X-ray computed tomography and its potential in ecological research: A review of studies and optimization of specimen preparation

Abstract

Imaging techniques are a cornerstone of contemporary biology. Over the last decades, advances in microscale imaging techniques have allowed fascinating new insights into cell and tissue morphology and internal anatomy of organisms across kingdoms. However, most studies so far provided snapshots of given reference taxa, describing organs and tissues under "idealized" conditions. Surprisingly, there is an almost complete lack of studies investigating how an organism's internal morphology changes in response to environmental drivers. Consequently, ecology as a scientific discipline has so far almost neglected the possibilities arising from modern microscale imaging techniques. Here, we provide an overview of recent developments of X-ray computed tomography as an affordable, simple method of high spatial resolution, allowing insights into three-dimensional anatomy both in vivo and ex vivo. We review ecological studies using this technique to investigate the three-dimensional internal structure of organisms. In addition, we provide practical comparisons between different preparation techniques for maximum contrast and tissue differentiation. In particular, we consider the novel modality of phase contrast by self-interference of the X-ray wave behind an object (i.e., phase contrast by free space propagation). Using the cricket Acheta domesticus (L.) as model organism, we found that the combination of FAE fixative and iodine staining provided the best results across different tissues. The drying technique also affected contrast and prevented artifacts in specific cases. Overall, we found that for the interests of ecological studies, X-ray computed tomography is useful when the tissue or structure of interest has sufficient contrast that allows for an automatic or semiautomatic segmentation. In particular, we show that reconstruction schemes which exploit phase contrast can yield enhanced image quality. Combined with suitable specimen preparation and automated analysis, X-ray CT can therefore become a promising quantitative 3D imaging technique to study organisms' responses to environmental drivers, in both ecology and evolution.

Keywords: anatomy; animal imaging; computed tomography; internal morphology; μ‐CT.

Figure 1

Conceptual figure showing procedural differences between computed tomography and physical sectioning of samples. CT (by means of several techniques) does not require sample destruction, and the resolution is identical in all orientations (isotropy) and enables visualization in different angles or axes. On the other hand, physical sectioning allows a wider variety of dying techniques for tissue recognition, but the plan is determined by the orientation of the sample; thus, also the z‐axis possesses a different (usually lower) resolution

Figure 2

Historical analysis of the usage of x‐ray computed tomography in scientific studies by means of Thomson‐Reuters's ISI on the Web of Science (all databases). Dark gray: total number (primary search terms), blue: biological sciences (primary search terms plus secondary filtering to exclude studies dealing with human and animal medicine and livestock production), dark pink: manually refined selection of ecological studies. Bars represent the annual count of publications and lines the cumulative sum. Results showed on the y‐axis (presented in logarithmic scale) were obtained using a combination of search term and research areas explained in the text. Beetle image © Alex Wild, used by permission

Figure 3

Quantitative analysis of ecological studies using x‐ray computed tomography. (a) Arthropoda, (b) Annelida, (c) Chordata, (d) Plantae (kingdom), (e) Cnidaria, (f) Mollusca, (g) Echinodermata, (h) Fungi (kingdom), (i) Porifera. μ‐CT: microcomputed tomography, SR‐μCT: synchrotron radiation microcomputed tomography

Figure 4

Experimental comparison of staining agents and scanning mediums using only ethanol as fixative in the cricket Acheta domesticus. After fixation for 24 hr in 70/30 solution ethanol/deionized water, samples were stained in phosphotungstic acid (PTA) or iodine (solution with ethanol) during 7 days and 24 hr, respectively. Posteriorly, the samples were scanned either in ethanol or air‐dried

Figure 5

Experimental comparison of fixatives and drying techniques using iodine as chosen staining agent in the cricket Acheta domesticus. After fixation for 24 in either 70/30 solution ethanol/deionized water, FAE (formaldehyde, acetic acid, and ethanol), or Bouin's solution (saturated aqueous picric acid, pure acetic acid, and 10% formaldehyde solution), the samples were air‐dried or submitted to CPD (critical‐point drying) before being scanned. Heads are presented in coronal planes and abdomens in sagittal planes

Figure 6

Segmentation process steps. Acheta domesticus female abdomen. Please click on the figure to activate the interactive 3D content and use the mouse to rotate the objects. Further functions (views, render modes, and model tree) are available in the menu