Design, Implementation, and Evaluation of a Fluorescence Laminar Optical Tomography Scanner for Brain Imaging

Abstract

Implementation of new instrumentation and techniques for neuroscience research in recent years has opened new avenues in the study of the dynamics of large-scale neural networks such as the brain. In many of these techniques, including fluorescence recordings and optogenetic stimulation, a combination of photonics and molecular genetic methods are exploited to manipulate and monitor neural activities. Such techniques have been proven to be highly efficient in unraveling the mysteries of data processing in the micro circuits of the brain and as a result these techniques are widely used nowadays in most neuroscience labs. In optogenetics, cell-types of interest are genetically modified by expressing light-sensitive proteins adapted from microbial opsin. Once these proteins are expressed, we are able to use light of appropriate wavelengths to manipulate, increase or suppress neural activity of specific neurons on command. With a high temporal resolution (in the order of milliseconds) and cell-type-specific precision, optogenetics is able to probe how the nervous system functions in real-time, even in freely-moving animals. Currently, whenever genetic modifications are employed in the study of nervous systems, fluorescence proteins are also co-expressed in the same cells as biological markers to visualize the induced changes in the targeted cells. Despite its importance to trace the signal of such markers in-vivo, capabilities of the developed fluorescence tomography instrumentation are still limited and researchers mostly document the fluorescence distribution and expression of proteins of interest after euthanizing the animal and dissection of the tissue. In this project, we present our effort in implementing a fluorescence laminar optical tomography (FLOT) system which is specifically designed for non-invasive three dimensional imaging of fluorescence proteins within the brain of rodents. The application of the developed technology is not limited to optogenetics, but it can be used as a powerful tool to help improving the precision and accuracy of neuroscience and optogenetic experiments. In this design, a set of galvanometer mirrors are employed for realization of a fast and flexible scanner while a highly sensitive camera records the produced fluorescence signals. Fluorescence laminar optical tomography (FLOT) scanner has shown promising results in imaging superficial areas up to 2mm deep from the surface, with the resolution of ~200µm. Details of the design of the hardware and reconstruction algorithms are discussed and samples of experimental results are presented.