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Biology Articles » Methods & Techniques » Real-time phase-contrast x-ray imaging: a new technique for the study of animal form and function » Figures
Figures
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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. (Click image to enlarge) |
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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. (Click image to enlarge) |
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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. (Click image to enlarge) |
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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. (Click image to enlarge) |
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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. (Click image to enlarge) |
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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. (Click image to enlarge) |
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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. (Click image to enlarge) |
rating: 1.00 from 1 votes | updated on: 11 Aug 2009 | views: 7370 |
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