MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues

Abstract

BackgroundComparative, functional, and developmental studies of animal morphology require accurate visualization of three-dimensional structures, but few widely applicable methods exist for non-destructive whole-volume imaging of animal tissues. Quantitative studies in particular require accurately aligned and calibrated volume images of animal structures. X-ray microtomography (microCT) has the potential to produce quantitative 3D images of small biological samples, but its widespread use for non-mineralized tissues has been limited by the low x-ray contrast of soft tissues. Although osmium staining and a few other techniques have been used for contrast enhancement, generally useful methods for microCT imaging for comparative morphology are still lacking.ResultsSeveral very simple and versatile staining methods are presented for microCT imaging of animal soft tissues, along with advice on tissue fixation and sample preparation. The stains, based on inorganic iodine and phosphotungstic acid, are easier to handle and much less toxic than osmium, and they produce high-contrast x-ray images of a wide variety of soft tissues. The breadth of possible applications is illustrated with a few microCT images of model and non-model animals, including volume and section images of vertebrates, embryos, insects, and other invertebrates. Each image dataset contains x-ray absorbance values for every point in the imaged volume, and objects as small as individual muscle fibers and single blood cells can be resolved in their original locations and orientations within the sample.ConclusionWith very simple contrast staining, microCT imaging can produce quantitative, high-resolution, high-contrast volume images of animal soft tissues, without destroying the specimens and with possibilities of combining with other preparation and imaging methods. Such images are expected to be useful in comparative, developmental, functional, and quantitative studies of morphology.

Quantitative Three‐Dimensional Analysis of Embryonic Chick Morphogenesis Via Microcomputed Tomography

“…A persistent challenge, however, was the limited ability to visualize and quantify soft tissues using Micro-CT. We and others have overcome this limitation with the optimization of osmium tetroxide (OT) as a CT dense contrast agent, showing dramatic visualization capability in mouse embryos without altering specimen geometry (Johnson et al, 2006;Metscher, 2009;Degenhardt et al, 2010). This study complements these by quantitatively analyzing embryonic chick morphogenesis.…”

“…More recently, we demonstrated using osmium tetroxide (OT) as a diffusive CT contrast agent that high resolution detailed visualization and segmentation of rodent embryos to newborns was possible with Micro-CT; these previous studies included early stage mouse and rabbit embryos (Johnson et al, 2006;Carney et al, 2007;Martinez et al, 2009). Investigators have since followed with related studies in model organisms to explore the effects of different chemical agents on soft tissue contrast enhancement (Metscher, 2009;Degenhardt et al, 2010).…”

Mechanical constraint from growing jaw facilitates mammalian dental diversity

“…To increase tissue contrast during the microCT scanning, soft tissues were stained overnight with 0.3% (wt/vol) phosphotungstic acid in 70% ethanol. Then samples were stored at 4°C in 70% ethanol (43). Samples were scanned in 70% ethanol.…”

Three-Dimensional Characterization of Tissue-Engineered Constructs by Contrast-Enhanced Nanofocus Computed Tomography

“…The latter is known to bind to connective tissues and more specifically to collagen and fibrin. 30 The former contains a negatively charged ioxaglate, which will be locally repulsed by the negative fixed charge density of the tissue; thus, all tissues that do not contain a negative charge will be stained. 31,32 Additionally, Hexabrix has been used for in vivo animal studies 33 and in a clinical setting 34 and, thus, is a potential candidate for noninvasive TE construct quality assessment.…”