On account of the low grain size three commercial emulsions GEVAERT NUC 307, ILFORD L4 and KODAK NTE have been investigated to assess their qualities for electron microscope microautoradiography. Grain size distribution curves were determined and a developer suitable for microautoradiography was selected after having tested different types of developers. In order to investigate the sensitivities of the three emulsions monolayer preparations were irradiated in the electron microscope using an energy of 5.7 keV, corresponding to the mean &#946;-energy of Tritium. After exposure the specimens were developed but left unfixed. The sensitivity may then be determined, using the ratio of developed to the total number of grains. For the formation of one latent image the ILFORD L4 emulsion must be hit on the average by 1-1.4 electrons per AgBr-grain; the corresponding figures for GEVAERT NUC 307 and KODAK NTE are 2-3 and 4-5, respectively. The problem of resolution of point and plane sources of radioactivity is discussed

Hülser, Dieter F.

Rajewsky, Manfred F.

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Investigations of Different Types of AgBr·Layers for Use in Electron Microscope Autoradiography. D. F. HiJLSER, M. F. RAmwsKY (*) (Max Planck·Institut fiir BiD-physik, Frankfurt/Main, Germany). ABSTRACT On account of the low grain size three commercial emulsions GEVAERT NUC 307, ILFORD lA and KODAK NTE have been investigated to assess their qualities for electron microscope microautoradiography. Grain size distribution curves were determined and a developer suitable for microautoradiography was selected after having tested different types of developers. IIi order to investigate the sensitivities of the three emulsions monolayer preparations were irradiated in the electron microscope using an energy of 5.7 keY, corresponding to the mean f!.energy of Tritium. After exposure the specimens were de~eloped but left unfixed. The sensitivity may then be detenp.ined, using the !"Ilia of developed to the total number of ~ains. For the formation of one latent unage the ILFORD 1A emulsion must be hit on the average by 1-1.4 electrons per AgBr-grain; the corresJ?Onding figures for GEVAERT NUC 307 and KODAK NTE are 2-3 and 4-5, respectively. . The problem of resolution of point and pJane sources of radioactivity is discussed. In biological and medical research nuclear track emulsions are often used for autoradiographical demonstration of radioDuclides in biological materials; for example loctilisation of labelled tracer molecules in cells, substructures of cells :md other small particles. With the presently available techniques the differences m resolution between light and electron microscope autoradiography (microauto-radiography) are relatively small. Nevertheless, we concentrate on microautoradio-graphy, as in the future sensitive emulsions with smaller grai . size, or methods such as the Use of solid state track detectors may increase a toradiographic re-solution. This would permit one to take advantage of the hi!,-' resolving power of the electron microscope in this sYstem. In our experiments we have used a technique modified after Caro et al.': Mai (*) Present address: Hygiene Institut der Stadt und Universitat Frankfurtl n, Paul·Bhrlich-Str. 40. 219 The emulsion is applied with a copper wire loop to the electron microscope grids which have been coated with an ultrathin « sandwich. of formvar, carbon, tissue and carbon, in that order (see Fig. 5). The limiting factors of the resolving power are, therefore: 1) Thickness of specimen; 2) Close contact of specimen and emulsion; 3) Energy and range of the radionuclide; 4) Method of developing; 5) Grain size and sensitivity of the AgBr-emulsion. In the present study. the last two points have been investigated using three different commercially available emulsions with small grain size. Fig. I shows the grain .size distribution measured for the emulsions GEVAERT NUC 307 ( .04 I')' ILFORD L4 ( .11 I') and KODAK NTE ( .04 I')' The grain size GEVAERT NUC 387 ILFORD 1A KODAK NTE FJc. 1 1 • .. i • KCRtIifDSSfN'I'EAlII..I.Ni ! . 11 .. I I . . I I ! I I DER EIrU..SIONEN ,~ NUC 301 • IIfcrd L4 D KadQk NTE distribution curves of ail three emulsions show half·width values of about 20 % of the mean. Whereas the IiFORD L4 and KODAK NTE emuisioDs were found to have the grain size stated by the manufacturers, the value found for tbe GEVA~! NUC 307 emulsion was about 40 % lower than that stated. There may be vanatJons of gniin size between different batches of this emulsion and it is. therefore. recommended to check the grain size before use. • A developer suitable for microautoradiography should produce typicallY shaped silver filaments which are as .small as possible. FiR. 2 demonstrates for the emulsion 22P ILFORD L4 (developed 1 minute) the types of silver filaments produced by three different developers. Physical development as used by Caro 1 and other authors was not used because of the likelihood of artefacts, a particularly serious problem in electron microscopy. MICRODOL X fine grain developer produced rather small silver filaments of ty-pical shape in all three emulsions, the fine end of a filament cOinciding with the locus of the original latent image in the AgBr grain. This developer was chosen for all subsequent experiments. FJC:. 2 In order to test the sensitivity of the three emulsions in question. monolayer preparations have been irradiated in the electron microscope and measurements of electron intensity have been made. The energy of the electron beam was main-tained at 5.7 keY, corresponding to the mean !>energy of Tritium, the radionuclide most frequently used in biological tracer studies. After irradiation in the electron microscope the specimens were' developed in MICRODOL X but left unfixed. This procedure permits the determination of the ratio of developed to the total number of grains. Fig. 3 shows the result of a typical experiment with ILFORD L4 emulsion and exposures to 0, I, 2 and 20 electrons per AgBr grain. Due to the short develop-iDg time of 1 minute not all latent images do grow out completely, as it takes longer to develop the internal latent image than those on the surface of the grain. The POints, however, where silver filaments are just starting to grow out from the grafus are clearly distinguishable. If we assume that electrons hit AgBr grains according to a Poisson distri-bution, we can calculate tbe number of grains which are hit by k and more electrons when the mean exposure is N electrons per grain. In Fig. 4 the percentage of developed AgBr grains is given on ' the ordinate, tbe number of electrons ne-cessary for the formation of a latent image can be read from the abscissa. OUr experiments show that for the formation of one silver filament an AgBr 221 ': .: ........... ; ..... !:," ,,- . '.-", .. .,. .... , '.:," .... :·0':. " .' •. • •• '. ·0 •. ....... : ...... '~ .. . ' ... ' .' . .... .. . • • ' .0 • 0 0 • • 0.0 • ••• '0 0" • '" ......... o •• eO .' . • ••••• 0 ••••• 0: •• ' !.-.. ~.-,", .0 .•. : .•. " •••• e" • •. •• • 0·.0 • • • . . ' . • .6 .5 " .2 l Fu;. 3 It:z. ............ . • AI .. ....,. .. MhkIt -~ z..._ a.trcNn ... ... -......... ...... 11 •••• ,.~W"UD~d.~ •• " -. rIC. , grain of the ILFORD LA emulsion must be bit on the average by 1~1.4 electrons. The coITeSponding figures for the GEV AERT NUC 307 and KODAK mE emulsions ..", 2~3 iind 4-5. respectively. From the energy loss in silver bromide for 5.7 keY 222 electrons it can be estimated that the ILFORD L4 and GEVAERT NUC 307 emuls· ions absorb about 800 eV for the formation of a latent image as compared to 1300 eV for the KODAK NTE emulsion. In sections of biological material we are often dealing with plane sources of radioactivity. In this case, the differences in sensitivity between the emulsions are not large enough to be important. What may be more critical, particularly where the localization of label in specific cell components is being studied, is the degree of resolution. The resolution may be visualized as a sphere with the diameter of the AgBr grain as its radius, the center of the sphere being the latent image. For MICRODOL X developed grains the thin end of the filament corresponds to the latent image and hence to the center of the sphere. If, as in the case of the ILFORD L4 emulsion, only one electron is required for the formation of a latent image in one grain. it will with great certainty have come from the sphere. For emulsions, however, which require several electrons for the formation of a latent image, the proba-carbon ti .. ue arbon .Fotmnr '" . .. bility for the formation of a latent image must be taken into account. This is demonstrated in Fig. 5. The probability (P,) for latent image formation is a function of the number of electrons (i), necessary for the production of one latent image, and of the probability for one AgBr grain to be hit by one electron. Thus, P, = ( r' ) i 4x' Nonnally one would assume that resolution goes up with decreasing grain size. As, however, the degree of resolution is proportional to the probabi~ty ~f latent image formation (P) the advantage of emulsions with smaller gralDS IS lost . because the smaller th~ grains are the greater will be the number of electrons (i) required for latent image formation. Regarding point sources this means that with emulsions requiring several electrons for latent image formation very little labelling will be obtained. In the case of line sources. as for instance membranes, the c electron collecting area» becomes so extended that the advantages of low grain size afe eliminated, as was mentioned above. Hence, giving a similar reso-223 JUlion as the GEVAERT NUC 307 emulsion , the ILOFORD L4 emulsion is prefer red, because with a considerably shorter autoradiographic exposure time, results are of comparable quality. In Fig. 6 the degree of resolution which can be obta ined u sing ILFORD L4 emulsion and MI CRODOL X developer is demonst rated in a microautoradiogram or a cell from the transplantable « Marshall » rat carcinoma. 3H-thymidine has been incorporated into the DNA o f this cell « in vitro . p H-t hymidine concentra-lion in the medium 20 [lc/ ml, incu bat ion time 90 minutes, exposure time 15 days, magnification 11 .000 x). The resolution is shown by circles around two si lver fi· laments). Flc. 6 It may be worth mentioning that another approach to produce suitable pho-tosensil i~'e silver bromide layers with vcry small grain size has been vacuum cvapo.ra tlon ~r metallic silver and subsequent exposure to bromium vapour, a techntque which has in principle been described by Silk et al. in 1961 ~. However, our .results wit~ this method indicate that although vcry small crystals of AgBr ru:e Indeed obt~Inable, ~he sensitivity of the grains is extremely low by comparison WIth commercially avaJiable autoradiographic em ulsions in gel form. F~(ure a~vances in microautoradiography will depend on the development of emulsIOns with lower grain sizes, but such improvem~nl must not be at the expense of sensitivity. On the othe ~ hand it seems to be of interes t to investigate the possibilities of the use of solld·st ate track detectors for high resolut ion microautoradiography. 224 ACKNOWLEDGEMENTS The authors are indebted to Dr. W. Lippert for valuable discussions and help with the eJectron microscopy. REFERENCES I. aJ CARO L G., VAN TUBERGEN R . P. and KOLB J. A.: J. Cell BioI., 15, 2, 173·188, 1962; bJ CARD L G.: J. Cell. BioI., 15,2, 189-199, 1962. 2. SILK M. H., HAWTREY A. 0., SPENCE 1. M. and GEAR J. H. S.: J. Biophys. Biochem. C)"lol., 10, 4, 577-587, 1961. 225 

Investigations of different types of AgBr-layers for use in electron microscope autoradiography

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Investigations of different types of AgBr-layers for use in electron microscope autoradiography

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