of rodent brain
activation in the visible. Sets of optimal wavelengths have been identified and validated in vivo for multispectral imaging
of the OB of rats following odor stimulus. We studied the influence of the wavelengths sets on the magnitude and time
courses of the oxy- and deoxyhemoglobin concentration variations as well as on the spatial extent of activated brain
areas following stimulation. Beyond the estimation of hemodynamic parameters from multispectral reflectance data, we
observed repeatedly and for all wavelengths a decrease of light reflectance. For wavelengths longer than 590 nm, these
observations differ from those observed in the somatosensory and barrel cortex and question the basis of the reflectance
changes during activation in the OB. To solve this issue, Monte Carlo simulations (MCS) have been carried out to assess
the relative contribution of absorption, scattering and anisotropy changes to the intrinsic optical imaging signals in
somatosensory cortex (SsC) and OB model.
Dynamic maps of relative changes in blood volume and oxygenation following brain activation are obtained using multispectral reflectance imaging. The technique relies on optical absorption modifications linked to hemodynamic changes. The relative variation of hemodynamic parameters can be quantified using the modified Beer-Lambert Law if changes in reflected light intensities are recorded at two wavelengths or more and the differential path length (DP) is known. The DP is the mean path length in tissues of backscattered photons and varies with wavelength. It is usually estimated using Monte Carlo simulations in simplified semi-infinite homogeneous geometries. Here we consider the use of multilayered models of the somatosensory cortex (SsC) and olfactory bulb (OB), which are common physiological models of brain activation. Simulations demonstrate that specific DP estimation is required for SsC and OB, specifically for wavelengths above 600 nm. They validate the hypothesis of a constant path length during activation and show the need for specific DP if imaging is performed in a thinned-skull preparation. The first multispectral reflectance imaging data recorded in vivo during OB activation are presented, and the influence of DP on the hemodynamic parameters and the pattern of oxymetric changes in the activated OB are discussed.
In vivo multispectral reflectance imaging has been extensively used in the somatosensory cortex (SsC) in anesthetized
rodents to collect intrinsic signal during activation and derive hemodynamics signals time courses. So far it has never
been applied to the Olfactory Bulb (OB), although this structure is particularly well suited to the optical study of brain
activation due to the its well defined organization, the ability to physiologically activate it with odorants, and the low
depth of the activated layers. To obtain hemodynamics parameters from reflectance variations data, it is necessary to
take into account a corrective factor called Differential Pathlength (DP). It is routinely estimated using Monte Carlo
simulations, modeling photons propagation in simplified infinite geometry tissue models. The first goal of our study was
to evaluate the influence of more realistic layered geometries and optical properties on the calculation of DP and
ultimately on the estimation of the hemodynamics parameters. Since many valuable results have been obtained
previously by others in the SSc, for the purpose of validation and comparison we performed Monte Carlo simulations in
both the SSC and the OB. We verified the assumption of constant DP during activation by varying the hemoglobin
oxygen saturation, total hemoglobin concentration and we also studied the effect of a superficial bone layer on DP
estimation for OB. The simulations show the importance of defining a finite multilayer model instead of the coarse
infinite monolayer model, especially for the SSc, and demonstrate the need to perform DP calculation for each structure
taking into account their anatomofunctional properties. The second goal of the study was to validate in vivo
multispectral imaging for the study of hemodynamics in the OB during activation. First results are presented and
discussed.
KEYWORDS: Luminescence, Tissues, Brain, Photons, Absorption, In vivo imaging, Monte Carlo methods, Scattering, Tissue optics, Auto-fluorescence imaging
Understanding the cellular mechanisms of energy supply to neurons following physiological activation is
still challenging and has strong implications to the interpretation of clinical functional images based on metabolic
signals such as Blood Oxygen Level Dependent Magnetic Resonance Imaging or 18F-Fluorodexoy-Glucose Positron
Emission Tomography. Intrinsic Optical Signal Imaging provides with high spatio temporal resolution in vivo
imaging in the anaesthetized rat. In that context, intrinsic signals are mainly related to changes in the optical
absorption of haemoglobin depending on its oxygenation state. This technique has been validated for imaging of the
rat olfactory bulb, providing with maps of the actived olfactory glomeruli, the functional modules involved in the
first step of olfactory coding. A complementary approach would be autofluorescence imaging relying on the
fluorescence properties of endogenous Flavin Adenine Dinucleotide (FAD) or Nicotinamide Adenine Dinucleotide
(NADH) both involved in intracellular metabolic pathways.
The purpose of the present study was to investigate the feasibility of in vivo autofluorescence imaging in the
rat olfactory bulb. We performed standard Monte Carlo simulations of photons scattering and absorption at the
excitation and emission wavelengths of FAD and NADH fluorescence. Characterization of the fluorescence
distribution in the glomerulus, effect of hemoglobin absorption at the excitation and absorption wavelengths as well
as the effect of the blurring due to photon scattering and the depth of focus of the optical apparatus have been
studied. Finally, optimal experimental parameters are proposed to achieve in vivo validation of the technique in the
rat olfactory bulb.
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