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

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 [1]. Magnetic resonance imaging (MRI) has been used to image insects [2], 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 [3] 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 [6]. 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 [7]. Such studies demonstrate the ability of synchrotron imaging to open up whole new avenues of scientific inquiry in biology.

Additional File 1. Rhythmic compressive movements in the tracheal system in the carabid beetle Platynus decentis, demonstrating the utility of phase-contrast synchrotron imaging for studies of respiratory dynamics in small animals. View (1.3 × 1.0 mm) is a dorsoventral projection through prothorax of a beetle (mass ~ 45 mg) using monochromatic x-rays (25 keV). The midline of the beetle lies on the right side of the video between the two coxae (large circular structures, bottom right). Collapse and reinflation of the air-filled tracheal tubes can be seen in the majority of the tubes in view. The smallest tracheal tubes that can be seen are about 10 μm in diameter; tracheoles (<1 μm diameter) are too small to be resolved. The circle and dark opaque spots the upper right are an air bubble and particles in the esophagus, respectively; note that they move anteriorly and posteriorly during the compression of the tracheal tubes. The white and dark spots that do not move with the beetle movement are artifacts due to the incident beam and detector system.

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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.

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 [8]):

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, mcith_08120902m01.gif, 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.

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.

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.

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.

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 [9]. Previous studies show that fruit flies (Drosophila melanogaster) [10] 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 [9] or hours [12]. At exposures greater than 2.5 kGy, insects do not recover, although it is unclear when death actually occurs [12]. Feeding patterns are affected after 600 (D. melanogaster) [13] to 1000 Gy (Bracon hebetor Say) [11]. In one study of D. melanogaster receiving doses of 600 Gy, metabolic rates were unaffected one day after irradiation [13]. 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 [14] and ageing [15]), 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.

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