In vivo X-ray cine-tomography for tracking morphological dynamics

Scientific cinematography using ultrafast optical imaging is a common tool to study motion. In opaque organisms or structures, X-ray radiography captures sequences of 2D projections to visualize morphological dynamics, but for many applications full four-dimensional (4D) spatiotemporal information is highly desirable. We introduce in vivo X-ray cine-tomography as a 4D imaging technique developed to study real-time dynamics in small living organisms with micrometer spatial resolution and subsecond time resolution. The method enables insights into the physiology of small animals by tracking the 4D morphological dynamics of minute anatomical features as demonstrated in this work by the analysis of fast-moving screw-and-nut–type weevil hip joints. The presented method can be applied to a broad range of biological specimens and biotechnological processes.The best method to study morphological changes of anatomic features and physiological processes is to observe their dynamics in 4D, that is, in real time and in 3D space. To achieve this we have developed in vivo X-ray cine-tomography to gain access to morphological dynamics with unrivaled 4D spatiotemporal resolution. This opens the way to a wide range of hitherto inaccessible, systematic investigations of small animals and biological internal processes such as breathing, circulation, digestion (1), reproduction, and locomotion (2).At the micrometer resolution range, state-of-the-art optical imaging techniques can achieve high magnifications to visualize tissues and even individual cells for 4D studies. These methods however are confined to transparent or fluorescent objects, or are limited either by low penetration depth <1 mm or poor time resolution (3). For optically opaque living organisms X-ray imaging methods are highly appropriate due to the penetrating ability of the radiation. Modern synchrotron radiation facilities provide brilliant and partially coherent radiation suitable for high-resolution volume imaging methods such as X-ray computed microtomography (SR-µCT). For static specimens SR-µCT has proven to be a powerful tool to study small animal morphology in 3D (4–6). The benefits of various physical contrast mechanisms, high spatial resolution, and short measuring times, as well as enormous sample throughput compared with laboratory X-ray setups, have led to its widespread use in life sciences.Real-time in vivo X-ray imaging with micrometer spatial resolution was realized so far by recording time sequences of 2D projection radiographs of different organisms (1, 6, 7), providing time information about functional dynamics but losing any information about the third spatial dimension.Recently, 4D in vivo X-ray experiments have been performed to study cell migration in frog embryos (8, 9) using tomographic sequences of a few seconds exposure time per tomogram interrupted by longer nonexposure time slots. In this way the authors followed relatively slow dynamics and morphological changes during embryonic development with 2-µm resolution over total time intervals of several hours. The fastest 4D time series yet reported were realized with a temporal resolution of 0.5 s and spatial resolution of 25 µm (10), applied to a living caterpillar used as test specimen for imaging, but without any analysis of dynamics.In this paper, we demonstrate the quantitative 4D investigation of morphological dynamics by in vivo X-ray 4D cine-tomography, introduced here as the combination of ultrafast SR-µCT and motion analysis procedures. Using this approach allows us to investigate previously inaccessible 3D morphological dynamics in small animals, presently with feature sizes in the micrometer range and with temporal resolution down to a few tens of milliseconds. In the past, ultrafast in vivo imaging was hardly possible for such applications, due to the strongly competing requirements for simultaneous high contrast, high signal-to-noise ratio (SNR), and concurrent low radiation dose, as well as the need for simultaneous high spatial resolution and maximum temporal resolution.In the following we describe how in vivo X-ray 4D cine-tomography meets the above challenges by optimizing image contrast, SNR, and spatial and temporal resolution in the ultrafast SR-µCT system and by establishing a dedicated data analysis pipeline, all within a unified framework (Fig. S1). We demonstrate the potential of the technique by investigating morphological dynamics in fast-moving weevils, focusing here on the exoskeletal joints.