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.
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