Light scattering properties vary across different regions of the adult mouse brain

Abstract

Recently developed optogenetic tools provide powerful approaches to optically excite or inhibit neural activity. In a typical in-vivo experiment, light is delivered to deep nuclei via an implanted optical fiber. Light intensity attenuates with increasing distance from the fiber tip, determining the volume of tissue in which optogenetic proteins can successfully be activated. However, whether and how this volume of effective light intensity varies as a function of brain region or wavelength has not been systematically studied. The goal of this study was to measure and compare how light scatters in different areas of the mouse brain. We delivered different wavelengths of light via optical fibers to acute slices of mouse brainstem, midbrain and forebrain tissue. We measured light intensity as a function of distance from the fiber tip, and used the data to model the spread of light in specific regions of the mouse brain. We found substantial differences in effective attenuation coefficients among different brain areas, which lead to substantial differences in light intensity demands for optogenetic experiments. The use of light of different wavelengths additionally changes how light illuminates a given brain area. We created a brain atlas of effective attenuation coefficients of the adult mouse brain, and integrated our data into an application that can be used to estimate light scattering as well as required light intensity for optogenetic manipulation within a given volume of tissue.

Figure 1. The experimental approach and sample data.

A: Basic experimental setup with the punch-through method. On an inverted microscope, an optical fiber was placed on a section of brain tissue such that light from the fiber would pass through the tissue and subsequently be imaged by an objective attached to a CCD camera. B: Optical transmittance as a function of tissue thickness. As the optical fiber was advanced through the section of brain tissue and repeated images such as the one in 1C were taken, the decrease in optical transmittance as a function of tissue thickness could be evaluated. The single measurements (“+” symbols) represent transmittance of blue light (453 nm) through a section of PPT at various thicknesses, while the solid line represents an exponential fit. C: An example of an original image captured by the CCD camera, showing light emitted from an optical fiber after it passed though a section of brain tissue.

Figure 2. Optical transmittance through different types of brain tissue.

2A: Measurements using the fiber punch-through technique were taken in seven different brain areas with blue (453 nm) light. In each case, optical transmittance decreased exponentially with tissue thickness; however, the exponential decreases observed varied greatly with the type of tissue. Single measurements are represented by the respective symbols while the solid lines represent exponential fits of the data. 2B: Effective attenuation coefficients with SEMs for the seven brain areas: VNTB 19.96+/−0.26; MNTB 18.16+/−0.69; LSO 17.92+/−0.80; PPT 15.26+/−0.78; OB 14.88+/−0.74; SC 13.91+/−0.83; Cerebellum 9.76+/−0.78; all units are 1/mm. 2C: Optical power values that would need to be fed into a 100 µm diameter optical fiber when 300 µm of tissue needs to be illuminated at intensities typically used for Channelrhodopsin activation. 2D: Same as figure C except that in this example the illumination was calculated to hypothetically activate Channelrhodopsin over a distance of 600 µm from the fiber tip.

Figure 3. Effects of wavelength on optical transmittance.

3A: Optical transmittance in the MNTB as a function of tissue thickness and optical wavelength. The three color-coded data sets represent corresponding measurements with light of three different optical wavelengths (blue (453 nm), green (528 nm), and red (940 nm)). Longer-wavelength light penetrates tissue deeper, resulting in a higher transmittance at any given tissue thickness. 3B: Effects of light wavelength on transmittance in two brain areas (MNTB and VNTB). The effective attenuation coefficient decreases with wavelength for the three wavelengths tested. MNTB measurements are represented by round symbols while VNTB measurements are represented by square symbols. Measurements in the three different colors are indicated by the color-code of the symbols.

Figure 4. Relating fiber punch-though measurements to brain atlas measurements.

4A: Image of a 300 µm coronal section of mouse brain stem, taken on a calibrated virtual microscopy system with monochromatic light. Areas with higher optical transmittance appear brighter on the image, while areas with lower transmittance appear darker. MNTB, VNTB, and LSO are outlined in red, orange, and yellow, respectively. 4B: Correlation in digital irradiance for brain areas tested with both the fiber punch-through and the virtual microscopy method. Digital irradiance was measured in six brain areas (MNTB (red), VNTB (orange), LSO (yellow), PPT (green), SC (light blue), and cerebellum (dark blue) with both the fiber punch through and the virtual microscopy technique. Results were normalized and plotted against each other. Each colored symbols represents the measurements from one brain area with two methods, the solid line indicates complete overlap between the measurements. The bars attached to each data point represent the standard error.