86 K. Mac Donald and G. Ravichandran Fig. 13.1 Compression loading device mounted to microscope stage with polyacrylamide specimen emission, Molecular Probes™) to produce a DVC speckle pattern. The polyacrylamide is cast into 13 mm diameter molds with a 2 mm height. Initial experiments are carried out on a continuous, homogeneous gel. Specimens with hard poly(methyl methacrylate) (PMMA) inclusions are also studied. A single inclusion is used to validate our methods against an analytical solution [6]. Multiple inclusion interactions are used to further explore the abilities of our methods. The second part of this study uses model granular packings of fluorescent microspheres. Specimens consist of 180– 212 m fluorescent red polyethylene microspheres (300–550 nm excitation, 605 nm peak emission, Cospheric) in a 13 mm inner diameter polylactic acid (PLA) ring bonded to a 25 mm diameter glass coverslip. These grains are highly spherical and on the scale of fine sand. 13.2.2 Experimental Design All experiments were carried out on a Zeiss inverted laser scanning confocal microscope (LSM 800) with Zen Blue System software for image stack capture. Images are captured via photomultiplier tubes (PMTs) and laser excitation is achieved using a 561 nm yellow-green diode laser. In these preliminary studies we used a 10 /0.30 M27 EC Plan-Neofluar objective. A uniaxial compression device mounted to a microscope stage insert, shown in Fig. 13.1, is used to apply load via micrometer to all specimens while imaging. Polyacrylamide specimens are mounted on the compression device using a 25 mm diameter glass coverslip as the base. Unidirectional compression is achieved by applying displacement incrementally with 6–8 min holds for image capture between loading steps. The loading (compression) was achieved in 0.05 mm increments in displacement and the unloading was in 0.01 mm increments. A total of six image stacks were taken for each loading and unloading cycle. The loading cycle stacks each consist of 318 2D images and the unloading cycle stacks consist of 393 2D images taken at 1.0 mdepth increments in both cases. This corresponds to a 317 mand a 392 m depth of view respectively. Granular specimens were mounted in the loading device in a similar manner as the polyacrylamide, but are confined by a PLA ring. To achieve confined compression loading, displacement is applied incrementally at 0.02 mm increments and with 3 min holds for image capture between loading steps. A total of 14 image stacks were taken during loading. Each stack consists of 109 2D images taken at 6.46 m depth increments, corresponding to a 704 m depth of view. 13.3 Preliminary Results Analysis of the images of the polyacrylamide specimens show that the achieved speckle pattern is suitable for DIC and DVC analyses. Improvements could be made by adjusting the gain and laser excitation intensity to achieve a more even intensity spread. During analysis of both polyacrylamide and granular specimens, application of point spread function (PSF) deconvolution to correct for optical refractivity effects will allow us to perform more accurate DVC analyses.
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