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This technique takes advantage of partially-coherent x-rays and diffraction to enable clear …

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

Our measurements show that a major change in CO2 emission pattern, probably indicating major damage to the central nervous system, occurred after about 2.4 kGy when the insect was exposed on the head or thorax. No change in CO2 emission was observed if the x-ray beam was incident on the abdomen. The TTRS was independent of mass and species. In ants, beetles and juvenile grasshoppers whose entire heads were irradiated, a cyclic or discontinuous gas exchange (DGC) CO2 emission pattern [24] occurred after the RS. Ants have also been shown to exhibit DGC after they are physically decapitated [25,26], supporting the hypothesis that the x-ray treatment caused major brain damage. In cases where the RS was observed in this study, it is likely that the very high, acute dose of radiation caused profound tissue damage, causing such problems as potassium leakage [27,28] and leading to effects akin to the 'central nervous system syndrome' known from mammals [29]. One puzzling result is that although grasshoppers were no different in TTRS at some power densities, they showed a surprising degree of behavioral control after long periods of irradiation, suggesting a greater tolerance of x-rays to the head. For these animals, whose heads were larger than the size of the x-ray beam, the positioning of the x-ray beam may have missed or only partially damaged parts of the central nervous system, including the major ganglia controlling respiratory and motor function. In particular, partial control of motor behaviors such as walking occur in ganglia in the thorax [30-32]. Many of the smaller insects received incidental radiation on the thorax due to geometry during nominal 'head only' trials and exhibited motor loss, lending further weight to this hypothesis.

Due to the many factors that contribute to the question of image quality versus survivorship, there is no single set of x-ray parameters that provide an optimal setting. Generally, one would like a very small source size to minimize image blur, and an efficient detector system so that a less intense x-ray beam can be used to maximize survivorship. In practice, for insect physiology, the first question is whether the particular internal dynamic or morphology can be visualized by this technique. Given the particular source and detector that is available, one usually starts with parameters that give superior image quality. Based on our experience with insects, this is usually with an x-ray energy of 10–20 keV and a sample-detector distance of 10–100 cm. After the desired feature is visualized, the experimenter can optimize the system based on the relative importance of image contrast, spatial resolution, and survivorship.

With our commercially available standard NTSC interlaced video camera (30 fps, Cohu 4920) and nominal incident fluxes of 2 × 1010 ph/s/mm2 at 25 keV, a 16.6 ms (1/60 s) exposure time is sufficient to produce a quality image and record many physiological functions. For body functions that require shorter exposure times (e.g., flight), higher incident beam fluxes are necessary (and are available), in which case insect survivorship will be correspondingly reduced. However, in many cases the total time needed to record such rapid phenomena will be lower. Nonetheless, because the current overall detection efficiency is still very low (< 10%) [33], there is ample room for technological improvement with the development of better detectors. In fact, during the course of this manuscript preparation, we acquired a new video camera with the same pixel numbers and sizes, but with twice the sensitivity; thus, we can now obtain high-quality images with only 1 × 1010 ph/s/mm2 incident beam flux (Figure 4b). This improvement should double the working time (from 5 to 10 minutes) before any x-ray related effect is observed.

Finally, although this study was targeted specifically at insects, these species were chosen primarily as exemplars to introduce the technique to the biological community. Synchrotron x-ray phase contrast imaging is broadly applicable to any organism with features on the micron scale and above. However, we urge caution when exploring new systems with this technique; it is crucial to understand the effects of the radiation on the organism when making biological interpretations.

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