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


Biology Articles » Methods & Techniques » Real-time phase-contrast x-ray imaging: a new technique for the study of animal form and function » Figures

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

mcith_08120902f01.jpg Figure 1 Full-field 2-D projection images created using phase-contrast synchrotron x-rays. Images were chosen to highlight the highest quality imagery currently obtainable (a, b) and corresponding stills from live video (c-l). (a) Carabid beetle (Pterostichus stygicus) in dorsoventral view with legs removed and sacrificed prior to imaging. Image is a high-resolution composite of multiple images. The air-filled tubes of the tracheal system can be prominently seen. The dark spots on the left side, mid-body are soil particles on the outer surface of the elytra. (b) Close-in view of one section of the prothorax, showing the branching pattern of tracheae. (c, d) One half-cycle of rhythmic tracheal collapse in a live carabid beetle (Platynus decentis) in dorsoventral view. Images are frame grabs from a video recording (See Additional file 1); time interval is 0.5 s. Total time of collapse and reinflation of the tubes is 1.0 s. (e-l) Visualization of internal food movement using labeled markers. (e) Schematic of the head and thorax of a butterfly (Pieris rapae) in lateral view. The foregut is shown in red; the square highlights the region of video stills in (f-h), and black arrow indicates the direction of food movement. (f-h) Video stills of passage of a food bolus posteriorly through the esophagus, moving through the frame from upper right to lower left (see Additional file 2). Red arrows indicate the leading (f) and trailing (h) edges of the bolus. Interval between frames is 0.5 s. Food is sugar water/iodine mixture. X-ray energy (33.2 keV) was tuned to just above the K-edge absorption band for iodine. (i) Schematic of a carabid beetle (Pterostichus stygicus) in dorsoventral view (legs removed). Circular structures in mid-body represent coxae; the gut is represented in gray and red. Square highlights video in (j-l), visualization of cadmium-laced food in the foregut (see additional file 3). Video stills (j-l) show movement of gut including anterior-posterior translation and squeezing of the crop (cr) and translation and rotation of the proventriculus (pr). The proventriculus is a valve that leads to the midgut [41]; here, it is closed. Note that only parts of the gut with contrast agent can be seen. Interval between frames: j-k, 2 s; k-l, 6 s. X-ray energy, 25 keV. Scale bars: a,b, 1 mm; c,d, 200 μm; f-h, 200 μm; j-l, 1 mm.

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mcith_08120902f02.jpg Figure 2 Experimental setup. (a) Schematic of phase-contrast imaging setup at the Advanced Photon Source. X-rays are produced by an undulator and monochromatized by a Si (111) double crystal monochromator. The partially coherent, monochromatic x-ray beam passes through an ion chamber and then the sample. The x-rays are converted to visible light by a scintillator screen, and the resulting image is recorded by a CCD image sensor. (b) Schematic of respirometry setup. MFC, mass flow valve and electronics control unit; S, CO2 scrubber; RC, respirometry chamber; CO2, CO2 analyzer; MFM, mass flow meter. (c) Typical plexiglass respirometry chamber. Yellow material is Kapton, used to provide an x-ray transparent window to the animal. Internal chamber volume is 0.25 ml.

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mcith_08120902f03.jpg Figure 3 Video image quality as a function of x-ray energy and sample-detector distance. Data are from an ant head (Camponotus pennsylvanicus) using a Cohu 4950 video camera. Within each column, the absorbed x-ray dose on the insect is constant. For all images, the photon flux was kept at approximately 2 × 1010 ph/s/mm2.

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mcith_08120902f04.jpg Figure 4 Image quality versus TTRS. (a) Plot of TTRS ('time to respiratory signal', which indicates major respiratory damage; see Figure 6) as a function of incident power density for all four species. At least three trials were performed per data point. A power law fit to the data gives: TTRS (s) = 90484 x-1.02, R = 0.97 where × is the incident beam power density in μW/mm2. TTRS measurements as a function of animal mass showed no correlation for the mass range 8.4–53.7 mg and 13.3–1473.5 mg for ants and grasshoppers, respectively. (b) Still images taken from video (16.6 ms exposure) footage of a dead fruit fly (Drosophila melanogaster) as a function of incident beam power density, which are, respectively from i-vi: 4, 8, 16, 36, 80, 103 μW/mm2. X-ray energy is 25 keV. At 80 μW/mm2, the photon density is 2 × 1010 ph/s/mm2. Field of view is 1.0 × 1.3 mm using a 5× objective lens. Head and thoracic air sacs and leg trachea can be clearly seen. These images are taken with our new camera (Cohu 2700), which is twice as sensitive as the camera used in the major part of this study. Although we subjectively consider (iv) to be a high quality image, usable images can be obtained using lower beam intensities.

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mcith_08120902f05.jpg Figure 5 R2 min as a function of incident beam power density. R2 min is the ratio of mean CO2 emission rate during the 2 minutes after 'beam on' divided by the mean CO2 emission rate during the 2 minutes before 'beam on'. Error bars denote standard deviation; numbers below each data point correspond to sample sizes. The 25 keV x-ray beam was incident on the head.

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mcith_08120902f06.jpg Figure 6 Representative CO2 emission traces for the four species used in this study. The x-ray beam (25 keV, 80 μW/mm2) was incident on the insect's head. No qualitative changes are seen immediately after beam on. A major change in CO2 emission pattern after 1000–1500 s of x-ray exposure is marked by RS (respiratory signature). The RS was based on CO2 emission patterns and was corroborated with x-ray video behavioral data; the RS is a major change in CO2 release pattern associated with shaking of the head or mouthparts. For the grasshoppers (Schistocerca gregaria), no behavioral change was observed at RS.

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mcith_08120902f07.jpg Figure 7 Comparison of x-ray impact on two different regions of the insect body. Representative CO2 traces are from two different ant specimens (Camponotus pennsylvanicus) with the x-ray beam targeted on the abdomen (a) and the head (b). Even though the abdomen-irradiated ant (a) received a higher x-ray flux (80 vs. 36 μW/mm2), it showed no discernible changes in CO2 emission pattern. In contrast, the head-irradiated ant (b) showed dramatic changes in CO2 emission, including a decrease in variance leading up to the RS (at which point the head stopped moving) and a cyclic pattern of release (similar to DGC in decapitated ants [25, 26]) thereafter.

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