Measuring Spatially- and Directionally-varying Light Scattering from Biological Material

The overall goal of this procedure is to measure and visualize the change in the direction of light scattered from a structurally complex material at multiple structural scales to control incident light and view directions. Place a material at the center of the spherical gantry with its lamp mounted on one arm and its camera mounted on another arm sequentially. Photograph the material while systematically moving the lamp through a series of discrete positions on a sphere, centered on the material in the software.

Select a region of interest on the material and extract the pixels comprising the region in each photograph. Then map the pixel values to their respective positions on the sphere to visualize the direction of the lights scattered from the selected region. Finally, use the data for planning additional measurements from multiple camera directions and with increased angular resolution.

Ultimately, this method helps researchers identify the relationship between organismal structure and directional visual signaling. The main advantage of this technique over existing imaging scatter geometry is our ability in software to visualize spatial variation in directional reflectance from complex materials over multiple biologically relevant scales. Although we're focused on avian directional signaling and plumage appearance, this method is valid for other optical systems exhibiting structural hierarchies of scale.

As a first step in the experiment, obtain a thin Ferris metal mounting plate with an aperture of about half an inch, surrounded by a ring of targets. Lay the feather to be studied against the back of the plate. Center the region of interest over the aperture.

Next, lay a sheet of magnetic film with a similarly sized aperture against the backside of the feather to press the feather against the plate. Align the film and plate apertures taking care not to shear the feather. This should result in the feather presenting a planar macro surface approximately coincident with the plate surface.

To configure the gantry. Begin by locating the center of the circular aperture at the origin of the gantry coordinate system. Place a light source on the gantry outer arm.

Aim and narrowly focus the light on the feather. Next, place a camera on the gantry inner arm. Adjust the camera distance and the focus of the macro lens until the ring of targets fills the width of the sensor.

At this point, the gantry arms should be calibrated and the camera focus and exposure should be configured. Start measurements by positioning the camera's optical axis normal to the surface plane. Place the light in the first of a series of positions that define the incident light directions.

The positions should be uniformly distributed on the sphere, centered on the feather in a dark room. For each light position, capture a raw image for each exposure time in the previously determined exposure bracket. Then move the light to its next position and repeat.

Once the data is collected, begin processing the images for each incident. Light direction. Integrate all low dynamic range exposures to a single high dynamic range color image.

These high dynamic range color images are used to create the data for visualization. To browse the process data, the custom simple browser application is used. It opens to a window containing the image of the feather illuminated by the first incident lighting direction.

Now select a region of the feather vein for analysis. Here, a rectangular region is chosen from the options available. Plot the average directional light scattering from the selected region, A plot window showing reflectance as a function of direction.

Co-sign opens adjacent to the image window, adjust the exposure of the color map. Using the software, it is possible to cycle the reflectance color map on the unit sphere between luminance RGB and chroma. Our GB is used in what follows.

To rotate the sphere, click on it. To enable the track ball interface, drag the interface to cause rotation. To view the reflectance hemisphere, return the sphere to its default position.

Rotate the sphere 180 degrees from the default position. To view the transmittance hemisphere for another view of the data, select the polar plot mode to see the radii of each direction on the unit sphere scaled by their respective luminance values. Change the color map of the luminance scaled sphere from RGB to chroma.

The direction of illumination of the displayed image is circled in red in the directional scattering plot. Click any other incident lighting direction to show the feather illuminated from that direction. Decrease the exposure of the image to correct for over exposure.

To investigate reflectance across a hierarchy of scales, return the plot mode to the unit sphere and use the RGB color map. Change the selection type from a rectangular region to a linear one. This will allow study of reflectance from individual fine scale structures in the rectangular region.

Plot the reflectance of the linear average in a new window while maintaining the rectangular average for reference. Here, the distal bar mules of the feather spanned by the linear region are seen to reflect light along the horizontal. Select one of the illumination directions in the linear plot to display the highly reflective distal bar mules in the image on the left to investigate the adjacent dark stripe.

Move the linear selection until it enters that region in the feather structure. It is here that the proximal ES branch from the rami in the linear average plot, the proximal ES are seen to reflect light vertically. Select one of the directions to display the highly reflective proximal pares in the image on the left.

Observe that the fine scale structures which reflect light horizontally and vertically in the linear plot combine to produce the far field signal seen in the rectangular plot. Having reviewed the basic steps for measuring and visualizing directional light scattering over a hierarchy of scale, the following Describe advanced camera calibration techniques in preparation to conduct experiments from multiple camera directions with a feather mounted clip, a checker patterned calibration target flat against the mounting plate. Place the camera with its access perpendicular to the plate.

Use any lighting sufficient for proper exposure to capture one image. Capture images of the plate for use in the boogey camera calibration toolbox within matlab. Nine images from camera positions within a 120 degree cone centered on the plates perpendicular axis have proven sufficient.

Once this is done, calibrate the camera position, including its distance Z to the calibration target. Next, remove the calibration target revealing the ring of targets surrounding the aperture. Use the camera mounted flash to capture two images of the targets, one from a direction perpendicular to the plate and the second from a grazing angle.

The two images will be used to calibrate the camera's distance to the target ring and the feather by solving for the translational offsets T one and T two. Now that we have calibrated the camera, we can measure light scattering from multiple camera directions using alternate directional sampling patterns. To begin, use simple browser to open a dataset containing a sparsely sampled sphere of incident light directions and a perpendicular camera direction.

View the directional distribution of the light reflected from the feather. Based on this review, refine the set of incident light directions to improve directional sampling. These positions should sample specular directions densely and non-secular directions.Sparsely.

Choose six additional camera directions distributed uniformly over half a hemisphere. For each direction, sample the reflectance hemisphere densely for specular directions and sparsely for nons specular directions For each incident light direction in each hemisphere. First photograph the target ring surrounding the feather with the camera mounted flash.

Second, photograph the feather at each exposure in the exposure bracket. Then integrate the exposures into a high dynamic range color image. Roughly rectify the flash illuminated photograph using gantry coordinates.

Then find and use its target centers to precisely project the HDR image of the feather as if it had been photographed from a perpendicular direction. After processing, use simple browser to visually browse the directional reflectance from the same region of the feather. In each of the seven non-uniform sampled hemispheres, arrange the directional reflectance plots for each of the camera directions On a polar coordinate system, shown in this polar plot are multiple camera position measurements made on the feather of a purple glossy starling.

The red arrows represent camera directions. The positions of the camera on the sphere are shown in the inset. In each of the viewing directions reflected light is gathered from hundreds of incident lighting directions.

The RGB color data revealed that the feather is iridescent changing from blue green at normal incidents to magenta at grazing incidents. The technique can be used for finer angular resolution studies when incident lighting and camera viewing directions are restricted to one dimension. Plotted in the inset is the chroma of the reflectance as a function of the half angle between the incident and viewing directions.

When these directions are in the plane perpendicular to the longitudinal axis of the distal bar, as the iridescent color arcs through chromaticity space, the hue shifts from blue-green to purple. The dominant wavelength of the reflectance as a function of the angle between the incident and viewing directions is depicted here. The red line corresponds to when the two directions are in plain with the longitudinal axis of the distal bar.

The shaded region is for when the directions are perpendicular to that axis. The color of the shading is the RGB color of the reflectance negative wave. The length values represent colors in a non spectral region.

In addition to the dominant wavelength, there is data on the percent chroma and percent luminance of the reflectance as a function of the angle between incident and viewing angle. Again, the red line corresponds to when the incident light direction and the viewing direction are in plain with the longitudinal axis of the distal bar. The shaded region is for when the directions are perpendicular to that axis.

Once mastered a typical measurement, using this technique can be acquired and processed in under 14 hours. This technique paved the way for researchers in the fields of ornithology and computer graphics to explore the relationship between complex plumage morphology and the directional effects of avian visual signaling.