References and Notes I J. Mendecki, S. Y. Lees, G. Brawerman, Bio- chemistry 11, 792 (1972); R. Sheldon, C. Jurale, J. Kates, Proc. Natl. Acad. Sci. U.S.A. 69, 417 (1972). 2. N. Sullivan and W. K. Roberts, Biochemistry 12, 2395 (1973). 3. K. N. Prasad, B. Mandal, J. C. Waymire, G. A. Lees, A. Vernadakis, N. Weiner, Nat. New Biol. 241, 117 (1973). 4. K. N. Prasad and A. W. Hsie, ibid. 233, 141 (1971); K. N. Prasad and A. Vernadakis, Exp. Cell Res. 70, 27 (1972); K. N. Prasad and S. Kumar, in Control of Proliferation in Animal Cells (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1974), p. 581. 5. H. Sheppard, G. Wiggan, W. H. Tsiem, Adv. Cyc. Nucleotide Res. 1, 102 (1972). 6. A 10 percent homogenate of chick brain in 0.32M sucrose was centrifuged (27,000g for 7 minutes). The supernatant was adjusted to The use of stable isotopes as tracers in biomedical and other fields is now of interest (1). In particular, the detection and localization of deuterium, the sta- ble isotope of hydrogen, would be use- ful. Since nonexchangeable hydrogen is contained in most organic molecules, deuterated organic compounds could be used as tracers in vivo. We show here that the D(T,n)4He reaction can be a sensitive indicator for the presence of deuterium and that plastic track detec- tors can be used to reveal the micro- scopic position of the deuterium (2). A general scheme for the localization of stable isotopes using nuclear reactions was proposed theoretically by Malmon (3). Conventional autoradiography (ARG) of radioactive nuclides is one of the most valuable microlocalization tech- niques available for studying the in- corporation and transport of molecules in living cell systems. The concomitant biological effect of the radiation on the living system under study may occa- sionally restrict the scope of informa- tion available from animal experiments, and certainly inhibits conventional ARG studies in human clinical investigation (4). For example, if one wished to trace autologous lymphocytes during biomedical studies of adoptive immuno- therapy (5), it would be preferable to use a stable isotope rather than tritium to label the cells in vitro before reinjec- tion. Tritiated lymphocytes could also be traced in the body after reinjection, 25 OCTOBER 1974 contain 0.02M KCI, 0.004M MgCl2, and 0.016M tris HCI (pH 7.8) and incubated with puro- mycin (125 ,tg/ml) for 20 minutes at 37°C. It was then brought to 1 percent sodium de- oxycholate and centrifuged at 100,OOOg for 90 minutes. This procedure causes complete release of messenger RNA from ribosomes (S. C. Bondy and J. L. Purdy, in preparation). The ribosomal pellet was suspended in 0.IM KCI, O.IM glycine, and 0.O1M EDTA, pH 9.5, and RNA was prepared as described in the text. 7. P. K. Sarkar, B. Goldman, A. A. Moscona, Biochem. Biophys. Res. Commun. 50, 308 (1973). 8. Supported by PHS grants NS 09603 and NS 09230 and by Foundations' Fund for Research in Psychiatry grant 70-487. We thank Dr. H. Sheppard of Hoffmann-La Roche for supply- ing R020-1724 compound. 25 April 1974; revised 19 June 1974 but they might be damaged by chronic low-level beta radiation. The deuterium is detected by bom- barding the sample with a triton beam to produce the D(T,n)4He reaction. The unique feature (6) of this reaction is a strong resonance with a peak total cross section of 5 barns at a low bom- barding energy of 160 kev and a high total energy released in the nuclear re- action (Q value) of 17.5 Mev. The full width at half maximum of this reso- nance corresponds to 2.5 ftm in tissue. The bombarding energy is so low that the yield of background alpha particles produced by triton bombardment of elements heavier than deuterium is neg- ligible. An alpha particle emitted in the forward direction has an energy of 4.7 Mev. This means that samples up to several micrometers thick can be used with an absorber to stop the incident triton beam. The alpha particles, after passage through a 6-,um-thick Teflon (7) absorber, have an energy of about 2 Mev, which is suitable for detection in plastic track detectors. In our experiments, an 8-ytm-thick cellulose nitrate plastic track detector on a I 00-pum Mylar backing for me- chanical support (8) is mounted on an aluminum slide (2.54 cm by 7.62 cm). A 6-utm Teflon film is fastened, wrinkle- free, to the surface of the track detec- tor. A drop of cells in suspension is evaporated to dryness on the Teflon. To minimize clumping of the cell speci- mens due to surface tension, the Teflon is coated with a layer of carbon 50 to 100 A thick by vacuum evaporation to complete the detector assembly (see Fig. 1). The slide is mounted in thermal contact with a metal block cooled with air circulating through a Dry Ice-alco- hol mixture during bombardment by a beam of 400-kev diatomic tritons (TT+) at the Research Van de Graaff acceler- ator of Brookhaven National Labora- tory. After bombardment, the detector is removed from the slide and etched in 6.25N NaOH at 40.0°C for 45 minutes to develop the alpha particle tracks as characteristic etched cones. The speci- mens and track detector can be ex- amined and photomicrographed before and after bombardment. Plastic track detectors (9) are very insensitive to other radiations, includ- ing photons, electrons, and neutrons produced during bombardment. The position and direction of the etched alpha particle tracks locate the spatial origin of the nuclear reaction to about 2 to 10 trm. The alpha particles which enter the track detector are detected with nearly 100 percent efficiency under these physical conditions. The effective reaction cross section, a, taking into account the target thick- ness and efficiency of the track detec- tor, is given by I1 xf = aW(X1 )G (nX7^XX2,X3)dn (1 ) where XI and X, are the thicknesses of the sample and of the absorber, respec- tively, X3 is the minimum detectable depth of alpha penetration into the de- tector, G is the geometrical efficiency, a is the nuclear reaction cross section, and the integration is carried out over the particle penetration distance -q (see Fig. 1). In the materials used and for the energies encountered, the range of an alpha particle is approximately pro- portional to energy. With this approxi- mation, the geometrical efficiency is (10) G (-o,X1,X2,X) - 1 I Xi1-X X2 XI 2 2 R -R2 R3J (2) where R,, R., and R3 are the calculated ranges (11) of reaction alpha particles in the sample, absorber, and detection plastic materials, respectively. The bio- logical samples analyzed were typically 1 to 3 [cm thick, the absorber was 6- ,um Teflon, and the minimum detectable 361 Deuterium Micromapping of Biological Samples by Using the D(T,n)4He Reaction and Plastic Track Detectors Abstract. A technique has been developed to micromap deuterium by using the D(T,n)-He reaction and plastic track detectors. Labeling of cells with subpicogram quantities of deuterium was demonstrated. The technique was used to localize human lymphocytes transformed in vitro. on May 15, 2013 www.sciencemag.org Downloaded from