For a given opsin gene, functional expression levels and the light power density selleck chemicals llc reaching the expressing cells will together determine the efficacy of light-based control (Figure 3A). To estimate this density of light reaching the targeted cells one must consider the propagation of light in tissue. Light propagation in biological tissue can be modeled as a combination of absorption and scattering, with scattering playing an especially important role in mature
myelinated brain tissue (Vo-Dinh, 2003). The transmission properties of light through the brain also depend strongly on wavelength, with longer-wavelength light scattering less and therefore penetrating more deeply (Figure 3). We have taken several HKI-272 complementary approaches to measuring and estimating the depth of light propagation under typical experimental conditions, specifically for the illumination of deep brain structures using thin optical fibers. In one approach (Aravanis et al., 2007), an optical fiber emitting a known
light power was lowered into a block of unfixed brain tissue, and light power was measured on the underside of the block, giving a transmission fraction for the tissue sample (nontransmitted light was either absorbed by or reflected out of the sample). This measurement was repeated for a range of tissue thicknesses by stepping the fiber through the block. These data were fit with standard equations for the propagation of light in diffuse scattering media (Kubelka-Munk model; Vo-Dinh, 2003), in order to estimate parameters that could be used to predict depth of transmitted light power in other experimental configurations. To estimate the light power density at a given distance from the fiber tip, the beam was modeled as spreading conically within the tissue, with an angle determined by the optical properties of the fiber.
This model, while involving a number of unrealistic assumptions including that the sample is a homogeneous, ideal diffuser illuminated from one side with diffuse light, and that reflection PAK6 and absorption are constant over the thickness of the sample, nevertheless allowed a good fit to measured data (Figures 3B and 3C; Aravanis et al., 2007) when used to estimate light power density at progressively deeper sites. Next, to directly observe the lateral spatial extent of the illuminated region at various distances from the fiber, we repeated the experiments above with the block of brain tissue placed on a thin diffusing layer revealing the two-dimensional pattern of illumination at the bottom of the block; this screen was imaged from below as the fiber was lowered through either brain tissue, or saline solution, and the resulting images were stacked to create a three-dimensional volume (Figures 3D and 3E).