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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 » Methods

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

Synchrotron x-ray phase-contrast imaging

Experiments were performed at the XOR-1ID and XOR-32ID undulator beamlines at the Advanced Photon Source (Figure 2). Synchrotron x-rays are produced here by a source with full-width half maximum dimensions of 35 μm (vertical) by 560 μm (horizontal) and source-to-sample distances of 60 m and 40 m, respectively. A Si (111) double crystal monochromator was used to select the x-ray wavelength. The incident beam flux (photons/s/mm2) was changed by varying the undulator magnetic gap and was monitored with an upstream ion chamber. Insects were mounted on top of a remotely controlled stage that enabled precise positioning in the x-ray beam. After passing through the insect, the x-rays were converted to visible light via a cerium doped yttrium aluminum garnet scintillator. The sample-to-scintillator distance was approximately 1 m; a distance of this magnitude is necessary for obtaining the phase-contrast effect. The visible light created by the scintillator was imaged onto a video camera (Cohu 4920 or Cohu 2700, Cohu, San Diego, CA, USA) or higher resolution CCD camera (SensiCam QE, Cooke, Romulus, MI, USA) using a 2× or 5× microscope objective. The field of view was 2.4 mm × 3.2 mm and 1.0 mm × 1.3 mm for the 2× and 5× objectives, respectively. Unlike most prior studies, the size of the x-ray beam was comparable to the size of the insect, and we only exposed parts of the insect to radiation.

Animals

We conducted the majority of our experiments on carpenter ant workers (Camponotus pennsylvanicus, n = 59), but also explored taxonomic diversity by examining beetles (Platynus decentis, n = 20), fruit flies (Drosophila virilis, n = 28), and grasshoppers (Schistocerca gregaria, n = 19). Ants and fruit flies were purchased from Carolina Biological Supply Company (NC, USA). Beetles were collected in the woods at Argonne National Laboratory, and grasshoppers were reared at one of the author's laboratory (JH). Insects were housed with free access to food and water prior to experimentation.

Survivorship and behavior

To determine the length of time that insects could withstand radiation on a particular part of the body (head, thorax, or abdomen), insects were monitored for CO2 release using flow-through respirometry while being observed with x-rays (Figure 2b). Insects were cold anaesthetized and placed individually in custom plexiglass respirometry chambers (volumes: 0.03, 0.25, 1.0, and 9.5 ml) with Kapton (Dupont, DE, USA) windows for x-ray transmission (Figure 2c). Because some insects actively moved away from the beam upon contact, cotton was used to fill in gaps within the chamber to constrain the insect within the field of view. The chambers were oriented such that the long axis of the body lay perpendicular to the beam path, providing either lateral or dorsoventral views.

CO2 emission was monitored by a flow-through respirometry system from Sable Systems International (SSI, Las Vegas, NV, USA). Room air was scrubbed of CO2 and H2O using a Drierite/Ascarite/Drierite column and pushed through the system using a pump (TR-SS3, SSI). Flow rate (100 ml/min for all species except D. virilis, 50 ml/min) was maintained via a mass flow controller (SSI MFC-2 using a Sierra Instruments mass flow control valve). CO2 exiting the insect chamber was measured by a gas analyzer (LI-7000, Li-Cor, Lincoln, NE, USA). Chamber washout times were on the order of 6–12 s. CO2 data were output to a computer via UI-2 (SSI) and recorded using ExpeData software (SSI). Pre-beam CO2 emission was typically recorded for 5–10 minutes before opening the x-ray shutter. For survivorship trials, insects were exposed to x-rays on the head, thorax, or abdomen until they clearly showed a respiratory signature that we infer to be respiratory function damage; otherwise, trials were ended after 2 hrs. CO2 emission was also monitored post-beam for up to 30 minutes. For behavioral trials, the insects were exposed for a fixed amount of time, then removed from the chamber and tested for the presence/absence of righting behavior, defensive behavior, and locomotor ability. All trials were conducted at room temperature (21–22°C). Data were analyzed using ExpeData and LabAnalyst X (Mark Chappell, University of California Riverside, CA, USA) software packages.

Feeding

To demonstrate the use of x-rays to visualize internal food movement during ingestion and digestion, beetles (Platynus decentis) were fed macerated insects mixed with fine particles of CdWO4, and butterflies (Pieris rapae) were fed sugar solutions laced with an iodine contrast agent (Isovue, Bracco Diagnostics, NJ, USA). Animals were held in place by securing the body to a microscope cover slip (beetles) or by a mounted clamp attached to the wings (butterflies). These examples illustrate the use of contrast agents to visualize internal food transport. The fine particles of CdWO4 had a significantly higher absorption over the entire x-ray energy range used in this study and appeared darker than the surrounding soft tissue. In the case of the iodine solution, differences in x-ray absorption at the nominal setting (25 keV) were minimal. To maximize contrast between the iodine and the surrounding anatomy, we used an energy just above the K-absorption edge (33.2 keV for iodine), where absorption increased dramatically. Because in general the use of higher energy x-rays results in an overall lower contrast for soft tissue (Figure 3), this technique is most applicable for cases where it is more important to track internal movements of food than to visualize clearly the surrounding insect anatomy. The use of simultaneous x-ray images above and below the K-edge to improve visualization of the contrast agent is possible, but would require a more complicated set of x-ray optics. Furthermore, it would imply a doubling of the x-ray dose to the animal.

For contrast agents, iodine is more suitable for fluids whereas CdWO4 is more suitable for solids. Although we have not investigated the toxicity of iodine versus CdWO4 in insects, we speculate that iodine is less harmful because Isovue is used for human medical diagnosis, and it is well known that cadmium is toxic [35]. We chose a cadmium compound for its convenience, but other high electron density materials in powder form (such as silica or lead) can be used to provide radio-opacity with lower toxicity.

Temperature

Possible change in temperature in the insect due to the absorption of x-rays was measured separately with two methods. First, an implanted 0.01 mm copper-constantan thermocouple and thermocouple thermometer (0.1 K resolution, Physitemp Instruments, Inc., NY, USA) were used to measure internal abdominal temperature in an adult grasshopper (Schistocerca gregaria) while it was irradiated for up to 10 minutes on the thorax. To test if local heating occurred at the site of irradiation, an infrared camera (Inframetrics 760, 0.1 K resolution, American Infrared, NY, USA) was used to visualize temperature change in the head of three beetles (Platynus decentis) and one grasshopper (Schistocerca gregaria).

Data analysis

We used two metrics to quantify the effect of the x-rays on the CO2 emission patterns. To assess immediate effects of the beam, we compared CO2 emission rates in the 2 minutes before 'beam on' to those 2 minutes after 'beam on' (Equation 2). We defined the ratio (R2 min) as:

08120902m02.gif

where 〈Epre-beam2 min and 〈Ebeam-on2 min are, respectively, the CO2 emission rates (μl/hr) averaged over the 2 minutes immediately prior and after the x-rays (25 keV) were turned on. Second, to assess the duration required for damage to occur due to x-rays, we identified a major change in the CO2 emission pattern within each species, which we refer to as the respiratory signature (RS; Figure 6). The RS was chosen for its repeatability; by the time of the RS, major (and likely irreversible) damage has occurred. This time interval, between when the beam first hit the insect and the RS, is the time to respiratory signature (TTRS).

Image quality and the corresponding photon fluxes and average power densities for 25 keV x-rays used in this study are shown in Figure 4. The incident photon fluxes were chosen for an approximate factor of two change in intensity between each setting. At 25 keV, a 1-mm thick water sample (over the entire beam area) would absorb about 3% of the incident beam energy [36]. Absorbed power for a volume of insect that is irradiated can thus be estimated by Equation 3:

Absorbed power = 0.03 × D × V     (3)

where D is the incident beam power density (μW/mm2) and V is the irradiated volume in mm3. Assuming an incident power density of 80 μW/mm2 and an ant head of volume 3 mm3, the estimated absorbed power is 7.2 μW and the absorbed dose rate (absorbed power per unit mass) is 2.4 Gy/s. X-ray absorbed powers were measured for each species (Table 1); these values are consistent with the theoretical estimates.

Table 2. Effect of duration of x-ray exposure to head on motor control abilities. m/n denotes m animals behaving normally out of n animal trials. Asterisks denote partially limited response (e.g., slow response).

Metabolic rate calculations were based on averages of all available prebeam CO2 recordings for each of the four species. Conversion from CO2 output to metabolic rate assumed respiratory quotients (RQ) of 1.0, 0.8, 0.7, and 1.0 and energy equivalences of 20.1, 24.5, 27.6 and 21.2 J/ml of CO2 for grasshoppers [37], beetles [38], ants [39], and fruit flies [40], respectively.


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