There is a trade-off between image quality and survivorship: higher quality images require greater exposures to radiation, which result in greater harm to the animal. With our video camera and beamline configurations, we found a satisfactory comprise between image quality and survivorship by using 25 keV x-rays at 80 μW/mm2 flux density (2 × 1010 ph/s/mm2) and 1 m sample-detector distance (hereafter referred as 'nominal' settings). With these settings, insects exhibited no negative behavioral effects for a period of about 5 minutes. X-rays on the insect's head or thorax caused major changes to the respiratory pattern by about 17 minutes (2.4 kGy). With the beam on the abdomen, no significant changes were observed on the respiratory pattern throughout the full 2-hr trials (17.3 kGy), or even in two trials that were extended to 4 hrs (34.5 kGy). No thermal effects of the x-rays were observed. Food transport and gut structures could be clearly seen using labeled food (Figure 1e–l). In cases where tracking food transport was more important than maximizing the clarity of internal anatomy, 33.2 keV x-rays were successfully used to visualize iodine-laced food. Although not explicitly tested, the shorter wavelength of the x-rays at this setting results in lower absorption and therefore lower impact on the animals. We observed insect feeding under irradiation for more than 30 minutes, depending on species and location of the x-rays on the insect.
Figure 3 demonstrates the advantage of phase-contrast imaging over conventional absorption-based imaging. At d = 5 cm, where the phase effects are minimal, image contrast is poor for all energies, consistent with the fact that the absorption is small. For d = 5 cm and E = 15 keV, although some differences due to absorption can be seen, it is the small phase-contrast edge enhancements that make the features easily discernible. At a fixed energy, increasing d clearly increases the image contrast, as predicted by Equation 1. A careful comparison of the image at d = 100 cm with d = 5 or 50 cm shows that the spatial resolution of the d = 100 cm image is poorer: the line widths at the edges of the air sacs are broader in the d = 100 cm image.
Thermal effects due to x-ray irradiation
Measurements from both the thermocouple and the infrared camera showed no change in temperature to the irradiated insect. This is not surprising, because the nominal power absorbed by the insect is, in the most extreme case, only about 20% of its unirradiated metabolic rate (Table 1).
X-ray irradiation effects on CO2 emission patterns
The effect of x-ray radiation dosage on metabolic rates was quantified by examining the effect of incident beam flux density on 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' (R2 min) (all measurements at 25 keV; Figure 5). Except for grasshoppers, the data show a slight but significant increase in average CO2 emission immediately after 'beam on'. This small increase is probably the result of movement of the insect (seen in the x-ray videos) as it tried to move away from the beam. However, increasing beam intensity to four-times nominal values had little effect on R2 min, suggesting that, although insects appear to sense the beam, there is a considerable safety margin in the capacity to absorb x-rays before major physiological damage occurs during the initial minutes of exposure.
Although CO2 emission patterns during the first minutes after 'beam on' are similar to those prior to irradiation (Figure 6), a major change in the CO2 emission pattern (respiratory signal, RS) was observed in all species after 1000–1500 s of irradiation. The RS was correlated with dorsoventral head-shaking in the ants and beetles, and a quivering proboscis in fruit flies, but was not correlated with any observable behavior in grasshoppers. We note that for ants, the RS is qualitatively similar to the 'mortal fall' signature observed for ants under thermal stress , even though in our case there were no measurable temperature changes (0.1 K resolution) in the animal. Shortly after the RS, the CO2 emission pattern became periodic for all ant samples, and for most of the beetle (12 of 15) and grasshopper (9 of 16) samples. For ants, these periodic patterns resemble discontinuous gas exchange (DGC) reported in decapitated ants , so we interpret the RS as indicating major brain damage.
Time to respiratory signal (TTRS) varied strongly with incident beam power density (Figure 4a); higher power densities resulted in lower TTRS for all species. The one exception was grasshoppers at the highest power density, which showed a higher TTRS than the other species. Figure 4b shows still images taken from the video corresponding to the different incident power densities. Together, Figures 4a and 4b provide a guide for an experimenter to gauge the compromise between image quality and physiological impact.
TTRS dependence on insect mass
Measurements at the nominal beam intensity showed no mass dependence on the TTRS for grasshoppers (N = 9, 13.3–1473.5 mg, Spearman ρ = 0.23, p = 0.51) and ants (N = 14, 8.4–53.7 mg, Spearman ρ = -0.24, p = 0.38). One possible explanation for this lack of pattern is that, in all cases, major portions of the brain were irradiated.
TTRS dependence on x-ray beam location on the insect body
From CO2 emission measurements in ants, there is no significant difference (Student's t-test, p = 0.4) in TTRS between having the nominal x-ray beam incident on the head (N = 3, TTRS = 1299 ± 177 s) or the thorax (N = 3, TTRS = 1092 ± 249 s) of the animal. In contrast, no change in CO2 emission pattern was observed with the x-ray beam incident on the abdomen (N = 3, Figure 7a), even after 4 hrs of exposure at the nominal beam intensity. These results are consistent with the fact that in ants, and most insects, most ventilatory activity is controlled by major ganglia of the central nervous system in the head and thorax .
X-ray irradiation effects on motor function
Using simple behavioral assays, we tested for the presence/absence of righting behavior, defensive behavior, and locomotor ability after a fixed duration of x-ray exposure on the head using nominal beam settings (Table 1). No changes in behavior were observed within the first 5 minutes of exposure. During 6–25 minutes of x-ray exposure, ants, beetles and flies progressively lost motor abilities, starting with leg twitches and ranging to full immobility. By contrast, after 2 hrs of exposure, the grasshoppers could still right themselves, hop, feed and fly (and were later observed to mate and lay eggs). One major difference between the grasshoppers and all other insects studied is that, because of their large size, only a part of the grasshopper's head was irradiated as opposed to the entire head in the other taxa. We note that, consistent with other studies [11,12,23], the loss of locomotor abilities observed in the insects at lower dosages was temporary, indicating radiation-induced lethargy. In many individuals, we observed recovery minutes to hours later, suggesting that radiation damage was at least partially repairable.