Protocol

Author

Siegfried Jahnke

BACKGROUND

Radioactive nuclides (= radionuclides) can be used for labeling of various compounds of interest either to follow their fate in metabolism, for example to elucidate biochemical pathways, or to identify their position or distribution on different scales from subcellular structures, cells, organs up to whole organisms. What can be detected and how, depends on the radionuclide, its nuclear rather than its chemical properties and the method of detection.

• Upon disintegration some radionuclides release charged particles such as e^–(= b^–-radiation or electrons) or e⁺(b⁺-radiation or positrons) which strongly interact with matter and may be absorbed by thin film materials through which autoradiographic images can be achieved. Radionuclides suited for biological experiments are ³H, ¹¹C, ¹⁴C, ³³P or ³⁵S, beside many others.
• Other radionuclides are emitters of g-rays (= g-radiation or photons) which have much weaker interaction with matter than charged particles and generally need solid (scintillation) material to be detected. The emitted g-rays may penetrate tissues with less attenuation the higher the energy of the photons. Imaging can be performed with gamma cameras, in particular with SPECT (Single Photon Emission Computed Tomography) providing 3D representation of the distribution of radionuclides.
• A special case is the emission of g-radiation by positron emitting nuclides. When such a radionuclide disintegrates a positron is released which then annihilates with an electron (its anti-matter) resulting in the emission of two 511 keV photons in opposite directions (`annihilation radiation’). This property can be used to localize the origin of the decay by Positron Emission Tomographic (PET) systems. Examples of suitable radionuclides for biological studies are ¹¹C, ¹³N, ¹⁵O, ¹⁸F or ⁵²Fe.

Based on the specific biological question and the requested spatial resolution or invasiveness of the method, the appropriate radionuclide or detection technique can be chosen each of which has its pros and cons (Table 1).

Table1: Pros and cons of different methods to image of radionuclides

Imaging method	Pros	Cons
Autoradiography	High spatial resolution from subcellular to organ level; noninvasive measurement possible but limited; most radionuclides suitable for plant research may be purchased	Not quantitative due to high attenuation of the b-radiation; provides 2D imaging only; generally invasive and time consuming
SPECT	Noninvasive and quantitative 3D imaging on organ level; coregistration with NMR or CT possible	No plant dedicated instruments available so far; limited number of suitable radionuclides for plant research; costly
PET	Noninvasive and quantitative 3D imaging on organ level; a few plant dedicated instruments already in use; coregistration with NMR or CT possible	Cyclotron for production of mostly short-lived radioisotopes needed; spatial resolution limited; costly

IMAGING METHODS

Autoradiography Depending on the size of an object and the scale of resolution this method can be generally performed as microautoradiography and macroautoradiography. Microautoradiography enables tracer localization down to (or even below) cell level and has been used, for example, to study import of ¹⁴C labeled sucrose by wheat grains ¹ or 14C labeled photosynthates in petioles and stems of poplar ². The technique is both time consuming and challenging in particular when water soluble compounds should be localized. (Macro)Autoradiography is used to visualize labeled compounds in thin-layer chromatography, e.g. by labeling proteins with ³⁵S and separating them on TLC plates ³, but also applicable to detect tracer distribution in plant organs. Examples for this are autoradiographs of ¹¹C tracer distribution in tissue slices (Fig. 1) or uptake of ¹⁸F FDG by roots of Sorghumplants ⁴.

Figure 1: Autoradiograph of a sugar beet disc showing sectorial distribution of ¹¹C labeled photoassimilates in different rings of the beet about 90 min after ¹¹CO₂was administered to a leaf of the plant. The image was obtained with a phosphor imaging plate and reprinted from ⁵. SPECT Single Photon Emission Computed Tomography (SPECT) is a well-established method in medical applications since there is a large number of, in particular ^99mTc labeled radiopharmaceuticals available for routine diagnostic scans in nuclear medicine ^6-8. Typical radionuclides for SPECT are ⁵⁷Co, ^99mTc or ¹²³I which are rarely used in plant studies. For SPECT only radionuclides are suited which emit g-rays of energies not higher than 250 keV since insufficient collimation would deteriorate imaging quality. PET Positron Emission Tomography is a modality used to image positron emitting radionuclides ⁹. In principle it is constructed as a ring comprised of a large number of small opposing scintillation detectors. These detectors are continuously measuring radioactivity and each detected event is digitized with a time mark; if two events in opposing detectors are in coincidence, which in practice means within a time window of e.g. <20 nsec ⁵, this is regarded to be due to an annihilation event caused by a positron-electron interaction. When a large number of such events is monitored the position or origin of radionuclides which have disintegrated can be calculated by using dedicated image reconstruction algorithms. When a PET system is not equipped with a full ring of detectors, detection efficiency is lowered but imaging can still be achieved ^5,10. Commercial PET systems are constructed for medical examinations or animal studies, and the detector rings are therefore in general horizontally arranged. For plants, however, it is much more appropriate to use vertically oriented systems. So far there are only two PET systems described which are dedicated to plant studies: the Positron-Emitting Tracer Imaging System (PETIS) ¹¹and the Plant Tomographic Imaging System (PlanTIS) ⁵. PETIS is a positron scanner which enables 2D imaging and has been used already in number of studies with various positron emitting radionuclides such as ¹¹C ¹², ¹³N ¹³, ¹⁵O ¹⁴, ¹⁸F ¹¹, ⁵²Mn ¹⁵, or ⁵²Fe ¹⁶used as tracer of different chemical compounds. PlanTIS is a PET system which has been shown to monitor allocation of ¹¹C -labelled photoassimilates in three dimension of bulky plant organs (Fig. 2) or roots growing in soil in particular when coregistered with NMR images ⁵. These systems are not only used to static imaging of tracer distribution in a plant or plant organs but to detect also temporal changes of tracer distribution enabling calculation of long-distance transport parameters such as transport velocity or lateral exchange rates of translocates in the phloem ¹⁷.

Figure 2: Images showing the distribution of ¹¹C -labeled assimilates in a radish bulb coregistered with MRI (grey) and PET (coloured). Left panel, side view of a radish bulb of which the lower part was growing in soil. Right panel shows a virtual cross-section through the bulb, modified after ⁵.

REFERENCES

1. Fisher and Wang (1993) A kinetic and microautoradiographic analysis of ¹⁴C sucrose import by developing wheat grains. Plant Physiology 101: 391-398.

2. Vogelmann, Larson, and Dickson (1982) Translocation pathways in the petioles and stem between source and sink leaves of Populus deltoides Bartr. ex Marsh. Planta 156: 345-358.

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4. Hattori, et al. (2008) Incorporation and translocation of 2-deoxy-2-¹⁸F fluoro-D-glucose in Sorghum bicolor (L.) Moench monitored using a planar positron imaging system. Planta 227: 1181-1186.

5. Jahnke, et al. (2009) Combined MRI-PET dissects dynamic changes in plant structures and functions. Plant Journal 59: 634-644.

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7. Biersack and Freeman, eds. (2007) Clinical Nuclear Medicine. Springer: Berlin.

8. Vallabhajosula (2009) Molecular Imaging. Radiopharmaceutical for PET and SPECT. Springer: Berlin

9. Phelps, ed. (2004) PET: Molecular Imaging and Its Biological Application. Springer: New York.

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12. Nakanishi, et al. (1999) Visualizing real time ¹¹C methionine translocation in Fe-sufficient and Fe-deficient barley using a Positron Emitting Tracer Imaging System (PETIS). Journal of Experimental Botany 50: 637-643.

13. Kiyomiya, et al. (2001) Real time visualization of 13N-translocation in rice under different environmental conditions using Positron Emitting Tracer Imaging System. Plant Physiology 125: 1743-1753.

14. Kiyomiya, et al. (2001) Light activates H₂¹⁵O flow in rice: Detailed monitoring using a positron-emitting tracer imaging system (PETIS). Physiologia Plantarum 113: 359-367.

15. Tsukamoto, et al. (2006) ⁵²Mn translocation in barley monitored using a positron-emitting tracer imaging system. Soil Science and Plant Nutrition 52: 717-725.

16. Tsukamoto, et al. (2009) ⁵²Fe translocation in barley as monitored by a Positron-Emitting Tracer Imaging System (PETIS): evidence for the direct translocation of Fe from roots to young leaves via phloem. Plant and Cell Physiology 50: 48-57.

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