Seminars Archive

Contrast-enhanced microCT imaging of biological samples: methods and applications

Abstract
X-ray microtomography is finding broader application in various areas of life sciences research, including microscopic analyses of unmineralized tissues at histological resolutions. The results obtainable with current lab-based microtomography systems can rival those commonly achieved by synchrotron facility users for whole organisms and other intact biological samples. Commercially available microCT systems can offer spatial resolutions down to less than 1µm in "micro" systems, which use projection-based imaging and microfocus x-ray sources, and to around 50nm in "nano" (or "ultra") systems, which employ x-ray focusing optics. For animal embryos and other whole tissue samples, contrast-enhanced microCT imaging is a powerful complement to other imaging methods, including LM, TEM, and SEM. With simple contrast staining methods, high-resolution volume images of unsectioned specimens can be produced routinely for morphological, embryological, and even molecular studies. Because animal and plant tissues generally have very low opacities to x-rays above about 1keV, contrast enhancement is a useful adjunct to microCT technology. Stains based on elemental iodine are especially versatile, and phosphotungstic acid imparts strong differential contrast to various types of animal tissues. Using a modification to the usual antibody detection schemes that employ horseradish peroxidase-mediated chromogen reactions, we have developed a method for imparting x-ray density to immunoprobe staining. By localized reduction of a soluble silver salt, the locations and quantities of a molecular probe can be visualized clearly in microCT images. Because tomographic imaging records quantitative spatial and object density information, it naturally generates data suitable for various kinds of modeling and analysis. Our current applications include measurements of developmental variation in zebrafish, correlating SPIM images of zfish cell proliferation with microCT images for development of asymmetric structure in the brain, and a new project to produce a 3D atlas of human embryos. We are also developing a new method for microCT imaging of molecular probes in whole-mount samples too large and opaque for fluorescence imaging.