Name: measuring spatially- and directionally-varying light scattering from biological material
Uploaded: 2013-05-20T12:00:00
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Description: Watch this Scientific Journal Video about Measuring Spatially- and Directionally-varying Light Scattering from Biological Material at JoVE.com

This research was funded by the National Science Foundation (NSF CAREER award CCF-0347303 and NSF grant CCF-0541105). The authors would like to thank Jaroslav K&#345;iv&aacute;nek, Jon Moon, Edgar Vel&aacute;zquez-Armend&aacute;riz, Wenzel Jakob, James Harvey, Susan Suarez, Ellis Loew, and John Hermanson for their intellectual contributions. The Cornell Spherical Gantry was built from a design due to Duane Fulk, Marc Levoy, and Szymon Rusinkiewicz.

<ol>
	<li>Nicodemus, F., Richmond, J., Hsia, J., Ginsberg, I., Limperis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Geometric considerations and nomenclature for reflectance. , (1977).</li><li>Marschner, S. R., Jensen, H. W., Cammarano, M., Worley, S., Hanrahan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+scattering+from+human+hair+fibers.">Light scattering from human hair fibers.</a> ACM Transactions on Graphics (TOG). 22 (3), 780-791 (2003).</li><li>Marschner, S. R., Westin, S., Arbree, A., Moon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+and+modeling+the+appearance+of+finished+wood.">Measuring and modeling the appearance of finished wood.</a> ACM Transactions on Graphics (TOG). 24 (3), 727-734 (2005).</li><li>Land, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physics+and+biology+of+animal+reflectors.">The physics and biology of animal reflectors.</a> Progress in Biophysics and Molecular Biology. 24, 75-106 (1972).</li><li>Durrer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colouration.">Colouration.</a> Biology of the Integument: Vertebrates. 2 (12), 239-247 (1986).</li><li>Brink, D., van der Berg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+colours+from+the+feathers+of+the+bird+Bostrychia+hagedash.">Structural colours from the feathers of the bird Bostrychia hagedash.</a> Journal of Physics D-Applied Physics. 37 (5), 813-818 (2004).</li><li>Kinoshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Structural colors in the realm of nature. , (2008).</li><li>Nakamura, E., Yoshioka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Color+of+Rock+Dove's+Neck+Feather.">Structural Color of Rock Dove's Neck Feather.</a> Journal of the Physical Society of Japan. 77 (12), 124801 (2008).</li><li>Westin, S., Arvo, J., Torrance, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+reflectance+functions+from+complex+surfaces.">Predicting reflectance functions from complex surfaces.</a> ACM SIGGRAPH Computer Graphics. 26 (2), 255-264 (1992).</li><li>Shawkey, M. D., Maia, R., D'Alba, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximate+bases+of+silver+color+in+anhinga+(Anhinga+anhinga)+feathers.">Proximate bases of silver color in anhinga (Anhinga anhinga) feathers.</a> Journal of Morphology. 272 (11), 1399-1407 (2011).</li><li>Maia, R., D'Alba, L., Shawkey, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+makes+a+feather+shine?+A+nanostructural+basis+for+glossy+black+colours+in+feathers.">What makes a feather shine? A nanostructural basis for glossy black colours in feathers.</a> Proceedings of the Royal Society B: Biological Sciences. 278 (1714), 1973-1980 (2011).</li><li>Dyck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+light+reflection+of+green+feathers+of+fruit+doves+(Ptilinopus+spp.)+and+an+Imperial+Pigeon+(Ducula+concinna).">Structure and light reflection of green feathers of fruit doves (Ptilinopus spp.) and an Imperial Pigeon (Ducula concinna).</a> Biologiske Skrifter (Denmark). 30, 2-43 (1987).</li><li>Yoshioka, S., Kinoshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+macroscopic+structure+in+iridescent+color+of+the+peacock+feathers.">Effect of macroscopic structure in iridescent color of the peacock feathers.</a> Forma. 17 (2), 169-181 (2002).</li><li>Osorio, D., Ham, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+reflectance+and+directional+properties+of+structural+coloration+in+bird+plumage.">Spectral reflectance and directional properties of structural coloration in bird plumage.</a> Journal of Experimental Biology. 205 (14), 2017-2027 (2002).</li><li>Stavenga, D. G., Leertouwer, H. L., Pirih, P., Wehling, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+scatterometry+of+butterfly+wing+scales.">Imaging scatterometry of butterfly wing scales.</a> Optics Express. 1 (1), 193-202 (2009).</li><li>Vukusic, P., Stavenga, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+methods+for+investigating+structural+colours+in+biological+systems.">Physical methods for investigating structural colours in biological systems.</a> Journal of Royal Society Interface. 6, S133-S148 (2009).</li><li>Stavenga, D. G., Leertouwer, H., Marshall, N. J., Osorio, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+colour+changes+in+a+bird+of+paradise+caused+by+uniquely+structured+breast+feather+barbules.">Dramatic colour changes in a bird of paradise caused by uniquely structured breast feather barbules.</a> Proceedings of the Royal Society B: Biological Sciences. 278 (1715), 2098-2104 (2010).</li><li>Irawan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Appearance of woven cloth [dissertation]. , (2008).</li><li>Irawan, P., Marschner, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specular+reflection+from+woven+cloth.">Specular reflection from woven cloth.</a> ACM Transactions on Graphics (TOG. 31 (1), 11:1-11:20 (2012).</li><li>Vukusic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+colour:+elusive+iridescence+strategies+brought+to+light.">Structural colour: elusive iridescence strategies brought to light.</a> Current Biology: CB. 21 (5), R187-R189 (2011).</li><li>Dana, K., Ginneken, B., Nayar, S., Koenderink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflectance+and+texture+of+real-world+surfaces.">Reflectance and texture of real-world surfaces.</a> ACM Transactions on Graphics (TOG). 18 (1), 1-34 (1999).</li><li>Chen, Y., Xu, Y., Guo, B., Shum, H. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+and+rendering+of+realistic+feathers.">Modeling and rendering of realistic feathers.</a> ACM Transactions on Graphics (TOG). 21 (3), 630-636 (2002).</li><li>Levoy, M., Zhang, Z., McDowall, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+and+controlling+the+4D+light+field+in+a+microscope+using+microlens+arrays.">Recording and controlling the 4D light field in a microscope using microlens arrays.</a> Journal of microscopy. 235 (2), 144-162 (2009).</li><li>Stevens, M., P&#225;rraga, C. A., Cuthill, I. C., Partridge, J. C., Troscianko, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+digital+photography+to+study+animal+coloration.">Using digital photography to study animal coloration.</a> Biological Journal of the Linnean Society. 90 (2), 211-237 (2007).</li><li>Kim, M. H., Harvey, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+imaging+spectroscopy+for+measuring+hyperspectral+patterns+on+solid+objects.">3D imaging spectroscopy for measuring hyperspectral patterns on solid objects.</a> ACM Transactions on Graphics (TOG). 31 (4), (2012).</li></ol>

No conflicts of interest declared.

Though the performance and function of many pigmentary and structural colorations are well recognized, the morphology of many integuments is so complex that their structural detail and function are poorly understood20. Integuments have developed specializations that vary spatially over the surface of the organism to differentially reflect light directionally toward the viewer. Directionality has received attention primarily in the study of iridescence due to its color shift with change of incident and viewing angle, and research into iridescence of biological integument has garnered primarily 1D and some 2D measurements8,12,17. But generalized 6D measurements have not been routine in the study of integuments21-23, iridescent or otherwise, and the literature on organismal color phenotypes is constrained by the lack of directional color data of the type our method provides.
The feather is an especially rich integumentary material comprising arrangements of milli-scale structure of the barb: rami, distal barbules, and proximal barbules. The small scale of the elements and their complex arrangements make it difficult to discern the light scattering performance of the individual elements. Our protocol successfully isolated milli-scale structure from the influence of macro-scale geometry. By characterizing the functional consequences of the directional expression of milli-scale structures to the far-field signature of the feather, we enabled inquiry into their adaptive consequences.
We faced practical tradeoffs between spectral, spatial and angular resolution. We chose high spatial, medium angular and low spectral for our studies. Other combinations could be used, but some (e.g. all high) lead to unworkably long measurement times. Attention must be focused where it is important for the particular phenomena being studied. In choosing to employ an RGB camera with a Bayer filter mosaic, we designed our protocol to match the human visual system. The RGB camera could be replaced and our protocol adapted to measure the relative color stimulus of any organism, e.g. sensitivity in the UV spectrum is needed to measure avian tetra-chromatic color24,25. A spectral imaging camera would provide the most general solution25.
We demonstrated our protocol with tertial wing feathers since they are colorful and easily flattened against a reference plate. Unfortunately, the aperture of the metal plate revealed only a fraction of the feather surface. If we could simultaneously measure the 3D shape of the feather surface while measuring its reflectance25, we could avoid mechanically flattening the feather and instead measure the entire feather in its natural, unflattened state.
Interactive, specialized, integrated tools for visualizing data provide substantial benefit to scientists exploring and interpreting large data volumes. The greater the integration and interactivity, the easier connections in the data are observed. In our software, a user can interactively plot average directional scattering as a function of surface position (Figure 4). Further development of our software could integrate other plotting functions (Figures 6, 7) to extend the interactive experience.

The color and pattern of an organism's integument play ecologically and socially critical functions in most animal taxa. These phenotypic properties are determined by the interaction of light with the structure of the integument, which can exhibit optical scattering that varies both spatially (across the surface of the integument) and directionally (with change in lighting and viewing direction). In complex biological materials, such as feathers, the direction of light scattering is influenced by the orientation of repeating milli-scale geometry. These milli-scale structures themselves may be embedded with nano-scale structures, such as melanin arrays, which often inherit the milli-scale orientation. From nano- to macro-scales, the structure of the integument has evolved functionally to increase the signaling capability of the organism. In order to assess the influence of the morphology of different scales upon the overall appearance, tools to measure and analyze the color of biological structures need flexibility to isolate directional light scattering at various scales of magnification.
We developed image-based measurement tools to study how the performance of a feather's complex and varied milli-scale morphology (barb rami, distal barbules, and proximal barbules) expands the range of expression possible from nano-scale structures alone. In a single image recorded by the camera, we observed that light reflected differently at different locations on the surface of the feather, that is, light reflectance was spatially-varying. When we moved the light and camera direction with respect to the feather, we observed the reflectance changed, that is, light reflectance was directionally-varying1. Following these observations, we designed a protocol to methodically move the light and camera around the subject using a spherical gantry2,3, with which we captured 2 dimensions of surface position (X and Y), 2 dimensions of light direction (latitude and longitude), and 2 dimensions of camera direction (latitude and longitude) (Figure 2). In software we visually explored the 6 dimensions of the scattered light as a function of position, illumination direction and view direction.
Previous research into the reflectance from integuments has too frequently discounted the contribution of directionality -- e.g. diffuse vs. specular or isotropic vs. anisotropic reflection -- to color expression. Most color measurements have fixed the incident light, object, and viewing geometry to carefully avoid directional effects. For instance, to eliminate specular reflection from color measurements, it is common to place the light normal to the surface and record the reflectance at 45&deg; from the normal. Studies that do link morphology to directionally-varying reflectance typically focus on the nano-scale and its iridescent consequences4-8. Few consider the contribution of micro-, milli-, and macro-scale geometries to the far-field optical signature8-11. It is therefore common to employ a light detector to aggregate reflectance across a single area of interest that may include multiple milli- and/or macro-scale components, such as barb rami, barbules, and even entire feathers6,8,11-17. When the region of interest is either smaller than the resolution limit of the detector or does not conform to the shape of the detector's field of view, the common protocol specifies specimen dissection to isolate the light scattering from the specific milli-scale element8,10,13,15.
We have developed a more encompassing protocol for measurement acquisition and visualization that encourages exploration of the many variables often ignored in other more focused studies. We measure light scattering over a sphere of directions and across a region of space using a massive set of high-dynamic range, high-resolution photographs taken from a systematic set of light and viewing directions. We employ a high-resolution imaging sensor with its 2D array of fine-scale pixel detectors. Aggregation in hardware occurs at the pixel-level, at a scale smaller than the milli-scale elements we are measuring. A second stage aggregates individual pixels in software as the user selects the shape and size of the region of interest. Accordingly, a single measurement set can be repeatedly analyzed in software to explore different aspects of light interaction with material at multiple biologically-relevant positions and scales. By eliminating dissection and measuring the entire feather, our protocol has the advantage of leaving the morphology of the feather vane intact, retaining natural context and function that is, light's interactions between constituent milli-scale elements.
Light scattering from organismal structure is multidimensional and difficult to quantify. Measured 6D light scattering cannot as yet be attributed to specific morphology within a hierarchy of scale with any singular instrument. But we have made an important step in this pursuit. We have developed a tool encompassing three complementary methods -- sampling reflectance using the gantry, exploring large data volumes in software, and visualizing data subsets graphically -- to extend our ability to measure 6D light scattering at any point on a material, down to the milli-scale. As protocols such as ours are employed, we predict biologists will identify a myriad of directionally- and spatially-varying traits and corresponding structural adaptations at multiple scales of development. Using our tools we are engaged in characterizing the signaling potential of the directional and spatial expression of milli-scale structures, and hope to shed light on their adaptive consequences. We address a range of questions, such as: from any given observation point, which fine-scale elements or gross-scale regions of the feather reflect strongly? How does the orientation of the fine-scale elements influence the direction of scattered light? What morphological conditions produce a satiny gloss vs. a sequined sparkle of the iridescent ornament? Do some species produce broader "windows" for observation of iridescence than others? These questions may be asked about birds and their feathers but also about any other organisms that have evolved a particular surface appearance for signaling, camouflage, or other reasons.

measuring spatially- and directionally-varying light scattering from biological material

Light interacts with an organism's integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the milli-scale structure of barbs and barbules largely determines the directional pattern of reflected light; and through the macro-scale spatial structure of overlapping, curved feathers, these directional effects create the visual texture. Milli-scale and macro-scale effects determine where on the organism's body, and from what viewpoints and under what illumination, the iridescent colors are seen. Thus, the highly directional flash of brilliant color from the iridescent throat of a hummingbird is inadequately explained by its nano-scale structure alone and questions remain. From a given observation point, which milli-scale elements of the feather are oriented to reflect strongly? Do some species produce broader "windows" for observation of iridescence than others? These and similar questions may be asked about any organisms that have evolved a particular surface appearance for signaling, camouflage, or other reasons.
In order to study the directional patterns of light scattering from feathers, and their relationship to the bird's milli-scale morphology, we developed a protocol for measuring light scattered from biological materials using many high-resolution photographs taken with varying illumination and viewing directions. Since we measure scattered light as a function of direction, we can observe the characteristic features in the directional distribution of light scattered from that particular feather, and because barbs and barbules are resolved in our images, we can clearly attribute the directional features to these different milli-scale structures. Keeping the specimen intact preserves the gross-scale scattering behavior seen in nature. The method described here presents a generalized protocol for analyzing spatially- and directionally-varying light scattering from complex biological materials at multiple structural scales.

The primary measurement of our protocol (Routine I in Figure 1) fixed the camera direction normal to the surface and only moved the light. Since light scattering adheres to the principle of reciprocity, the result is the same whether we hold the camera constant while moving the light over the hemisphere or vice versa. When we fix either the camera or the light, the complete 4-dimensional direction set is undersampled. A fuller picture of the scattering behavior is observed when, unlike the primary measurement, both light and camera are moved away from the surface normal and in a multiplicity of directions. Ideally, we could measure light scattering from many camera directions, even as many as the number of incident light directions, to yield a symmetrical data set. In practice, this would require far too many exposures. In our experience, we can obtain sufficient information about different viewing positions by moving the camera a few times assuming 180&deg; rotational symmetry about the surface normal. During the secondary measurement phase, we acquired measurements from 7 viewing directions distributed over the hemisphere and within 60&deg; of the zenith18,19 (Routine II.A in Figure 1).
In the figures of this paper, we show representative data measured from a feather of Lamprotornis purpureus (Purple Glossy Starling), the reflectance of which is iridescent, glossy, and anisotropic (Figure 5). In each of the 7 viewing directions, reflected light is gathered from hundreds of incident lighting directions on the hemisphere. The directions form a narrow band orthogonally oriented to the central axis of the feather (see feather image in Figure 4). The iridescence color shift is subtle (bluish-green at normal incidence and greenish-blue at grazing incidence) when the feather is viewed normal to its surface as seen in the {0&deg;,0&deg;} RGB plot of Figure 5. As the viewing angle approaches grazing, the angles between the viewing direction and the grazing incident directions are maximized, leading to a more striking color shift (bluish-green at 0&deg; and magenta at 240&deg; between incident and viewing directions) as seen in the {60&deg;,0&deg;} RGB plot in Figure 5.
We can afford to step the light and camera at much finer angular resolution when we restrict the movements to 1 dimension. Figure 6 shows the chromaticity of the reflectance of L. purpureus plumage as a function of the angle between the incident and viewing directions, where the incident and viewing directions are in the plane containing the specular band, which is perpendicular to the longitudinal axis of the distal barbule. As the iridescent color arcs through chromaticity space, the hue shifts from bluish-green to purple.
Spatial variation in the directional reflectance is visible where different (X,Y) coordinates of the integument correspond to different milli-scale structures. In the case of L. purpureus only one structure -- the distal barbule -- is visible over most of the area. By contrast, in C. cupreus, three milli-scale structures -- the rami, distal barbules, and proximal barbules -- are clearly distinguished in the data; we can observe that reflectance from the feather is oriented with respect to the longitudinal axis of each structure (Figure 8).
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50254/50254fig1highres.jpg" src="/files/ftp_upload/50254/50254fig1.jpg" /> 
Figure 1. This schematic overview depicts two mounting methods, the spherical gantry coordinate system, types of acquisition sampling and their respective results. <a href="https://www.jove.com/files/ftp_upload/50254/50254fig1large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/50254/50254fig2.jpg" /> 
Figure 2. The flattened feather is visible through an aperture in a metal plate surrounded by a ring of targets. A spherical gantry can be posed to measure light scattering from a feather at multiple incident lighting and viewing directions. L=Light arm (latitude). C= Camera arm (latitude). B=Camera Base (longitude). T=Turntable (longitude). F=Feather.
<img alt="Figure 3" src="/files/ftp_upload/50254/50254fig3.jpg" /> 
Figure 3. Average directional scattering may be computed from a point, line or rectangular region of feather vane.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50254/50254fig4highres.jpg" src="/files/ftp_upload/50254/50254fig4.jpg" /> 
Figure 4. Example of directional scattering plotting functions (R*=Reflectance, T*=Transmittance, P*=Top, F*=Front, S*=Side, A*=Arbitrary) and color schemes (*1=Luminance, *2=RGB, *3=Chromaticity). <a href="https://www.jove.com/files/ftp_upload/50254/50254fig4large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 5" fo:content-width="4.5in" fo:src="/files/ftp_upload/50254/50254fig5highres.jpg" src="/files/ftp_upload/50254/50254fig5.jpg" /> 
Figure 5. The luminance (top) and RGB color (bottom) of the hemispherical reflectance in direction cosine space as viewed from the (elevation angle, azimuth angle) coordinate pairs: {0&deg;,0&deg;}, {30&deg;,0&deg;}, {30&deg;,90&deg;}, {60&deg;,0&deg;}, {60&deg;,45&deg;}, {60&deg;,90&deg;}, and {60&deg;,135&deg;}. The reflectance is averaged from a 25&times;25 pixel rectangular region of the lateral vane of a tertial L. purpureus (Purple Glossy Starling) feather. The red arrows represent camera directions. <a href="https://www.jove.com/files/ftp_upload/50254/50254fig5large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 6" fo:content-width="6in" fo:src="/files/ftp_upload/50254/50254fig6highres.jpg" src="/files/ftp_upload/50254/50254fig6.jpg" /> 
Figure 6. Chromaticity of the reflectance as a function of the half-angle between the incident lighting and viewing directions: CIE 1976 Uniform Chromaticity Scales (USC) with magnified region. <a href="https://www.jove.com/files/ftp_upload/50254/50254fig6large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 7" fo:content-width="6in" fo:src="/files/ftp_upload/50254/50254fig7highres.jpg" src="/files/ftp_upload/50254/50254fig7.jpg" /> 
Figure 7. Reflectance as a function of the angle between the incident lighting and viewing directions, in-plane with (red) and perpendicular to (shaded) the longitudinal axis of the distal barbule: (A) Dominant wavelength, (B) Percent chroma, (C) Percent luminance. The color shading in plot A is the RGB color of the reflectance. Negative wavelength values represent colors in the non-spectral purple triangle. <a href="https://www.jove.com/files/ftp_upload/50254/50254fig7large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 8" fo:content-width="5in" fo:src="/files/ftp_upload/50254/50254fig8highres.jpg" src="/files/ftp_upload/50254/50254fig8.jpg" /> 
Figure 8. Average directional reflectance of distal barbules and proximal barbules between two adjacent rami of the C. cupreus (African Emerald Cuckoo).
<img alt="Figure 9" fo:content-width="6in" fo:src="/files/ftp_upload/50254/50254fig9highres.jpg" src="/files/ftp_upload/50254/50254fig9.jpg" /> 
Figure 9. (A) Non-rectified image illuminated by gantry lamp, (B) Non-rectified image illuminated by flash on camera, (C) Filtered target candidates on affine-transformed, flash-illuminated image, (D) Acceptably sharp targets within depth of field, (E) Rectified lamp-illuminated image, (F) Rotated feather tip up, cropped and masked. <a href="https://www.jove.com/files/ftp_upload/50254/50254fig9large.jpg" target="_blank">Click here to view larger figure</a>.

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

We present a non-destructive method for sampling spatial variation in the direction of light scattered from structurally complex materials. By keeping the material intact, we preserve gross-scale scattering behavior, while concurrently capturing fine-scale directional contributions with high-resolution imaging. Results are visualized in software at biologically-relevant positions and scales.

Light interacts with an organism's integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the ...

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Fabrication of Spatially Confined Complex Oxides

Complex materials such as high Tc superconductors, multiferroics, and colossal magnetoresistors have electronic and magnetic properties that arise from the inherent strong electron correlations that reside within them. These materials can also possess electronic phase separation in which regions of vastly different resistive and magnetic behavior can coexist within a single crystal alloy material. By reducing the scale of these materials to length scales at and below the inherent size of the electronic domains, novel behaviors can be exposed. Because of this and the fact that spin-charge-lattice-orbital order parameters each involve correlation lengths, spatially reducing these materials for transport measurements is a critical step in understanding the fundamental physics that drives complex behaviors. These materials also offer great potential to become the next generation of electronic devices 1-3. Thus, the fabrication of low dimensional nano- or micro-structures is extremely important to achieve new functionality. This involves multiple controllable processes from high quality thin film growth to accurate electronic property characterization. Here, we present fabrication protocols of high quality microstructures for complex oxide manganite devices. Detailed descriptions and required equipment of thin film growth, photo-lithography, and wire-bonding are presented.

We describe the use of pulsed laser deposition (PLD), photolithography and wire-bonding techniques to create micrometer scale complex oxides devices. The PLD is utilized to grow epitaxial thin films. Photolithography and wire-bonding techniques are introduced to create practical devices for measurement purposes.

Complex materials such as high Tc superconductors, multiferroics, and colossal magnetoresistors have electronic and magnetic properties that arise from ...

Measuring Material Microstructure Under Flow Using 1-2 Plane Flow-Small Angle Neutron Scattering

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is presented. The SANS shear cell consists of a concentric cylinder Couette geometry that is sealed and rotating about a horizontal axis so that the vorticity direction of the flow field is aligned with the neutron beam enabling scattering from the 1-2 plane of shear (velocity-velocity gradient, respectively). This approach is an advance over previous shear cell sample environments as there is a strong coupling between the bulk rheology and microstructural features in the 1-2 plane of shear. Flow-instabilities, such as shear banding, can also be studied by spatially resolved measurements. This is accomplished in this sample environment by using a narrow aperture for the neutron beam and scanning along the velocity gradient direction. Time resolved experiments, such as flow start-ups and large amplitude oscillatory shear flow are also possible by synchronization of the shear motion and time-resolved detection of scattered neutrons. Representative results using the methods outlined here demonstrate the useful nature of spatial resolution for measuring the microstructure of a wormlike micelle solution that exhibits shear banding, a phenomenon that can only be investigated by resolving the structure along the velocity gradient direction. Finally, potential improvements to the current design are discussed along with suggestions for supplementary experiments as motivation for future experiments on a broad range of complex fluids in a variety of shear motions.

A shear cell is developed for small-angle neutron scattering measurements in the velocity-velocity gradient plane of shear and is used to characterize complex fluids. Spatially resolved measurements in the velocity gradient direction are possible for studying shear-banding materials. Applications include investigations of colloidal dispersions, polymer solutions, and self-assembled structures.

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is ...

Using Neutron Spin Echo Resolved Grazing Incidence Scattering to Investigate Organic Solar Cell Materials

The spin echo resolved grazing incidence scattering (SERGIS) technique has been used to probe the length-scales associated with irregularly shaped crystallites. Neutrons are passed through two well defined regions of magnetic field; one before and one after the sample. The two magnetic field regions have opposite polarity and are tuned such that neutrons travelling through both regions, without being perturbed, will undergo the same number of precessions in opposing directions. In this case the neutron precession in the second arm is said to &#34;echo&#34; the first, and the original polarization of the beam is preserved. If the neutron interacts with a sample and scatters elastically the path through the second arm is not the same as the first and the original polarization is not recovered. Depolarization of the neutron beam is a highly sensitive probe at very small angles (&#60;50&#160;&#956;rad) but still allows a high intensity, divergent beam to be used. The decrease in polarization of the beam reflected from the sample as compared to that from the reference sample can be directly related to structure within the sample.
In comparison to scattering observed in neutron reflection measurements the SERGIS signals are often weak and are unlikely to be observed if the in-plane structures within the sample under investigation are dilute, disordered, small in size and polydisperse or the neutron scattering contrast is low. Therefore, good results will most likely be obtained using the SERGIS technique if the sample being measured consist of thin films on a flat substrate and contain scattering features that contains a high density of moderately sized features (30 nm to 5 &#181;m) which scatter neutrons strongly or the features are arranged on a lattice. An advantage of the SERGIS technique is that it can probe structures in the plane of the sample.

Progress has been made in utilizing spin echo resolved grazing incidence scattering (SERGIS) as a neutron scattering technique to probe the length-scales in irregular samples. Crystallites of [6,6]-phenyl-C61-butyric acid methyl ester have been probed using the SERGIS technique and the results confirmed by optical and atomic force microscopy.

The spin echo resolved grazing incidence scattering (SERGIS) technique has been used to probe the length-scales associated with irregularly shaped ...

Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus have potential applications in nonlinear optics, which requires extraordinary optical intensity. One of the most studied nonlinearities in plasmonics is nonlinear absorption, including saturation and reverse saturation behaviors. Although scattering and absorption in nanoparticles are closely correlated by the Mie theory, there has been no report of nonlinearities in plasmonic scattering until very recently. 
Last year, not only saturation, but also reverse saturation of scattering in an isolated plasmonic particle was demonstrated for the first time. The results showed that saturable scattering exhibits clear wavelength dependence, which seems to be directly linked to the localized surface plasmon resonance (LSPR). Combined with the intensity-dependent measurements, the results suggest the possibility of a common mechanism underlying the nonlinear behaviors of scattering and absorption. These nonlinearities of scattering from a single gold nanosphere (GNS) are widely applicable, including in super-resolution microscopy and optical switches. 
In this paper, it is described in detail how to measure nonlinearity of scattering in a single GNP and how to employ the super-resolution technique to enhance the optical imaging resolution based on saturable scattering. This discovery features the first super-resolution microscopy based on nonlinear scattering, which is a novel non-bleaching contrast method that can achieve a resolution as low as l/8 and will potentially be useful in biomedicine and material studies.

Saturable and reverse saturable scattering were discovered in isolated plasmonic particles and adopted as a novel non-bleaching contrast method in super-resolution microscopy. Here the experimental procedures of detecting and extracting nonlinear scattering are explained in detail, as well as how to enhance resolution with the aid of saturated excitation microscopy.

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus ...

Experimental Methods for Investigation of Shape Memory Based Elastocaloric Cooling Processes and Model Validation

Shape Memory Alloys (SMA) using elastocaloric cooling processes have the potential to be an environmentally friendly alternative to the conventional vapor compression based cooling process. Nickel-Titanium (Ni-Ti) based alloy systems, especially, show large elastocaloric effects. Furthermore, exhibit large latent heats which is a necessary material property for the development of an efficient solid-state based cooling process. A scientific test rig has been designed to investigate these processes and the elastocaloric effects in SMAs. The realized test rig enables independent control of an SMA&#39;s mechanical loading and unloading cycles, as well as conductive heat transfer between SMA cooling elements and a heat source/sink. The test rig is equipped with a comprehensive monitoring system capable of synchronized measurements of mechanical and thermal parameters. In addition to determining the process-dependent mechanical work, the system also enables measurement of thermal caloric aspects of the elastocaloric cooling effect through use of a high-performance infrared camera. This combination is of particular interest, because it allows illustrations of localization and rate effects &#8212; both important for efficient heat transfer from the medium to be cooled.
The work presented describes an experimental method to identify elastocaloric material properties in different materials and sample geometries. Furthermore, the test rig is used to investigate different cooling process variations. The introduced analysis methods enable a differentiated consideration of material, process and related boundary condition influences on the process efficiency. The comparison of the experimental data with the simulation results (of a thermomechanically coupled finite element model) allows for better understanding of the underlying physics of the elastocaloric effect. In addition, the experimental results, as well as the findings based on the simulation results, are used to improve the material properties.

Experimental methods for investigation of solid state cooling processes and characterization of elastocaloric material properties of Shape Memory Alloys (SMA) are presented. A custom-built test rig has been designed for controlling and comprehensive monitoring of elastocaloric cooling processes. Furthermore, it provides a validation platform for thermomechanically coupled modeling approaches.

Shape Memory Alloys (SMA) using elastocaloric cooling processes have the potential to be an environmentally friendly alternative to the conventional vapor ...

Scanning Light Scattering Profiler (SLPS) Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light scattering from intraocular lenses (IOLs) using goniophotometer principles. This protocol describes the SLSP platform and how it employs a 360&#176; rotational photodetector sensor that is scanned around an IOL sample while recording the intensity and location of scattered light as it passes through the IOL medium. The SLSP platform can be used to predict, non-clinically, the propensity for current and novel IOL designs and materials to induce light scatter. Non-clinical evaluation of light scattering properties of IOLs can significantly reduce the number of patient complaints related to unwanted glare, glistening, optical defects, poor image quality, and other phenomena associated with the unintended light scattering. Future studies should be conducted to correlate SLSP data with clinical results to help identify which measured light scatter is most problematic for patients that have undergone cataract surgery subsequent to IOL implantation.

Scanning Light Scattering Profiler SLPS Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

This protocol describes the scanning light scattering profiler (SLSP) that enables the full-angle quantitative evaluation of forward and backward scattering of light from intraocular lenses (IOLs) using goniophotometer principles.

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light ...

High-speed Continuous-wave Stimulated Brillouin Scattering Spectrometer for Material Analysis

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as aqueous solutions and biomaterials, with fast acquisition times. Here, we discuss the assembly and operation of a Brillouin spectrometer that uses stimulated Brillouin scattering (SBS) to measure stimulated Brillouin gain (SBG) spectra of water and lipid emulsion-based tissue-like samples in transmission mode with &lt;10 MHz spectral-resolution and &lt;35 MHz Brillouin-shift measurement precision at &lt;100 ms. The spectrometer consists of two nearly counter-propagating continuous-wave (CW) narrow-linewidth lasers at 780 nm whose frequency detuning is scanned through the material Brillouin shift. By using an ultra-narrowband hot rubidium-85 vapor notch filter and a phase-sensitive detector, the signal-to-noise-ratio of the SBG signal is significantly enhanced compared to that obtained with existing CW-SBS spectrometers. This improvement enables measurement of SBG spectra with up to 100-fold faster acquisition times, thereby facilitating high spectral-resolution and high-precision Brillouin analysis of soft materials at high speed.

We describe the construction of a rapid continuous-wave-stimulated-Brillouin-scattering (CW-SBS) spectrometer. The spectrometer employs single-frequency diode-lasers and an atomic vapor notch-filter to acquire transmission spectra of turbid/non-turbid samples with high spectral-resolution at speeds up to 100-fold faster than those of existing CW-SBS spectrometers. This improvement enables high-speed Brillouin material analysis.

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as ...

Determination of the Excitation and Coupling Rates Between Light Emitters and Surface Plasmon Polaritons

We have developed a unique method to measure the excitation and coupling rates between the light emitters and surface plasmon polaritons (SPPs) arising from metallic periodic arrays without involving time-resolved techniques. We have formulated the rates by quantities that can be measured by simple optical measurements. The instrumentation based on angle- and polarization-resolved reflectivity and photoluminescence spectroscopy will be described in detail here. Our approach is intriguing due to its simplicity, which requires routine optics and several mechanical stages, and thus is highly affordable to most of the research laboratories.

This protocol describes the instrumentation for determining the excitation and coupling rates between light emitters and Bloch-like surface plasmon polaritons arising from periodic arrays.

We have developed a unique method to measure the excitation and coupling rates between the light emitters and surface plasmon polaritons (SPPs) arising ...

Synchrotron X-ray Microdiffraction and Fluorescence Imaging of Mineral and Rock Samples

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder microdiffraction two-dimensional (2D) maps at beamline 12.3.2 of the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. Measurements can be performed on any sample that is less than 10 cm x 10 cm x 5 cm, with a flat exposed surface. The experimental geometry is calibrated using standard materials (elemental standards for XRF, and crystalline samples such as Si, quartz, or Al2O3 for diffraction). Samples are aligned to the focal point of the x-ray microbeam, and raster scans are performed, where each pixel of a map corresponds to one measurement, e.g., one XRF spectrum or one diffraction pattern. The data are then processed using the in-house developed software XMAS, which outputs text files, where each row corresponds to a pixel position. Representative data from moissanite and an olive snail shell are presented to demonstrate data quality, collection, and analysis strategies.

We describe a beamline setup meant to carry out rapid two-dimensional x-ray fluorescence and x-ray microdiffraction mapping of single crystal or powder samples using either Laue (polychromatic radiation) or powder (monochromatic radiation) diffraction. The resulting maps give information about strain, orientation, phase distribution, and plastic deformation.

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder ...

Measurement of Dynamic Force Acted on Water Strider Leg Jumping Upward by the PVDF Film Sensor

This study aimed to make an explanation for the phenomenon in nature that water strider usually jumps or glides on the water surface easily but quickly, with its peak locomotion speed reaching 150 cm/s. First of all, we observed the microstructure and hierarchy of water strider legs using the scanning electron microscope. On the basis of the observed morphology of the legs, a theoretical model of the detachment from water surface was established, which explained water striders' capability to slide on water surface effortlessly in terms of energy reduction. Secondly, a dynamic force measurement system was devised using the PVDF film sensor with excellent sensitivity, which could detect the whole interaction process. Subsequently, a single leg in contact with water was pulled upward at different speeds, and the adhesion force was measured at the same time. The results of the departing experiment suggested a deep understanding of the fast jumping of water striders.

The protocol here is dedicated to investigating the free and quick maneuvering of water strider on water surface. The protocol includes observing the microstructure of legs and measuring the adhesion force when departing from water surface at different speeds.

This study aimed to make an explanation for the phenomenon in nature that water strider usually jumps or glides on the water surface easily but quickly, ...