We present our effort in implementing a fluorescence laminar optical tomography scanner which is specifically designed for noninvasive three-dimensional imaging of fluorescence proteins in the brains of small rodents. A laser beam, after passing through a cylindrical lens, scans the brain tissue from the surface while the emission signal is captured by the epi-fluorescence optics and is recorded using an electron multiplication CCD sensor. Image reconstruction algorithms are developed based on Monte Carlo simulation to model light–tissue interaction and generate the sensitivity matrices. To solve the inverse problem, we used the iterative simultaneous algebraic reconstruction technique. The performance of the developed system was evaluated by imaging microfabricated silicon microchannels embedded inside a substrate with optical properties close to the brain as a tissue phantom and ultimately by scanning brain tissue in vivo. Details of the hardware design and reconstruction algorithms are discussed and several experimental results are presented. The developed system can specifically facilitate neuroscience experiments where fluorescence imaging and molecular genetic methods are used to study the dynamics of the brain circuitries.
Predicting the distribution of light inside any turbid media, such as biological tissue, requires detailed information about the optical properties of the medium, including the absorption and scattering coefficients and the anisotropy factor. Particularly, in biophotonic applications where photons directly interact with the tissue, this information translates to system design optimization, precision in light delivery, and minimization of unintended consequences, such as phototoxicity or photobleaching. In recent years, optogenetics has opened up a new area in deep brain stimulation with light and the method is widely adapted by researchers for the study of the brain circuitries and the dynamics of neurological disorders. A key factor for a successful optogenetic stimulation is delivering an adequate amount of light to the targeted brain objects. The adequate amount of light needed to stimulate each brain object is identified by the tissue optical properties as well as the type of opsin expressed in the tissue, wavelength of the light, and the physical dimensions of the targeted area. Therefore, to implement a precise light delivery system for optogenetics, detailed information about the optical properties of the brain tissue and a mathematical model that incorporates all determining factors is needed to find a good estimation of light distribution in the brain. In general, three measurements are required to obtain the optical properties of any tissue, namely diffuse transmitted light, diffuse reflected light, and transmitted ballistic beam. In this report, these parameters were measured in vitro using intact rat brain slices of 500 μm thickness via a two-integrating spheres optical setup. Then, an inverse adding doubling method was used to extract the optical properties of the tissue from the collected data. These experiments were repeated to cover the whole brain tissue with high spatial resolution for the three different cuts (transverse, sagittal, and coronal) and three different wavelengths (405, 532, and 635 nm) in the visible range of the spectrum. A three-dimensional atlas of the rat brain optical properties was constructed based on the experimental measurements. This database was linked to a Monte Carlo toolbox to simulate light distribution in the tissue for different light source configurations.
Optogenetics is the science where recent progresses in the field of photonics are combined with the techniques in
molecular genetics to develop a methodology for modulation of neural activities.<sup>1-9</sup> Despite enormous enthusiasm
in using optogenetics for brain studies, little has been done on the engineering side such as technology development
for light delivery or realization of reliable systems for optical monitoring of the induced activities. In this
project, we have implemented a Digital Micromirror Device based microprojection system capable of delivering
illumination patterns through a high-resolution imaging fiber bundle that guides the pattern to the region of
interest on the surface or within the brain tissue. The system is also equipped with an imaging path for detection
of calcium signals and monitoring the induced patterns of cellular activities. A very interesting application of the
system is extracting topographic computational maps of cortex or cellular receptive fields in-vivo. It is known
that such maps are the engine of information processing in the cortex. Better understanding of the structure
of such maps will help to unravel the mysteries of brain higher level computations. Another application of this
system is related to the high-resolution stimulation patterns that cannot be produced with electrode arrays.
Production of high-resolution patterns is important in the study of specific modes of brain activities. We report
the details of our optical design, preliminary results produced by testing the system on tissue, and we discuss
our strategy to extract new data from the brain tissue.