Real-time phase-contrast x-ray imaging: a new technique for the study of animal form and function

Abstract

BackgroundDespite advances in imaging techniques, real-time visualization of the structure and dynamics of tissues and organs inside small living animals has remained elusive. Recently, we have been using synchrotron x-rays to visualize the internal anatomy of millimeter-sized opaque, living animals. This technique takes advantage of partially-coherent x-rays and diffraction to enable clear visualization of internal soft tissue not viewable via conventional absorption radiography. However, because higher quality images require greater x-ray fluxes, there exists an inherent tradeoff between image quality and tissue damage.ResultsWe evaluated the tradeoff between image quality and harm to the animal by determining the impact of targeted synchrotron x-rays on insect physiology, behavior and survival. Using 25 keV x-rays at a flux density of 80 μW/mm-2, high quality video-rate images can be obtained without major detrimental effects on the insects for multiple minutes, a duration sufficient for many physiological studies. At this setting, insects do not heat up. Additionally, we demonstrate the range of uses of synchrotron phase-contrast imaging by showing high-resolution images of internal anatomy and observations of labeled food movement during ingestion and digestion.ConclusionSynchrotron x-ray phase contrast imaging has the potential to revolutionize the study of physiology and internal biomechanics in small animals. This is the only generally applicable technique that has the necessary spatial and temporal resolutions, penetrating power, and sensitivity to soft tissue that is required to visualize the internal physiology of living animals on the scale from millimeters to microns.

Correlated patterns of tracheal compression and convective gas exchange in a carabid beetle

“…Phase-enhanced images were created using monochromatic x-rays (25 keV), a scintillator screen (cerium-doped yttrium aluminum garnet), and a sample-to-scintillator distance of ~0.5 m. For more details of this method, see Socha et al (Socha et al, 2007). To minimize potential harm to the animal, the lowest possible incident beam flux to form a viewable image was used; in some trials, shutters were used to reduce the field of view to the tracheal tube of interest, further reducing x-ray flux.…”

“…To determine the connection between internal dynamics and external gas exchange, we imaged live beetles with synchrotron x-rays while simultaneously recording gas exchange patterns. Although many natural behaviors such as breathing and feeding are performed by insects in the synchrotron x-ray beam, this method can lead to a number of deleterious effects (including eventual death), depending on the location, energy, intensity and duration of exposure (Socha et al, 2007). Therefore, an important technical issue is whether the tracheal compressions observed with synchrotron imaging vanish or change their characteristics in undisturbed insects not being xrayed.…”

“…Providing micrometer-scale resolution of millimeter-sized subjects, it enables an experimenter to view real-time events in living animals (Socha et al, 2007). More commonly used as a tool by physical science communities (Fitzgerald, 2000;Nugent et al, 2001), it has been applied only recently to questions of organismal biology (Westneat et al, 2008).…”

Rheinische Friedrich‐Wilhelms‐Universität Bonn

“…This applies especially to sea urchins, where the strong X-rays have to penetrate the calcite endoskeleton twice, making the analysis of soft tissues in this taxon with conventional desktop µCT equipment practically impossible. However, more advanced CT methods such as synchrotron radiation tomography and very-high-resolution X-ray computed tomography have been shown to be capable of soft tissue imaging in specimens with strong endo-and exoskeletons (Tafforeau et al 2006, Betz et al 2007, Socha 2007. Unfortunately, the allocated scanning times at synchrotron facilites are extremely limited.…”

Solute Transport in Cyclically Deformed Porous Tissue Scaffolds with Controlled Pore Cross-Sectional Geometries

“…Recent developments in high-speed X-ray microimaging may be sufficiently fast to accomplish CT scans in seconds. 54 Another potential solution to this problem may be the recently developed cryostatic micro-CT method 55 in which specimens are snap-frozen during the solute transport process and scanned while frozen.…”