Design and validation of a convolutional neural network for fast, model-free blood flow imaging with multiple exposure speckle imaging.

Multiple exposure speckle imaging has demonstrated its improved accuracy compared to single exposure speckle imaging for relative quantitation of blood flow in vivo. However, the calculation of blood flow maps relies on a pixelwise non-linear fit of a multi-parametric model to the speckle contrasts. This approach has two major drawbacks. First, it is computer-intensive and prevents real time imaging and, second, the mathematical model is not universal and should in principle be adapted to the type of blood vessels. We evaluated a model-free machine learning approach based on a convolutional neural network as an alternative to the non-linear fit approach. A network was designed and trained with annotated speckle contrast data from microfluidic experiments. The neural network performances are then compared to the non-linear fit approach applied to in vitro and in vivo data. The study demonstrates the potential of convolutional networks to provide relative blood flow maps from multiple exposure speckle data in real time.

Obesity in Midlife Hampers Resting and Sensory-Evoked Cerebral Blood Flow in Mice.

OBJECTIVE: This study aimed to investigate the effects of a high-fat diet (HFD) and aging on resting and activity-dependent cerebral blood flow (CBF). METHODS: To run a comparison between obese and age-matched control animals, 6-week-old mice were fed either with regular chow or an HFD for 3 months or 8 months. Glucose tolerance and insulin sensitivity were assessed for metabolic phenotyping. Resting and odor-evoked CBF at the microvascular scale in the olfactory bulb (OB) was investigated by multiexposure speckle imaging. Immunolabeling-enabled imaging of solvent-cleared organs was used to analyze vascular density. The ejection fraction was studied by using cardioechography. Olfactory sensitivity was tested by using a buried-food test. RESULTS: Glucose intolerance and compromised odor-evoked CBF were observed in obese mice in the younger group. Prolonged HFD feeding triggered insulin resistance and stronger impairment in activity-dependent CBF. Aging had a specific negative impact on resting CBF. There was no decrease in vascular density in the OB of obese mice, although cardiac function was impaired at both ages. In addition, decreased olfactory sensitivity was observed only in the older, middle-aged obese mice. CONCLUSIONS: OB microvasculature in obese mice showed a specific functional feature characterized by impaired sensory-evoked CBF and a specific deleterious effect of aging on resting CBF.

Multiple speckle exposure imaging for the study of blood flow changes induced by functional activation of barrel cortex and olfactory bulb in mice.

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.

Simulation of nanoparticle-mediated near-infrared thermal therapy using GATE.

Application of nanotechnology for biomedicine in cancer therapy allows for direct delivery of anticancer agents to tumors. An example of such therapies is the nanoparticle-mediated near-infrared hyperthermia treatment. In order to investigate the influence of nanoparticle properties on the spatial distribution of heat in the tumor and healthy tissues, accurate simulations are required. The Geant4 Application for Emission Tomography (GATE) open-source simulation platform, based on the Geant4 toolkit, is widely used by the research community involved in molecular imaging, radiotherapy and optical imaging. We present an extension of GATE that can model nanoparticle-mediated hyperthermal therapy as well as simple heat diffusion in biological tissues. This new feature of GATE combined with optical imaging allows for the simulation of a theranostic scenario in which the patient is injected with theranostic nanosystems that can simultaneously deliver therapeutic (i.e. hyperthermia therapy) and imaging agents (i.e. fluorescence imaging).

Experimental assessment of the safety and potential efficacy of high irradiance photostimulation of brain tissues.

Optogenetics is widely used in fundamental neuroscience. Its potential clinical translation for brain neuromodulation requires a careful assessment of the safety and efficacy of repeated, sustained optical stimulation of large volumes of brain tissues. This study was performed in rats and not in non-human primates for ethical reasons. We studied the spatial distribution of light, potential damage, and non-physiological effects in vivo, in anesthetized rat brains, on large brain volumes, following repeated high irradiance photo-stimulation. We generated 2D irradiance and temperature increase surface maps based on recordings taken during optical stimulation using irradiance and temporal parameters representative of common optogenetics experiments. Irradiances of 100 to 600 mW/mm2 with 5 ms pulses at 20, 40, and 60 Hz were applied during 90 s. In vivo electrophysiological recordings and post-mortem histological analyses showed that high power light stimulation had no obvious phototoxic effects and did not trigger non-physiological functional activation. This study demonstrates the ability to illuminate cortical layers to a depth of several millimeters using pulsed red light without detrimental thermal damages.

PIXSIC: A Wireless Intracerebral Radiosensitive Probe in Freely Moving Rats.

The aim of this study was to demonstrate the potential of a wireless pixelated ß+-sensitive intracerebral probe (PIXSIC) for in vivo positron emission tomographic (PET) radiopharmacology in awake and freely moving rodents. The binding of [(11)C]raclopride to D2 dopamine receptors was measured in anesthetized and awake rats following injection of the radiotracer. Competitive binding was assessed with a cold raclopride injection 20 minutes later. The device can accurately monitor binding of PET ligands in freely moving rodents with a high spatiotemporal resolution. Reproducible time-activity curves were obtained for pixels throughout the striatum and cerebellum. A significantly lower [(11)C]raclopride tracer-specific binding was observed in awake animals. These first results pave the way for PET tracer pharmacokinetics measurements in freely moving rodents.

Characterization, comparison, and choice of a commercial double-clad fiber for nonlinear endomicroscopy.

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.

Extension of the GATE Monte-Carlo simulation package to model bioluminescence and fluorescence imaging.

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.

Experimental and analytical comparative study of optical coefficient of fresh and frozen rat tissues.

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 µa 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.

Multispectral reflectance imaging of brain activation in rodents: methodological study of the differential path length estimations and first in vivo recordings in the rat olfactory bulb.

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.

Reconstruction of field excitatory post-synaptic potentials in the dentate gyrus from amperometric biosensor signals.

A new feasible and reproducible method to reconstruct local field potentials from amperometric biosensor signals is presented. It is based on the least-square fit of the current response of the biosensor electrode to a voltage step by the use of two time constants. After determination of the electrode impedance, Fast Fourier Transform (FFT) and Inverse FFT are performed to convert the recorded amperometric signals into voltage and trace the local field potentials using a resistor-capacitor circuit-based model. We applied this method to reconstruct field evoked potentials from currents recorded by a lactate biosensor in the rat dentate gyrus after stimulation of the perforant pathway in vivo. Initial slope of the reconstructed field excitatory postsynaptic potentials was used in order to demonstrate long term potentiation induced by high frequency stimulation of the perforant path. Our results show that reconstructing evoked potentials from amperometric recordings is a reliable method to obtain in vivo electrophysiological and amperometric information simultaneously from the same electrode in order to understand how chemical compounds vary with and modulate the dynamics of brain activity.

Imaging odor-evoked activities in the mouse olfactory bulb using optical reflectance and autofluorescence signals.

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain. Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex(1). IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group(2). The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues(3), it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group(4). The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of ˜100µm(3) constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB(5, 6, 7) which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet. Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

Autofluorescence imaging of NADH and flavoproteins in the rat brain: insights from Monte Carlo simulations.

There has been recently a renewed interest in using Autofluorescence imaging (AF) of NADH and flavoproteins (Fp) to map brain activity in cortical areas. The recording of these cellular signals provides complementary information to intrinsic optical imaging based on hemodynamic changes. However, which of NADH or Fp is the best candidate for AF functional imaging is not established, and the temporal profile of AF signals is not fully understood. To bring new theoretical insights into these questions, Monte Carlo simulations of AF signals were carried out in realistic models of the rat somatosensory cortex and olfactory bulb. We show that AF signals depend on the structural and physiological features of the brain area considered and are sensitive to changes in blood flow and volume induced by sensory activation. In addition, we demonstrate the feasibility of both NADHAF and Fp-AF in the olfactory bulb.

The potential of a radiosensitive intracerebral probe to monitor 18F-MPPF binding in mouse hippocampus in vivo.

UNLABELLED: As mouse imaging has become more challenging in preclinical research, efforts have been made to develop dedicated PET systems. Although these systems are currently used for the study of physiopathologic murine models, they present some drawbacks for brain studies, including a low temporal resolution that limits the pharmacokinetic study of radiotracers. The aim of this study was to demonstrate the ability of a radiosensitive intracerebral probe to measure the binding of a radiotracer in the mouse brain in vivo. METHODS: The potential of a probe 0.25 mm in diameter for pharmacokinetic studies was assessed. First, Monte Carlo simulations followed by experimental studies were used to evaluate the detection volume and sensitivity of the probe and its adequacy for the size of loci in the mouse brain. Second, ex vivo autoradiography of 5-hydroxytryptamine receptor 1A (5-HT(1A)) receptors in the mouse brain was performed with the PET radiotracer 2'-methoxyphenyl-(N-2'-pyridinyl)-p-(18)F-fluorobenzamidoethylpiperazine ((18)F-MPPF). Finally, the binding kinetics of (18)F-MPPF were measured in vivo in both the hippocampus and the cerebellum of mice. RESULTS: Both the simulations and the experimental studies demonstrated the feasibility of using small probes to measure radioactive concentrations in specific regions of the mouse brain. Ex vivo autoradiography showed a heterogeneous distribution of (18)F-MPPF consistent with the known distribution of 5-HT(1A) in the mouse brain. Finally, the time-activity curves obtained in vivo were reproducible and validated the capacity of the new probe to accurately measure (18)F-MPPF kinetics in the mouse hippocampus. CONCLUSION: Our results demonstrate the ability of the tested radiosensitive intracerebral probe to monitor binding of PET radiotracers in anesthetized mice in vivo, with high temporal resolution suited for compartmental modeling.

Combining the radiosensitive Beta MicroProbe to nuclear magnetic resonance: theoretical approach for in vivo studies in small animals.

In vivo small animal imaging with multiple modalities has become an important tool in modern biomedical research. Indeed, combining exploratory techniques allows simultaneous recording of complementary data, which is required to elucidate complex physiopathological mechanisms. In this field, because of strict technical constraints in vivo, an exciting challenge remains in the combination of Nuclear Magnetic Resonance (NMR) and Positron Emission Tomography (PET). Coupling NMR with a radiosensitive Beta MicroProbe offers therefore a very interesting technical alternative. Here, we assessed the feasibility of this new combination by theoretically evaluating the ability of the Beta MicroProbe to monitor radioactivity in a magnet. To that aim, we modelled with Geant4 the effect of an intense magnetic field on the probe field of view and showed that the field should not have an impact on the global efficiency of the probe.

Arterial input function measurement without blood sampling using a beta-microprobe in rats.

UNLABELLED: The evaluation of every new radiotracer involves pharmacokinetic studies on small animals to determine its biodistribution and local kinetics. To extract relevant biochemical information, time-activity curves for the regions of interest are mathematically modeled on the basis of compartmental models that require knowledge of the time course of the tracer concentration in plasma. Such a time-activity curve, usually termed input function, is determined in small animals by repeated blood sampling and subsequent counting in a well counter. The aim of the present work was to propose an alternative to blood sampling in small animals, since this procedure is labor intensive, exposes the staff to radiation, and leads to an important loss of blood, which affects hematologic parameters. METHODS: Monte Carlo simulations were performed to evaluate the feasibility of measuring the arterial input function using a positron-sensitive microprobe placed in the femoral artery of a rat. The simulation results showed that a second probe inserted above the artery was necessary to allow proper subtraction of the background signal arising from tracer accumulation in surrounding tissues. This approach was then validated in vivo in 5 anesthetized rats. In a second set of experiments, on 3 rats, a third probe was used to simultaneously determine 18F-FDG accumulation in the striatum. RESULTS: The high temporal resolution of the technique allowed accurate determination of the input function peak after bolus injection of 18F-FDG. Quantitative input functions were obtained after normalization of the arterial time-activity curve for a late blood sample. In the second set of experiments, compartmental modeling was achieved using either the blood samples or the microprobe data as the input function, and similar kinetic constants were found in both cases. CONCLUSION: Although direct quantification proved difficult, the microprobe allowed accurate measurement of arterial input function with a high temporal resolution and no blood loss. The technique, because offering adequate sensitivity and temporal resolution for kinetic measurements of radiotracers in the blood compartment, should facilitate quantitative modeling for radiotracer studies in small animals.

In vivo quantification of localized neuronal activation and inhibition in the rat brain using a dedicated high temporal-resolution beta +-sensitive microprobe.

Understanding brain disorders, the neural processes implicated in cognitive functions and their alterations in neurodegenerative pathologies, or testing new therapies for these diseases would benefit greatly from combined use of an increasing number of rodent models and neuroimaging methods specifically adapted to the rodent brain. Besides magnetic resonance (MR) imaging and functional MR, positron-emission tomography (PET) remains a unique methodology to study in vivo brain processes. However, current high spatial-resolution tomographs suffer from several technical limitations such as high cost, low sensitivity, and the need of restraining the animal during image acquisition. We have developed a beta(+)-sensitive high temporal-resolution system that overcomes these problems and allows the in vivo quantification of cerebral biochemical processes in rodents. This beta-MICROPROBE is an in situ technique involving the insertion of a fine probe into brain tissue in a way very similar to that used for microdialysis and cell electrode recordings. In this respect, it provides information on molecular interactions and pathways, which is complementary to that produced by these technologies as well as other modalities such as MR or fluorescence imaging. This study describes two experiments that provide a proof of concept to substantiate the potential of this technique and demonstrate the feasibility of quantifying brain activation or metabolic depression in individual living rats with 2-[(18)F]fluoro-2-deoxy-d-glucose and standard compartmental modeling techniques. Furthermore, it was possible to identify correctly the origin of variations in glucose consumption at the hexokinase level, which demonstrate the strength of the method and its adequacy for in vivo quantitative metabolic studies in small animals.

SIC, an intracerebral beta(+)-range-sensitive probe for radiopharmacology investigations in small laboratory animals: binding studies with (11)C-raclopride.

UNLABELLED: Our aim was to show the ability of a recently developed beta(+)-range-sensitive intracerebral probe (SIC) to measure, in vivo, the binding of radioligands in small animals. METHODS: The potential of the device for pharmacokinetic studies was evaluated by measurement of the dynamic striatal binding of (11)C-raclopride, a well-documented D(2) dopaminergic receptor ligand, in rat brain after intravenous injection of the labeled compound. The effects of preinjection of the unlabeled ligand (raclopride, 2 mg/kg intravenously) and of increasing the synaptic dopamine level (amphetamine treatment, 1 mg/kg intravenously) or of depleting synaptic dopamine (reserpine pretreatment, 5 mg/kg intraperitoneally) on in vivo (11)C-raclopride binding were monitored by SIC. RESULTS: The radioactivity curves measured as a function of time were reproducible and consistent with previous studies using PET imaging (ratio of striatum to cerebellum, 2.6 +/- 0.3 after 20 min). Further studies showed significant displacement of (11)C-raclopride by its stable analog. Finally, the device proved its capacity to accurately detect changes in (11)C-raclopride binding after a sudden (amphetamine) or a gradual (reserpine) modulation of endogenous dopamine levels. CONCLUSION: These results show that the new device can monitor binding of PET ligands in anesthetized rodents in vivo, with high temporal resolution.