The ability to visualize the internal anatomy of living animals is fundamental to our understanding of biology and medicine. Although imaging systems for respiratory, circulatory and musculoskeletal systems are available for large animals, real-time visualization of the internal processes of small animals has been limited by scaling factors and imaging technology. In order to visualize internal physiological mechanisms of millimeter-sized animals in real-time, a probe must have the following features: (1) ability to penetrate the opaque exterior, (2) spatial resolution in the 1–10 μm range, (2) temporal resolution below 100 ms, and (4) sensitivity to soft tissue. Visible light microscopy (conventional or confocal) is not broadly applicable for intact, live animals due to animal opacity and size limitations. Near-infrared (NIR) microscopy has been tried, but with limited success due to poor spatial resolution . Magnetic resonance imaging (MRI) has been used to image insects , but the best resolution obtained so far is about 50 μm, and images must be averaged over seconds to minutes. For sufficient penetration, spatial resolution of ultrasound imaging is wavelength-limited  to about 100 μm. Conventional x-ray imaging relies on absorption as the contrast mechanism, which is ineffective at visualizing soft tissue. For example, at 25 keV, the maximum absorption contrast of a 100-μm diameter air-filled trachea in water is only 0.3%, smaller than the Poisson noise for a high-end 16-bit CCD camera (0.4%).
Compared to these other techniques, synchrotron x-ray phase-contrast imaging [4,5] is ideal for visualizing well-defined internal structures that have different mass densities. Tracheal tubes, in particular, show up extremely well (Figure 1a–d; see also Additional file 1), with edge contrast in a 100-μm diameter trachea that can be more than 50%. For example, this technique has been recently used to observe directly tracheal compression dynamics in opaque insects . This research, which examined detailed networks of tracheal tubes down to tubes of 10 μm in diameter in living specimens, has revealed a mechanism of breathing that was previously identified with only a single species of translucent flea . Such studies demonstrate the ability of synchrotron imaging to open up whole new avenues of scientific inquiry in biology.
The basis of the x-ray phase-contrast imaging described here is Fresnel diffraction. For samples with minimal absorption, true for insects at the x-ray energies used here, the intensity of an image at a distance d downstream of the sample can be approximated by Equation 1 (see also ):
I(x, y) = Iinc (1 + 1.3 × 10-6 × d × λ2 × ∇2 [∫ ρ(x, y, z)dz]) * R(x, y) (1)
where Iinc is the incident beam intensity, λ (in Å) is the x-ray wavelength, ρ (in g cm-3) is the sample density, R(x, y) is the effective detector resolution, x-y is the image plane, z is the beam direction, and * denotes a convolution. R(x, y) depends on the detector properties (scintillator, lens and pixel size) and the projected source size, , where σs is the source size, d is the sample-detector distance, and L is the source-sample distance (Figure 2). Increasing either the x-ray wavelength (lowering x-ray photon energy) or the sample-detector distance increases contrast (Figure 3). However, using longer x-ray wavelengths results in higher absorption, which is detrimental to the living animal. Similarly, increasing the sample-detector distance results in a loss of spatial resolution due to the increase in projected source size. Higher incident beam intensities give brighter and less noisy images (Figure 4b), but cause more harm to the insect. Given the complex interplay of these physical and biological factors, there is no a priori prescription for how best to optimize synchrotron phase-contrast imaging for organismal studies. Thus, one of the objectives of this study is to examine multiple experimental parameters to provide biologists a framework for using synchrotron phase-contrast imaging.
A major concern in using synchrotron x-rays to study physiological processes in small animals is the effect of the x-rays on the animal. Radiation causes molecular damage, including protein and lipid oxidation and gene transmutation; however, the effects depend on dose . Previous studies show that fruit flies (Drosophila melanogaster)  and wasps (Habrobracon and Bracon hebetor) [11,12] temporarily lose motor control after a dose of about 1–2 kGy, but recover to normal behavior within minutes  or hours . At exposures greater than 2.5 kGy, insects do not recover, although it is unclear when death actually occurs . Feeding patterns are affected after 600 (D. melanogaster)  to 1000 Gy (Bracon hebetor Say) . In one study of D. melanogaster receiving doses of 600 Gy, metabolic rates were unaffected one day after irradiation . In summary, the literature suggests that there are no observable physiological effects at doses less than 500 Gy, a temporary loss of motor control is observed after ~1.5 kGy, and a more permanent loss of motor control occurs at doses greater than 2.5 kGy. However, in most prior studies of radiation effects on insects (concerned primarily with insect control  and ageing ), animals have been subjected to full body irradiation; the few studies that examined localized x-rays have used low levels of radiation [16-19]. Thus it is unknown how insects are affected by intense, targeted radiation – such as in a synchrotron x-ray beam – on specific parts of the body. Furthermore, previous studies focused primarily on effects that occur on a relatively long time scale, usually days after irradiation, and few studies have examined immediate radiation effects. This study strives to answer two questions: what combination of x-ray beam parameters optimizes image quality while minimizing damage to the animal? And under these conditions, how much time is available before the insect is negatively impacted? We varied x-ray parameters and used both CO2 emission patterns and motor behaviors as proxy indicators to assess physiological damage in four insect species. In addition, we demonstrate the range of studies that can be addressed using this technique by showing examples of high-resolution still imagery and real-time movement of food during ingestion and digestion.