UNIVERSITY OF MICHIGAN Ann Arbor REPORT HIGH RESOLUTION DETECTION OF RADIATION By HENRY JP:..G-OMBERG, Principal Investigator MARVI.N lASS WILLIAM KERR REUBENA RABEZZANA MILTON SCELESINGER GEORGE Cc TOWE CAROL UHLENDORF PROJECT AT( g I -) -O70 -No January, 1954

...:-.:::...: i.. -. *???'

TAHBE T OF CONT.ENTS Page LIST OF FIGURES... B -iii INTRODUCTION... 1 RESEARCH RESRETTS 6 I. INSTRMENTATION - THE BETA-RAY MICROSCOPE..... 6 II ~ AUTORADIOGRAPHY A. Wet-Process Autoradiography Applied to Metall.r gical Studies 9.............. B. Dry Film Version of the Wet-Process. o. 12 C. Com mercial Stripping Film.m..........e 14 III. NEW TECHNIQUES A. Modified Wet-Process Film for the Electron Microscope 17 B3 Radiation Induced Polymerization...be.....e 1.8 C. Spark Discharge Tests.............. 24 IV APPLICATIONS A. The Biochemical Synthesis of Melanin Pigment by Cells in Tissue Culture b..e... b... 27 B. Autoradiography Applied to Metallurgical Problems 29 C. Studies of Incorporation of P- 2 in Onion Root Tip Cells 29 PROPOSED RESEARCH I. Be ta-Ray Microscope........... 3 II. Autoradiography................. 34 III New Techniques.. 1.... 1..........-.4 IV e.Applica-tions..... 9. e. b.... 35 REFERENCES................. t. 6 ii

LIST OF FIGURES Figure Page 1. Coincidence circuit for the Beta-Ray Microscope 7... 2 Optical System and phototube holder for the Betaa-Ray Microscope, I...... o s e. ~ ~ 5- Autoradi ograpshs of the same ce'mentite crystals at different exposure ti-mes......... 11I 4. Bright field and polarized li.ght photomicrogra)phs of carbon -l.4 in iron....................... 1 5. Wet-Process autoradiograph of rad.ioactive rait thyroid... 13 6. Response of dry collodion film to radioactive thalliuma. 1..5 7. Stripping film autoradiograph of mouse skin tissue incorporated with casirbon-14 Dopa.............. 1^ 8. Radioactive thallium source - mouined on edge - used for radiation induced polymerization.......... 20 9. Radioactive thallium's source mounted flat in luIcite 20 10. eSelective formation of plastic over radioactive thallium edge 22 1.! Platinum-nic;kel-63 wire before and after application of plastic (TEGMA)......a............. 23 12. Spark Discharge Counter Assembly........... 26 135. Autoradiograph localiz?1ing carbon-14 t- melanin areas of chick skin cell.s.2 14- Stripping film autor.adiograph showing grain "boundary diffusion of radioactive nickel into iron 0.. g... 0 15. Stripping film autoradiograph showing diffusion of radi-oactive nickel into iron. a...... 30 16. Stripping film autoradiograph of copper containing radi.oactive bism.uth.....l...... 31 1t7 Stri.ppi-ng f ilm autoradiograph showing graino bou ndary diffusion of radi.oactive bis:muth into copper.o. @ a 3 1 111t

PROGRESS.:REPORT HIGH RESOLUTI.ON DETECTILONi OF RADIATION Pro ject AT( 11-1 ) - o70-.No l. INTRODUCTION The major purpose of'the Unliveirsity of Michigan Project on High Resolution Radiation Detection is the di scovery, developmentl and application of new methods o I ing ocopc urces of fradia tion. These sources mayy'be incorporated in organic systems such as radioactiv'e phosphorus used to tag specific cell. bodies, or in inorganic systems such a s the tagged carbon compounds in steel, In keeping with this purpose, research studies have been continued or initiated in the following areas, (1) The Radiation Microscope This is a new device, making possible -the direct measurement of rad.ioactivity wiithin the lfeld of an optical microscope^ A given area of a suitable specimen can'be examined optically at 1000 diameters and the radio activity of the areaa under observation determined directly and instantaneously. Both observat-ion and radiLoacti-vity meascuremen.t (by scintillation techniques) make use of the came opti.cal system without moving the object bei.ng studied so tha.t there can be no question as to egister and correltion beteen the eld obrved and the radioactivity measurement. The first and second of the microscope models have now been buiLlt and tested. successfullyat at 970 diameters., ith activities of less sthan one microcurie per square centimeter (Cobalt-6o). A third model, suitable for use by laboratoIry personnel concerned with tr acert research and not trained in special` insotrumentation tec'hniques is no w under consitruction0 (2 ) Au.t oad i o graphy The wet process of autoruadiography yin which a.radiation sensitive

layer is created chemically- directly on the surface to be studied, has been carriedd as far as it would appear worth going for its use as a general ttechnique Some advances have been -made in imnproving resolution (now 5 microns or better1 ) and sensitivity, but since the rcent development of the commercially available stripping films with comparable resolution, there is Iess need for the wet process technique as sucho There are still special features of the wet process such a8s a) Very low background fog - as low as one part in -40 compared to stripping fi m. b) Extreme mechanical stability and freedom from slipping, peeling, reticulation, etc. The sensitivity, however, is somewhat lower and the chemical stability is much lower than that of the stripping film. Where short exposures (24 —48 hours) are possible there are advantages in the wet process'which can outweigh the technical difficulties of its application However regardless of its use as an autoradiographic method to be used in connection with optical microscopy, it hars opened a new possibility of autoradiography combined with electron microscopy. This is discussed further in section III A, (3) New Techniques a) Modified Wet Process Film for Electron Microscopy The use of tracers in systems requiring electron microscopy for observation has been a continuing challenge. An approach to this problem has been made through the possibility of sensitizing the collodion screens similar to those used for supporting electron microscope specimens. The specimen preparation can be activ.ated applied to a sensitized collodion screen and left there long enough to cause some direct exposure in the screen. The screen and preparation are now treated to remove any unexposed detector material (silver halide) and then the screen and prepa-raton arse studed n an electron microscopee

Because of the excellent- scattering power of sil'ver, a very small a-mount in the support screenh-rought down'by the original tracer ra.dioactivity, should show up well atnd corr.elate with the specimene resting on it. Using techniques based on the original- wet process:s and the dry-film modification described in our last report, prelimin.ary studies have been made on the formation of extremely Jthin sensitized collodion screens Results to date are given in secti.on III A. This work is being carried out in co-operation with DPr. 0 I- Bergold, of the Laboratory of Insect Pathology, Canadian Department of Agriculture, b) Plastic Pot.ymerizatfion as a Method of Detecting Radiation Sources. Some monomers bwhich are stable at room temperature and under ord inary light conditions wil.l polymerize when subtjected to ionizing radiation. These monomer. as liquids' or as solids in solution, are capable of penetrating tissue blocks of moderate dimensions. If significant sensitivity to ionizing radiation can be developed. it should be possible to prepare not only two dimensional maps of rad ioacLtivity by coating active surifaces. but three dimensional. mps by local polyme.erization of the monomer- due -to entra ined radioactivity in a ti.ssue blocks Early coat3ing tests, on activated thallium specimens were very encouraging, but he- -the results had. to be discarded when -it was found that -thallium metal could be catalyzing the polymerization chemically. A new series of tests have been started, using X-rays to determine the best conditions for inducing polymerization. These re reported in section I B. of the report and indicate that under proper conditions, adequate sensitivity for tracer studies can be achieved. The results of these tests will be used to guide new tissue impregnation tests. c) Spark Discharge Tests IThe ionizat r ion pr> oduced by a particle leaving a'adioactive surface can, under proper conditioLns' be u.sed to initiate a spark discharge. If this spark can be made to strike the surface in the neighborhood of the ionized reg on

.s 4k - produced by the partic le, i.t should ma rk or burn the suirIacec so that it can be identified later Thus, a microscopic burned spot should mark a zone of radioactivity. Preliminary tests using this principle have been ade without conclusive results' The conditions under which the spark is initiated primarily by the presence of an ionizing particle have not yet bee n achieved, Because of the desire to look into some of the other processes descr,iobed, work on this idea has been temporarily suspended Description of the tests is given in section III C. (4) Application and Co-operative Problems As a continuing test of our methods and ideas we underttake co-aope ative research with other MUniversity groups requiring high resolution radiation detector techniques, as well as carrying out studies in ouri own). laboratories' The principal problems so studied during the past year were: (a) Continued study of melanin synthesis from radioactlive precursorsz Autoradiographic tests lend no support to the hypothesis that tyros e an tryptophane are not melanin precursors' New tests have been mde using C-14 labelled Dopa as the substrate. These tests, carried out in co-operation with Dr. Clement Markert, are reported in more detail in the body of the report. (b) Continued co-operative research with Watertown Arsenal Metallurgy Study Project~ While no AEC funds were spent directly on this work, it was done in our laboratories, using the same techniques and Phoenix Project equipment mad available to the AEC research project. Detailed studies on the diffusion of nick:el into steel, bismuth into copper, ni.ckel in-to copper, anld on. the formation of iron sulfide in iron have been made. Some sample autoradiaographs and some description is incorporated in the body of -the report in section. IV B (c) Uptake and Loca liza.tion of P-32 in onion root tips in correlation withi their mitotic cycle ~

This is being carried out as an independ.ent study problem'by our own. laboratory personnel. A similar problem, using Vicia faba seedlings. was reported. by Howard and. Pelc (5). The status and resul ts are given in section IV C. The activity of the past year also included lectures on autoradiography, given at Purdue University, and the University of Rochester, and also participation through lectures and discussion in the Oak Ridge Instlitute for Nuclear Studies' intensive course in autoradiography this past summerr In addition paepers have been presented on the Radia.tion Microscope at the Nuclear Engineering Conference at Berkeley, California^ September 1953., and the Scintill ation Counter Conference in Washington in January 1954. A report on the microscope was presented in Science, Volume 119, No. 3080, in January 1954Two doctoral theses were published during the past year. These consisted. of the Radiation Microscope (Dr, William Kerr) and Metallurgical Application of Wet Process Autoradiography (Dr George C Towe)~ Two more will'be completed this year.

RESEARCH RESULTS I. BETA RAY MICROSCOPE The initial. development of the Beta Ray Microscope was discussed in a special report (Dr, Kerr's thesis) submitted in Septeerb ei, 19535 On comple-'tion of this initial phase, it was decided to expand our activ1ity in this area in order to bring the development to fruition with minimum delayr What follows i.s a report on work started since October 1, 1953 Work was'begun orT improvements in the micr0oscope along two general lines: (1) Improved mechanicall packaging, (2) Increa.sed flexibility of the phosphor-optical system. The improved mechanical packaginng has been aimed both at the electronic circuitry and. -the photootube-optical systemr A coincidence circuit (Figure 1) has been designed and built which is much more compact than that used ir..n the original model The preamplifier-photottube mount has been redesigned to allow the photocathode to be more easily al1igned with the optical system (Figure 2). An improved -version of the optical system has been constructed;which will make the switch from visual viewing to isotope detection easier to accomplish~ One revision included an attempt to light the specimen by light transmitted through the objective. At present this attempt has not been successful, apparently becauose of reflecttion at the rear surface of the objec-tive. Further investigation is being made. Design of a background shutter is being consideredu Design of a complete ighiLt tight system is continuing. Tests are being'tun on plastic phosphors furnished by a comnme-rcial upplier. The phosphor was supplied in small sheets 7 microns thick Those tested exhibit aaen effiiency in detecting betas from ThallIum-20 of about 50 per cent of that of anthracene in the am thickness' Investigati on of the grow-ing of thin anthracene crysttals by ubli mat~ion

Fig. 1X New Coincidence Analyzer is at left. Unit at right is old Coincidence Analyzer with its power supply. Fige 2~ Optical system and phototube holder for Beta-ray microscope

from a large crystal and condensation of the resulting vapor is being continued A new dense plastic phosphor has been secured from Dr~ Cowan) at Los Alamos and will be tested as soon as the technique of forming thin films has been mastered. Both RCA and Dumont promise that a, sall diameter hea.d.-on cathode phototube will be availa.ble in the nea future. This tube should simplify the arrangement of the phototube-optical system, Consideration is being given to the design of a light tight optical system to eliminate the need for enclosing the phototube-optical system within a light tight box.

II AUTORAD OCRAPEY A. Wet Process Autoradiography WMr George Towe completed a detailed s tudy of the wet process technique of autoradiography, and his results were subsequently published in a doctoral thesis. An abstract of the thesis follows: Wet-Process Autoradiography Applied To Metallurgical Studies The purpose of this investigation was to (1) adapt wet-process au.toradiography to metallurgical studies, (2) evaluate and improve the process^ and (3) obtain metallurgical information using this high resolution autoradiographic method, Wet-process autoradiography involves the prepara/tion of a collodion. film on the surface of a radioactive metallographic sample. The collodion film contains soluble halides which Torm silver halides when immersed in a silver nitrate solution. The insoluble silver halide, held in place by the collodion film, is subject to ionization by the radiation from the metal samtple. Following the exposure, the radiation affected silver bromide in the collodion emulsion is photographically developed and fixed. The developed silver grains are located directly over the radioactive region.. Physical development'is used with the wet collodion process, The developing solution must contain i silver ions as well as a reducing agent. The silver: of the photographic latent image nucleates the reduction of the silver ions in solutionA It is possible to control the developed silver grain size by varying the time and temperature of development and by changing the composition of the developer. The developed silver grains are frequently about one micron in diameter. Since the photographic chemicals must be kept from contacting the metal specimen, it was necessary to develop suitable protective layers. Gold and silver vacuum evaporated films and many plastic materials were investigatedA For high resolution autoradiography, this protective layer must be very thine The Vinylite layer recommended is less than one micron thick. High resolution autoradiography requires the use of a thin heavily loaded detecting photographic layer in close contact with the sample surface. The -thickness of the collodion "emulsion." is controlled largely by the amount of solvent used to dilute the collodionm Suitable layers, containing about 90 weight per cent silver brom.ide, were prepared approximately 4 microns thick, Preparation and processing of the detecting layer were the subject of close study Sensitizer s were added with the hope of improving radiation sensitivity The te mperatures of the several. solutions were varied from room temperature to 20 C. Photographic background fog is minimized when the autoradiographic exposure takes place a, temp eratures near 20 C. A fog level of less than 1000 sxlver grains per square mxillimeter is readily achieved.6 This s lomwer by a f actor of perhaps 10 r 100 tha e fog level btaine with commercial emulsions

10 A comnrparison is made of results obtained with wet-process autorad io.graphy and with a coml'lercial autoradiographic emulsiono The rmetallurgical parti cul ar are reviewed, Under favora able conditions of suitable radiation and propemr u enmulsion processing, a:resolution of better th an. ten microns can be obtained with wetprocess autoradiography. The autoradiograph i.s examined in place on the metallurgical spec imen at magnifications as high as 1000 X, Standard radioactivre samples were prepared including carbon-r carburized iron and steei nickel63 electroplated on platinum or siiver, a nickelDsilver powder mixture containing nickel-63t, and a copper-antimony alloy containing copper-614. A complex alloy (type N-155) conta ninr tung ten-l185 as also prepared The tungsten distribution in this alloy was unknown. Figure 3 illustrates an experiment noting the sensitivity of the wetprocess techniq ue Various experiments were carried out during the past year to attempt to increase the sensitivity of -the wet process and to decrease fog level. The description and results of these projects are listed as follows: (1) Studies wxith the pyrazole derivative of L, Jenny, as noted in our last report, provided no great difference in its ability to sensitize the system. (2) Tests with the anti-foggant, 6-nitro-benzidi mazole, resulted in smaller grain size, but little or no good anti-fogging action was detec-tble9 The wet process as it exists now, is useful for biological and metallurgical samples of high activity. The process is relatively s.imple to carry out (I), but extrerme conditions of cleanliness are required, particularly when working with metallurgical samples' F urthermore, exposures are limited to about 48 hours. The process, however, has the advantage of variable grain size and no danger from the film slipping awary'from the specimen. This technique has been used with some success in our laboratories in,carrying out routine autoradiographic studies f Samples of radioactive thyroid and meta,l samples "bearing radioactive materia-ls gave successful autoradiogra.phs (Figures 4 and 5)- Further work on development of the wet process has been

Alllliilllllliil IllIIlIlIilII I l IlllllDllll''lillI~l-l l' ~jI ]4 l ll ~l I llll — I II 4 H I' PS il-i~lllll-lli~illllllli1tIiI^ nI I —~-~ —I"I I~, —I I IF lll'-ltl^"l^-:l c-F -Ft —F-4c:t:.-.-~II II ^~~ ~~ ^ 0 0 ^n N~^"l^ — 4^1H S ^Sc~ I CIII CDI I Ic II I4I I4. c-I HIIOIII CD rn " — -— " — 1 dI II-.:, -. I tI I IIr YI ^ ~ ~ ~~~~~ ~~~~ - I —-.. —-. —~::~.~. -, I~-. I,~% ~~-. CIDI IH D D I H,~~~~~~, O d— 11-8S^ I I- 1 1 1-1 -1..~...~.11-.~I: I..`, ~ I OII OHI, iill~lll::llllll~lllll.Il@^ll.gll,4 C)1.~,I,Ii~:i:~ "1 "r ~: H' - ~.. ISF - O A~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ cF - 44 Xe ~, ~:,,I,.,, 1:.,I~ ICD D x..I., I.~, - ~.1,I I. 1 I I.,..,,. CO.. ~ I:~:~ ~~~~ AT," "ll I..I~ -I-.: +-..~.1 ~.I

- 12 curtailed, and research has been primarily devoted, to working with new technioues BT Dry Film Version of the Wet Process During the past year further rork was devoted, to adapting the wet process autoradiography to a dry film. Carried out by Mr. Marvin Hass and described. in part in the last annual report, this technique consisted of coating the collodion wet plates with a preservative so as to render them stable to dryness., This process uses the solutions of bromi)dized collodion and silver nitrate described in the wet process (1) -he preservative follows the formula of H. 0. Klein in "Colloidion Emulsion" A. W Penrose & Co. Ltd. 2nd. Edition, London, 1910, p. 110. It consists of 6 gramslsugar, 6 grxms gum arabic, and 1 gram Tannin, dissolved in 220 ml of distilled water. The procedure is outlined below: Emulsions are prepared on a coated. slide in a manner similar to the wet process. The slides are immersed in silver nitrate for one hour, then carefully removed and immersed in cold distilled water for two minutes' They are next dipped in fresh cold wash for five minutes, after which the slides are placed in the cold preservative solution for at least five minutes, They aJre then removed and dried at room temperature for thirty to forty-five minutes. During exposure perio they are tored in a cold climate in the dark. Development of the slides is carried out with dilute D-19, 1., for thirty seconds, The slides are then dipped in the acid stop bath for a few seconds, followed by hypo for a few seconds, followed by acid stop, or preferably water. The clearing time is quite fast. Figure 6 shows the response of a film prepared in this way to radioactive thallium sour~ces ~ Attempts;were made to sensitize this dry version with agold sensitizer, ammonium aurous t iocya.nate (2) ~The anti-foggant, 6-~nitro-benzidima zale.

.13 - Bright field Polarized light Fig. 4-. Autoradiographs showing silver grains over radioactive carbides' Carbon-I4 in iron, Picral etch. XiOOO m~ m - M - ^~'^^^^^^^^^^ W Fig. 5. Wet process autoradiograph ofl radioactive rat thyroids Localization of radioactivity is noted in the colloidal tissue of the thyroid foliicle^ X~tl5

was also atried. Neither of these proved to oe successful. While this process is a satisfactory adaptatlion 0of the twet process it is felt that existing commercial stripping film and liquid emulsions are equally as sensitive and capable of the resolution auttained here9 in addition to being iore reliable and relatively easier to work with. Therefore, further work along the line of that described above has been curt'ailed C. Commercia.l Stripping Film In the -application of autoradiography as carried out in this laboratory-, we have relied on the permeable base autoradiographic stripping film supplied by Eas.tman Kodak Company, We have found that the following method of application results in good autoradiographs: (1) Slides to be used are dipped in 2% Saran, dried under an. infra-red lamnp and then baked at 1500C. for half an hour. (2) Tissue sections are mounted on the Saran coated slides, stained by routine procedures, dipped in 2% Saran again', and dried under the infrared lampe This method of drying avoids any clouding of the Saran as usually occurs on humid days. This technique was found to be superior to a previous method in which the slides were dipped in Saran and dri ed in vacuo (5) The activity of the specimen is determined by use of an end window Geiger counter t (y) The stripping film is applied, and the slides stored in black bakelite'boxes (sealed with black electrical tape) in the refrigerator for the duration of the exposure time, The exposure time is determined pri - manrly by running severr specimens and developing them at various times' Through experience we have been able to corre].ate activity as determined by Geger counter wth exposure time for P-52 aMCA-i

Fig. 6. Response of dry collodion film to radioactive Thallium discs placed immediately over the film. Exposure time was 12 days. Activity of the discs ranged from 0 03 to 10 microcuries/cm2 Some chemical fog is observed in the upper half of the slide tissue incorporated'with carbon -) I Dopa Pic75 microns ~ X415

(5) The slides are developed, dried, and cover slips applied using Canadian Ba lsarfat In some cases the protective layer of Saran coating (steps I and 2) may be eliminatedi'but the processing of control specimens Is essential under these conditions The difficulty with stripping film peeling off the specimens during exposure, as noted in our last report, seemi to have been overcome. The stripping film is floated on a 5% solution of glycerine, allowed to stcnd for a minute, and then is picked up on the slide The film seem to dry more smoothly, although somewhat more slowly, than when pure water is used., We have stored film on slides for two months with this technique withbut the film peeling off the specimen. The stripping film still hasa tendency to slip during the processing - and specimens must'be carefully examined for indication of this action A good example of this is seen in Figure 7. Some work was also attempted using commercial liquid emulsion. Exceedingly high fog level was noted in these cases.

. 17.. III. NEW T-HNIQUES GENERAL In the investigation of new techniques of high resolution autoradiography we are seeking the following objectives: (1) The system should require a vanishingly thin film. (2) It should respond to high energy particles while having low sensitivity to light photons and chemical artiacts (3) It should have high chemical stability in the unexposed state. A- A Modified Wet Process Film for Electron Microscopy Late in the year we received a communication from DrL G. H~ Bergold, of the Laboratory of Insect Pathology of the Canadian Department of Agriculture) inquiring as to the possibilities of adapting the wet process to a dry thin film suitable for work with the electron microscope. As presently conceived, the idea is to sensitize, with silver halide, the collodion screen used to hold the sample when placed in the electron microscope. The sample, containing a radioactive tracer, would be placed on the sensitized screen for a sufficient exposure period in the dark~ The screen may be developed but this will be done only if the amount of reduced silver produced by direct radiation action is insufficient after fixing to cause electron scatter. "Fixing" with or without development is done to remove the unreacted silver halide. The screen is then placed. in the electron microscope and the scattering of electrons by the silver particles correlated with the specimen. This then would be the electron-microscope autoradiograph. The initial requirements for such a scheme consists of obtaining a very thin film highly loaded with a uniform concentration of silver halide~ Thin films of Angstrom thickness can be obtained by applying a drop of 2% parlodion in amyl acetate solution on water ~ By dissolving cadmium iodide in the amyl acetate and using water containing the silver nitrate, a thin film loa ded -with silver halide crystals is formed. We have worked with various concentrations

-.1.8 - of silver nitra-te in water arid cadmium iodide in amyl acetatn We Ihave al t done some preliminary work varying the tem perature of the solutions, and using alcohol-water mixtures of the silver nitrate. Thus far, our best films have been. formed using the following solutions: (1) 5% cadmium iodide in awyl acetate plus 4% parlodion in amyl acetate (35:1) (2) 5 grams silver nitrate in 20 ml of distilled water~ Ten drops of solution (2) are placed in 25 ml of distilled water in a watch glass and mixed. One drop of solution (1) is placed in the wa.ter. The filrm is allowed to dry, washed with water, and picked ip on a. glass slidei' Results to date have not produced a high degree of uniformity^ nor a sufficiently high density. However, the prospects of this type of dry version of the wet process appear quite promising as a potential method for electron microscope autoradiography, and research is continuing on this problem. B. Radiation Induced Polymerization One approach to a method of high resolution detection of radioactive material initiated during the past year war the investigation of monomer-polymer chemical systems that could oe influenced by high energy radiation. Considerable evidence exists describing the effects of high energy radiation on the initiation of polymerization. This reaction proceeds as a result of the free radicals formed under the action of the high energy radiation. Of particular interest was work performed by Schmitz and La'wton (5) in which they described a monomerr-polyrner system in which the polymerization initiated by high energy electrons wa found to'be dimensionally specific. Although relatively high dose rates are reported, for such reactions, it ws felt that I uch a sstem might prove poten tially adaptable for a means of high resotution autoradiogr aphy. The monomter, tetra ethylene g lycol dimethacrylate wcas chsosen to wor k ~with at the outsets This material polymnerizes by cr'oss-linking, and its di

19 tunc'tfional grouping lea. d s to a greatGer suscep)tibii..it..ity in poly erizat ion as c mi pared to monomers containing only one double bond. Furermore t he polymer i.s insoluble i-n the monomer and would thus "dissolve" out- This miateri.al a.is obtalned from the Monomer-Polyamer Company amnd washed free of irnnhibitor by- runniiing the material through an activated Alumina column~ Other ma.terials which are being studied include ethylene glycol dimethacrylate, methacrylam. ide, a <nd EIN-' methylene bis-acrylam:.i de. The latter two are solids at room temperature. The others are'viscous liquids which polymerize to a clear solid materal when heated The radioactive specimens that were used in the initis-l- experime.nts consisted of thal lium 2(o electroplated on copper plates and supplied by Harwell Laboratories in England Sma.ll sections of these plates were mounted edgewi-se in baklelite blocks along with a strip of plain copper to act as a control (Figure 8)~ The strips were apI roximr-ately 300 " wide and the thallium layer was of the order of o30-40 p in width Samples of activities ranging from u.005 itc/cm2 to 100 p.c/cm2 were used.. In later work, discs of these sources were punched out and mounted flat in lucite mBiolds (Figure 9). Other sources utilized consisted of small pieces of uranium in bakelite, and metallurgical, mounts of nickel 63 and. C-_it Thyroid tissue containing 1-1351. was used as a biological specimen for this work. The first experiments were carried out by merely spreading the monomer over the thallium samples that had. been polished and cleaned. The samples were stored. at room temperature and pressure. After varying exposure times, the unpolymn.erized material was washed off with benzene. The initi.al results of this technique indicated that some reaction was proceeding - as indicated by a cloudn like formation over the radioactive edge of the metal strip after about 6-8 hours exposure (Figure 10) These results however, were sporadic and inconsistent with respec.t to thte varying ac.tivities of the s pecimen. Furthermore

Fig. 8. Typical radioactive Thallium source — mounted on edge -used in the first experiments with radiation-inducted polymerization. The strips here are of Thallium 204 vwith activities from 0.03 to 30 microcuries/c-m2 A plain coppert strip serves as a control. X2 Figa 9 Radioactive Thal lium source mounted flat in lucite Metal lies directly under surface X2

21- no results could be detected when the uranium sarmples were used as sources, Ono successful reaction was noted for a nickel 63 specimen (figure 11), but th"i snot reproduci ble Negative resul ts were also forthcoming when placing -131 thyroid tissue in the monomer and, also when spreading -the monomer over radioactive 1th -hyroid tissue sections whTich had a. rela tively h-igh specific activity. Attempts were then made to first, obtain consistent results with the thallium samaples and second, to determine whether the reaction noted was chemical rather than radiation-tnduced in natu're A variety of techniques and solutions were tr-ied in seeking a consistent reaction. These included working with wetting agents, increasing the viscosity of the monomer) and exxposing the material at low temperatu.res and under nitrogen. To date we have not'beer.n ab.L.e to get consistent results. Two approaches were undertaken to settle the question of chemical action, one was to use a protective layer over the sources the other was to run our tests with stable materials A protective laye'r3 proved t-o be difficult to achieve as the monomer diss olves aw a-y most materials that have heretofore offered acceptakble protection. Recently we have found- that mylar film and polyethylene film appear impermeable to the monomer, but these mater ials must be molded to the surface or,t.he liquid monomer seeps und.er the laver. Experiments with these protected sources are now in progress. Late in the year we obtained stickis of stable thallium and mounted some of this metal in bakelite Results of the monomer placed over this specimen show a reaction similar to that seen over the radioactive material,'but here again results have been sporadic These inconclusive results suggest a more detailed a me dtudy o the action of solutions of this monomer under vatious doses of external ra-diation~ We ar. e now workThing with solutions oftis h monomer preparted, in vials and placed

* 22 - Fig, lO1 Top: Selective formation of plastic (Tetraethylene glycol dimethacrylate) over radioactive thallium edge~ 6Exposure' was for 20 hours' followed by washing in benzene. Dark field (Ultrapak) X780 Bottom: Same specimen before application of plastic DTrk field (Ultrapak) X780

.23 Fig, 1 a) Platinrumi-Nilckei-63 wi.re specimren before application of moTnomer (TEGMA)} Specimen iS made up by plating nri..kei-63 on platinum wire, f-ollowed by a protecti. v e silver plate Dark field (Ultrapak) il tumination XlOOO Fig, 11 b) FormLation pf plastic ( TEGMA) over section of Platinum-Nickel-63 wire' Exposure"' was for six days, followed by benzene wash. Dark field (Ultrapak) illumination. XIOO0

24 under various atmospheric- conditionss Addition of chemical sensitizers is also being investigated The other onomers noted a:bove are al so being studied under these conditions, in an attemptr to determine the optimum conrd.iti.on - s for raadiationinduced polymrerization It is then intended to work with the metalJ. urgical and'biological materials using the monomers under these conditions C. Spaprk Discharge Tests Another potential technique for obtaining hi igh resolution autoradoiograp hy wa investigated during the past year. This work was concerned with adapting a, spark counter to obtain au-toradiograp.hs Work by K. S. Lion (4) at M. IT. showed that photographic sensitivity could be affected. by anr eIlectritcal discharge. This method essentially make' use of an electr-ical. discharge which is tr iggereu3d, by incide-nt radiation and acts upon a phiotographic emullsion, For adapting this principle to our work, we had pl.anned -to use the emitting radiation from a. radi.oactive material -to initiatee h lectrical discharge which;h could 1then effect some detection device such as sparking a smnall hole in a thin paper For the preliminary experiments a piece of photogtraphic paper was used and placed over a radioactive thallium source These were placed in an ai -rti.ghlt lucite' box fitted with brass plates - one of them adjustable for varying the plate distances (Figure 12)., Experiments were run at various pressures and voltsages, in an attenmpt to determine whether sparking would occur selectively to "the radioactive SOur7ce These trials proved unsuccessful, as our control. specimens were of a different material a nd, hence, different in their threshold, potentials for spark initiation. In addition, we encountered considerable dificulXty wit.h respect to field nonuniformity arising from irregular surfaces and. edge effects.

25 Considerable further work needs to be done here w ith 3regard to improving the counter itself and finding a suita ble means of detecting the spark action a Research was temporarily interrupted in order to devote fulls attention to the polymerization reactions HEowever, it is planned to go ahead again with this project in the near future.

26 Leads to Lucite Gasket Anode 2ti_~~~C~~~~~.., Lock Nut Filmn for a" ",/ \./,/?/ /M/ ~Ac. —- D etecting Spork Action / _^X^ ^, N =ItInsulated Spacer Radioactive a Control Metal Specimens Mounted in Baklite Fig, 12a. Sketch of Spark Counter Fig. 12b Spark counter disassembled showing lucite containers plates, thin film, and photographic paper. The latter two were used for detection of spark action.

IV. APPLICATIONS A. The Biochemical Synthesis of Melanin Pigment by Ce1ls in Tissue Culture. Work has been continued on the cooperative project with Dr, C.lement L. Markert, Director of Phoenix Project No. 56, and Dr. Glenn Fischer The objecrt of this study, as outlined in last year's report, is to determine the manner in which genes produce their effects during development It is hoped. that'by identifying partiAcua.r substrastes used in -the synthesis of melanin pi. gments by cells of different genetic makeup, correlation ca-n be drawn between gene structure and enzyme specificity as revealed by the nature of the substrate accted upon. Las t year's report outlined the experiments usling ra dioactive. tyros'ine and tr rptophane in tissue culture's cc ntaining cells which synthesized melanin. Results noted by autoradiography indicated that these substances were not precursors However an autoradiograph showing resolution of the radioactivity to the parts of the cell containing melanin was obtained (Figure 13) Readioactive tyrosine was incubated with tyrosinase under conditions which should lead to an accumulation of oxidized intermediate products on the way to melaninT format ion T-is radioautograph was obtained when. uch a solution -.was cult ured with chick skin. The autograph was probably due to a precursor of melanin produced by the enzymatic oxidnation of tyrosine. Durming the past year, tests for melanin synrthesis have been carried out using C-14 Dopa as the substrate. Experiments have been completed using mouse s kin in vivo and in vitro frog embryo^ chick epidermi-s and skin, and piogmented layers of the chick retina. The procedure follows that described in last year' s reports Results to date obtained by autoradiography suggest that Dopa is not a precursor of m.elanin Research is continuing on'the problem,

28 Fig. 13 Autoradiograph localizing carbon-14 to melanin areas of chick-skin cells. The results seen here are believed to be due to a nonmelanin product of tyrosine oxidation by tyrosinase. xg60

B, Autoradiography Applied to Metallurgical Problems The autoradiographic technique has also 1been widely utili.ze.d by groups of the University studying various metallurgical problems We have been able to offer advice and assistance 1in adapting the techniques of autcoradiography to some of these problemsr Two specific investigations are noted. belo'w In a study of the diffusion of nickel intlo iron. as noted by autoradioggraphy, the gamma state of the nickel was seen to penetrate through the boundaries of the small grains of austenite. Figures i.4 and 1.5 clearly ill ustrate this action. No metallographic evidence of this structure sensitive diffusion has been previously detected. to as great a depth as is evidenced here Work on the copper-bismruth system showed that bismuth exists at'the grYain boundari.es of the copper On subsequant cold rolling and recrystallization the bismu.th did not shift to the new boundarties but remained i.n situ and appears at the center of new grains. HFere again, the autoradiographic technique is far superior to metaallographic technique for indicating the presence of bism3fut-h in the system, as noted in fi. gures 16 and 17 Studies on this systemrn ha.ve also showin that the girain bound.ary angle is'the control3lin.g factor in detertmin'.ing the depth to which bismuth will diffuse into coppe.r-. C, Studies of Incorporation of P-32 in Onion Root Tip Cells, In -the latter part of the year^ an experiment was undertaken in'this laboratory to correlaate the upte,ke of P-32 in plant cells with'the mitotic activity of these cells, as determined by the techniques of autoradiographyThe original work in this study' was reported by Howard and Pelc (5)'who used. Vicia farba seedlings. We are working with onion root tips and follow - ing the general procedure described by Pelc~ The cells are incubated in NaH2PP-'04 soiution - with 16 mg/l of carrier phosphate. The activity is

- 350 Fig. 14 Stripping film autoradiograph showing grain boundary diffusion of radioactive nickel into iron at 18000 F X500 Fig. 15o Diffusion of radioactive nickel into iron at 13000 Fp Stripping film autoradiograph X500

' ~' "'.- I.... ~ L~~~~~~~~~~.,'"~.'. -~',r' tDL'~~~~~~~~~~~.,:,"'":'" ~. ".,... 0%..'"00 ~ ~~~~~~~~~~~~I:.,.~. I. ~'. ~ ~ ~ ~ - ~~.."~.''.. -1 -.. P,0 ~~I ~ ~ ~, ~~~ ~, ~~~ ~.,, I:.'Z::::..,,::::'i:? iI I. - -... -.., "..:,I..::."...,~~~~~ 1., ~ ~.,,.,~. 1..1"':-~..': ~..,:,,. ~.X.,...:.",i.:.,,.~,:".'":, II - "..'~ -,'." 11'.::~ -:.::. 1" 0',, -. -.''.,i..~:. —:i~~~ ~~: "i:.-.:I i'::.'' "......'::, ~..,,...~, ~ N.,,. ~.., ~~~~~, ~ X:_ — t~~'' ~....',:,.::''..,,:,..' I,,. ~::., - ~,.,~,': -, "':.r. -.,~,. II0~~.~ ~~,~ ~~~~~~~~~~~~~~~~~~~~~~~i:.:},.:.-'.L,:,l...:,..:~. -r.~ Io - ~,~~~~~~~~~~~~~~~~~~~~~,''~~~~~~~~~~~~~~ ~ ~ ~ ~ ~ ~ ~:.,..:x,,..,.:,~~~~~~~~,. ll~~~~~~~-....,,.., l,', "..,,.,,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ I I 1-.:(.....''1.':;..'.'. I:....."..:.. -:.. i~; i..i i-::,.: n:. ~ ~:. ~. ~. I I.

32 - adjusted so that concentration (microcuries) x time (hours) is 4t8-> After incubation the tips are fixed -in:1. solution of acetic acid and ethanol, hydrolyzed in 1 N HC1 in alcohol (l:!), and reimtmertsed in a fresh solution of 3:1 acetic acid and ethanol. They are then stained. with aceto-orcein and squashed on a, saran-cocated slide under a thin film of polystylyrenes Whien dry the polystyrene is peeled off and the slides are dipped in 2% saran solutionX The samples are then counted with an end window GtM~ tube and st.ripping -,film applied Good autoradiograpphs have been obtained from cells a.fter incu1bati)on of 3 and 6 hours. Quantitative analysis and correlation with mitotic activity will be carried.. out,

III. PROPOSED RESEARCH General Introduction The ever widening use of autoradiography in the study of fundtament al scientific problems has led -to a need for continued research in the improvement and refinement of this technique Of equal importance is the need for -the investigation of new methods of high resolution detection uti-liz-,1ing the miechanisllis associated with the high energy radiation of radioactive tracers The possibility of adapting the autoradiographic method to electron microscopy work and o oobtaining three dimensional type autoradiographs has recenrtlty presented itself through research in our laboratories. It is felt that work should be continued on these projects h There is also need for continued research on the radiation microscope presently in the advanced stages of development. The specific projects planned are presented below0 A~ Microscope Investigation of the Beta-Ray IMicroscope beyond that describAed in COO-51 has continuedS The details of- this investigation to date have been described in two Progress Reports dated February 4, 1954, and March 12, 1954 t is proposed to con-tinue this investigation along the following lines for the next year: (1) The optical system. will -be modified to allow the placement of the field defining diaphragm ahead of the light splitter t his involves forming an image ahead of the light spli.tter and will probably require a special lens design. This problem has been discussed with the American Optical Company and Bausch and Lomb. Both have indicated. that since it is off the beaten path, they are not in a posit-on to undertake it. Corr:espondence with foreign optical companies is pl..anned (2) "The investigation of phosphors w'ill continue This will include

34 - both the characteristics of new phosphors, such as the mercutrated. -polyst yrene developed by Cowan^ anrd methods of preparing thin crystals of anthraceune. (3) Work is being done toward making that pat of the op tical sys tert used in radiation detection, light tight, This would eliminate the need for enclosing all of the detector in the presently-gtusedt light-tighg t box, (4) The electronic circuits used in the system are being consolidated into a more compact arrangements Additional workk will be done on electrxic f ilters designed to decrease the external pickup, and consequently in'crease the sensitivity of the detector. Effort is being made to secure a small headon cathode-ty-pe pho totube from both RCA and Dumont as this shout d simplify the problem of getting light from optical system to photocathode. B3. Autoradiography During the coming year we will continue to work with the stripping film techniqu e - trying to inriroe our methods for obtaining goo autora diograplh We have recently obtained some stable and radioactive chlorella and plan to use these materia>ls as test specimens for checking techniques' The Sa-an protecting layer as no utsed has recently led to poor observation of -the tissue preparations - and work to eliminate this problemn s plannedd C New Techniques In our research with new techniques of high resolut ion auttorad.iography, the following projects are planned for the coming yeart (1) We will continue efforts toward obtaining the very thin dry collodion films for applicat bon to electron microscope autorad-iographyL A variety of solutionsn and techniques believed useful for loading these films with high uni.rform densities of silver halide is planned. (2) Work with the radiation induced pol ymeriza.tion systems will be carried:further during the coming year I' tio water soluble mater ia-ls, Nmethylene-'lbi sacrylnamuide anud m-tehacrylamideid are being investigated, in addition

to the liquids noted earlier These m.aterials will be studied using x-irradiation, protected radioactive thallium sources, and radioactive maetallurgical and'biological specinens. We plan to do research with regard to determining ithe optimum conditions for radiation induced polymerization, such as atmospheric pressure, presence of oxygen, pH of solution and introduction of sensitizers Various -techniques for the application of these mnterials to radioactive specimens for obtaining autoradiographs will 8aso be consideredm (3) The preliminary research utilizing the principl.e of spark di.s charge will'be extended. We plan'to atte mpt first to duplicate the results of LionJ, as noted earlier, and go on from there to using radioactive sources instead of the x-irradiation used by himi. D. Applications Aplications of the autoradiographic techniques to several biological problems are also planned. We will continue the experiment now in progress concerned with the uptake of P-32 into plant cell nuclei, obt aining quantitative data correlating radioactive cells with the piarticular stages of the umitoti.c cycle. Further aresearch. related to plant ph-yslology as determined by autoradiography technique is contenplatedm An experiment is also planned to study the luptake of C-l4 in leukocytes as indicated by au-toradiography in work related to that of Skipper (6). This laboratory, in the past, has offered assistance and advice to rmembers of the various departments of the Univers- ty who are instrested in applying autoradiography to their particular r esearch problems We will continue to act'in this capacity during the coming year.

- 3 - REFERENCES (1) Gorberg, Ht J. "A New High Resolution System of Autor.adiography" Ntuceonics 9, No 4N. (28), 1951. (2) Hoerlin. IHi. F.W.H Mueller'. Opt. S oc. Amn 4.O No. 4 246-251, April 1950. (3) Schmitz, J.V, E.S. Law-ton. "Initiation of Vinyl Polymeriwzation by Means of High Energy Electrons' Science 113 No 2947 (718), 1951(4) Lion, K.S~ "A Method of Irncreasing Photographic Sensitivity by Electrical Discharget' Je Applied Physics, 24? No, 3, (367) ) 19539 (5) Howard, A. S.RPelc. Isotopes in Biochemistry Churchill, London, pp 138-151, 1951. (6) Skipper, HWE. et al tpPreferenti al Incorpora-tion of Formate Carbon into Leukemic Blood Cells as Indicated by Autoradiography'Y Proc Sock. Biol> and Med 77, (849), 1951