Recent Developments in Light and Electron Microscope Radioautography

Recent Developments in Light and Electron Microscope Radioautography G. C. BUDD Department of l’l?ysiologv, Medical ColCege of Ohio,Toledo, Ohio

I. Introduction . . . . . . . . . . . . . . . . . . . . . . 11. Radioautography of Diffusible Materials . . . . . . . . . . . A. General Considerations . . . . . . . . . . . . . . . . . B. The Freezing of Tissues . . . . . . . . . . . . . . . . C. Treatment of Tissues after Freezing. . . . . . . . . . . . D. Diffusible Substance Radioautography and Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . 111. Electron Microscope Radioautography . . . . . . . . . . . . A. General Considerations . . . . . . . . . . . . . . . . . B. The Measurement of Efficiency . . . . . . . . . . . . . C. Resolution Measurement . . . . . . . . . . . . . . . . D. Absolute Quantitation . . . . . . . . . . . . . . . . . IV. Light Microscope Radioautography with Thin Sections and Emulsion Layers . . . . . . . . . . . . . . . . . . . . V. Electron Microscope Radioautography of Diffusible Substances VI. Summary . . . . . . . . . . . . . . . . . . . . . . . . Refcrences . . . . . . . . . . . . . . . . . . . . . . . .

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I. Introduction The principal aim in radioautography is to localize, with the aid of a photographic (ionographic) emulsion, the precise position of radioactive atoms that have been introduced into a specimen. Ionizing radiations emitted from radioactive atoms within an histological section induce the formation of latent images in a closely apposed layer of emulsion. The latent images are then converted chemically into permanent grains of silver which are usually observed with a microscope. The observer must determine from the distribution of grains the most likely distribution of radioactive sources in the section. There are two facets of the radioautographic procedure. One aspect is technique, the other being the interpretation of the results. I n recent years there have been a number of new developments in the technical aspect, including the use of thin sections and the introduction of successful methods for detecting soluble radioactive substances which were not retained in earlier procedures. In addition, techniques for preparing radioautographs that can be viewed with an electron microscope have been introduced. These methods are providing powerful tools for the cytologist, especially in the study of such 21

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problems as macromolecular synthesis and turnover, quantitative enzyme localization, and drug metabolism. I n addition to the development of new techniques and improvement in established methods, new information has been obtained that can help an investigator more fully interpret his observations. This includes the measurement of absolute sensitivity (or efficiency) and resolution. Together, these provide the basic information required for determining the quantitative uptake of radioactivity into defined areas of a specimen. Because it allows the invcstigator to calculate the number of radioactive sources within a defined region of a cell or intracellular organelle, radioautography has undoubtedly become one of the most valuable quantitative tools available to the cytologist. The continued development of new or improved radioautographic techniques is stimulated by the constant need to relate biochemical events to the morphological and physiological observations of cytologists. I n common with other cytochemical techniques, it is possible, with the aid of radioautographs, to localize known biochemical reactions to specific intracellular sites without disrupting the cells. Radioautography has the special merit, however, of allowing the localization to be made quantitatively. It also can be used to localize reactions for which there are no specific cytochemical staining methods. Radioautography is particularly useful for localizing sites of synthesis or areas with the capacity for binding physiologically or pharmacologically important ions and molecules. Very often it is possible to distinguish between initial binding or receptor areas and regions to which the products of synthesis are translocated. Radioautography thus affords greater specificity than many cytochemical staining methods in that it allows new synthetic products to be distinguished from accumulated stores. This chapter is mainly concerned with describing and evaluating recent methods for visualizing diffusible radioactive materials within and between cells and with developments in quantitative radioautography at both the light and electron microscope levels. For a more complete survey of the history, methods, and applications of radioautography, the reader is referred to the monographs by Boyd (17) j), Rogers (1767), and Baserga and Malamud (1769). and the symposium volume on radioautography of diffusible substances edited by Roth and Stumpf (1769).

11.

Radioautography of Diffusible Materials A.

GENERAL CONSIDERATIONS

The main disadvantag,e of the standard liquid emulsion (BClangcr and Leblond, 1946; Joftes and Warren, 175j; Kopriwa and Leblond, 1762) and

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stripping film (Pelc, 1947; Doniach and Pelc, 19jo; Pelc, 1 9 ~ 6 techniques ) of radioautography is their limitation to the study of radioactive materials that are insoluble in histological fixing, dehydration, and infiltrating solvents. This fact considerably reduces the value of these techniques in investigating the histological distribution of ions, lipids, many physiologically important compounds that are soluble, and most drugs. This disadvantage may be overcome if the diffusible radioactive materials are immobilized in situ prior to and during exposure to the radiation-sensitive emulsion. Several methods have been introduced recently which allow diffusible substances to be located in sections or in whole cells. Diffusible materials are usually immobilized initially by rapidly freezing labeled tissue to below -7o'C. Subsequently, the tissue may be freeze-dried and exposed only to nonaqueous solvents prior to infiltration and sectioning (Wilske and Ross, 1 9 6 ~ Stirling ; and Kinter, 1967; Nadler e t al., 1969), or it may be sectioned while frozen (Ullberg, 1934; Eckert, 1968; Appleton, 1964; Stumpf and Roth, 1964). Single cells may also be exposed to dry emulsion directly after drying, without subsequent sectioning (Miller e t al., I 964). Most of the recent methods for radioautography of diffusible substances have been developed for use with the light microscope and are extensions of the original method of Ullberg ( 1 9 ~ 4 )for radioautography of whole animals and large organs. In addition, recent attempts have been made to develop a method for diffusible substance radioautography which can be used with an electron microscope (Appleton, 1969 ; Christensen, 1969; Eckert, 1969). Before attempting to evaluate these methods, it is necessary to consider the factors that are likely to determine the success or failure of a high-precision method for locating diffusible substances in cells or tissues. Simply stated, there are three requirements that should be met to ensure success: ( I ) The diffusible radioactive material under consideration must be rapidly immobilized within the specimen. (2) The immobilized radioactive material must not diffuse during any subsequent procedures until an image (or latent image) has been formed in a radiation-sensitive emulsion layer which is in close apposition to the specimen. (3) The specimen and emulsion must retain the same position relative to each other from the time they are first placed in contact until the developed image and specimen have been examined microscopically. The first of these requirements, namely, that diffusible substances be rapidly immobilized, is most easily met by rapidly freezing the specimen (suspended cells or fresh tissue) to a temperature close to that of liquid nitrogen (-196°C). Attempts have been made to meet the second and third requirements in a variety of ways with variable success. An account of these investigations is given later in this section and in subsequent sections of the chapter (Sections II,C and V).

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B.

THEFREEZING OF TISSUES

The formation of ice crystals appears to be inevitable when living tissues or solutions of physiological concentration are frozen (Meryman, 1966). Ice crystal formation can be prevented only by the addition of substantial amounts of solute. T o minimize the size of intracellular ice crystals, tissues intended for histological study are usually frozen rapidly to lower than - I 30°C in a few seconds by immersion in a solvent (isopentane, propane, or Freon 12) which is cooled by liquid nitrogen at -196°C. There is recent evidence that the vitrification temperature for pure water is approximately -I 46°C (Yannas, 1968) which is below the minimum temperature at which isopentane remains liquid (- I 30'C). In rapid freezing in liquid nitrogen-cooled propane at -I 8ooC, it may be expected that vitrification would be favored. Even after the most rapid freezing rates, however, intracellular ice crystal formation has been observed (Trump, 1969). which results in variable amounts of disruption to organelles. The disruptive effects can be reduced by pretreating the tissues with cryoprotective agents, including glycerin and dimethyl sulfoxide (DMSO), which may displace some of the intracellular water, acting as partial dehydration agents (Rebhun, 1965; Bullivant, 1961). Because of their solvent action, the use of such cryoprotective agents is unlikely to be applicable to radioautographic studies of diffusible substances. We are therefore left at the present time with rapid low-temperature freezing of fresh tissue as the only available method for instantaneously immobilizing most diffusible substances for radioautography. The disruption of cell morphology by intracellular ice crystals implies the possibility that even during the few seconds required for rapid freezing there may be movement of solutes from one site to another as the ice crystals form. The extent to which this affects the interpretation of solute distribution in living cells based on the radioautographic image remains to be determined. The prolonged storage of rapidly frozen tissues at temperatures above that employed for initial freezing should be avoided because of the danger of ice recrystallization (Meryman, 1956; Appleton, 1967). C. I.

TREATMENT OF TISSUES AFTER FREEZING

The Use of Nonagueoux Solvents

The preparation of frozen tissue or cell suspensions for diffusible substance radioautography depends on the substance being studied. The use of any extraneous solvent either before or after freezing is generally undesirable if translocation of soluble substances is to be avoided. There are, however, special instances in which nonaqueous solvents have been used after initial freezing with some success in experiments designed to localize specific materials

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that are soluble in normal physiological solutions (Wilske and Ross, 1965; Stirling and Kinter, 1967; Nadler et a/., 1969). The usual procedure in these instances involves freeze-drying the tissue and direct infiltration with epoxy embedding agents. Such a procedure can result in adequate intracellular retention of several substances that are soluble in physiological solutions yet insoluble in embedding agents, but it is always necessary to verify independently that there is minimal loss of radioactivity from the tissue. Loss of radioactivity could occur in the vapor phase during freeze-drying or by solution in the embedding material. Such loss is easily detected with standard radiation counting methods, but there remains the possibility that translocation of the radioactive material occurs during freeze-drying and also during infiltration with the embedding medium. The extent to which such translocation occurs in practice is not always known, but it does not appear to be significant in some studies (Stirling and Kinter, 1967). Sections of freeze-dried and epoxy-embedded tissue may be cut with a glass knife, collected on water, placed on a microscope slide, and covered with emulsion for light microscope radioautography. Using this method, Wilske and Ross (1965) were successful in localizing tritium-labeled aspirin, which is highly soluble in organic solvents and moderately soluble in aqueous solutions, within blood vessels of the rat. In a similar study, Nadler e t al. (1969) were successful in localizing inorganic iodine-I z j within rat thyroid follicular epithelium. In both cases the freeze-dried, labeled tissue was vapor-fixed with osmium tetroxide in a sealed desiccator prior to infiltration with embedding medium. As a part of the iodine study, experiments were conducted to assess the extent to which radioactivity was retained in the tissues during freezing, drying, infiltration, and sectioning. Interestingly, there was very little loss of radioactivity prior to sectioning ( 3 . 8 %), but there was approximately 10yo loss from sections floated on water after cutting. These investigators suggested that the loss of radioactivity from floating sections may be partly induced by the knife used during sectioning. Sections of freeze-dried and epoxy-embedded tissues show considerable ice crystal damage when viewed in the electron microscope, even when rapid freezing and fixation in the vapor phase with osmium tetroxide are employed. The maintenance of organelle structure is adequate enough, however, for localization of radioactivity using electron microscope radioautographic methods (Stirling and Kinter, 1967; Eckert, 1969; see also Figs. 1-3). In a study of the distribution of tritiated galactose, mannitol, and phlorizin, Stirling and Kinter (1967) prepared both light microscope and electron microscope radioautographs of freeze-dried, epoxy-embedded intestinal epithelium. They observed that leaching of these water-soluble substances sometimes occurred when sections were collected over water, resulting in diffusion artifacts in the radioautographs. These artifacts were prevented, however, by

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FIG. I . Electron micrograph of rapidly frozen, freeze-dried, osmium tetroxide vaporfixed, epoxy-embedded, and sectioned exocrine pancreas of a rat. Some ice crystal damage is visible in the nucleoplasm, but preservation of endoplasmic reticulum (see inset) and other cytoplasmic organelles is excellent. x Z J O O ;inset X 15,000.(Kindly provided by Dr. Helmut Eckert, Sandoz Ltd., Bade, Switzerland.)

addition of 1 % silicone fluid (Dow-Corning silicone fluid zoo) to the epoxy infiltration medium. These investigators suggest that potential channels for water permeation were wetted or filled by the hydrophobic silicone compound. Even when obvious diffusion artifacts (halos of developed grains over and around the sections) were avoided, I -p sections lost about 27 %, of their radioactivity to the bath water. How much of this loss may be attributable to the cutting action of the microtome knife was not determined. Using a similar technique, Eckert ( I 969) studied the intracellular distribution of acetyldigoxin3H in the rat intestine. After freeze-drying and fixation in osmium tetroxide vapor, the amount of radioactivity lost in the embedding medium was less than O . Z O / ~ of the total activity in the embedded tissue. Conventional fixation in aldehyde and osmium solutions, followed by dehydration in ethanol and embedding in plastic, however, resulted in the loss of 90% of the tissue radio-

FIG. 2. Electron micrographs of proximal renal tubules prepared as in Fig. I . Microvilli, lysosomes, mitochondria, and plasma membrane infoldings are well preserved (A and B). The glomerular basement membrane (C) does not show the usual three layers present in conventionally fixed and dehydrated material. Possibly the differencc reflects ice crystal damage. x 15,000.(Kindly provided by Dr. Helmut Eckert, Sandoz Ltd., Bask, Switzerland.)

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FIG. 3, Radioautographs of freeze-dried, osmium tetroxide vapor-fixed, epoxy-embedded rat kidney visualized I minute after injection of inulin-SH. In both, the light microscope (inset) and electron microscope radioautograph of the diffusible substance is localized preferentially in the lumen of a proximal tubule and in the region occupied by microvilli. x 7000; inset x 1000.(Kindly provided by Dr. Helmut Eckert, Sandoz Ltd., Basle, Switzerland.)

activity. Clearly, the low solubility of the labeled substance after freeze-drying in the infiltration medium is a necessary requirement for its in sits localization with this technique. There are many water-soluble physiologically and pharmacologically important materials in addition to those mentioned above that are likely to be insoluble in embedding media and could be profitably studied with the freezedrying and Epon-embedding method. In each case it is necessary to confirm

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independently that there is no loss or translocation of radioactivity during drying and infiltration with the embedding agent. In addition, allowances must be made for possible loss of radioactivity during sectioning or while sections are floating on water. The possibility that diffusion of radioactive material can occur during application of warm o r cold emulsion to the sections should also be considered. Chang (1767) has proposed the application of a freeze-substitution technique for dehydrating frozen sections at dry ice temperature. Although acetone is an excellent solvent for many water-soluble materials, it is possible that this method has advantages over conventional methods involving aqueous fixation when radioautography is to be used for localizing water-soluble but acetoneinsoluble proteins, or when radioactive cytochemical reagents are to be localized. For radioautography of water-soluble materials, the main advantages of using sections of embedded tissues prepared by freeze-drying or freezesubstitution is that emulsions are applied in accordance with standard radioautographic procedures. No unusual precautions must be used when applying a layer of emulsion to the sections and it is possible to store and process the radioautographic preparations together with radioautographs of tissue prepared in a conventional way using aqueous fixation and organic solvent dehydration. 2.

Sectioning Froxen Tisstre

Many of the ions and small molecules in cells and tissue fluid are very soluble in organic solvents and the infiltration media used for embedding tissue. In order to localize these substances, alternative techniques that require the sectioning of frozen tissue and storage of radioautographs at low temperatures have been developed. The initial step in all these procedures after the radioactive tissue has been rapidly frozen involves cutting sections at a temperature of - I j "C or lower. The earliest method widely used today involves sectioning frozen pieces of tissue or whole animals at - I 5 O C and attaching each section to a piece of adhesive tape (Ullberg, 1954; Hammarstrom et a/., 1965 ; Ullberg and Appelgren, 1969). After drying, the sections are directly apposed to dry photographic emulsion which is usually presoaked in a glycerol-ethanol solution and allowed to dry. This treatment improves adhesion between the sections and the emulsion. After exposure but before processing, the tape is removed by dissolving the adhesive in xylol. Recently, a modification of this method for use with sections cut at temperatures as low as -75OC was proposed (Wedeen and Jernow, 1968). Some investigators have picked up sections onto strips of plastic or onto coated glass slides and allowed them to dry under reduced pressure either before or after pressing the sections against an emulsion

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layer (Roberts etal'., 1964; Stumpf, 1964; Eckert, 1968). This method resembles the apposition technique originally introduced by Lacassagne and LattCs (I 924) for conventionally prepared sections. T o ensure the adherence of section to emulsion during photographic processing, it may be necessary to apply an adhesive to the sections after storage but before immersion in developer solution (Roberts e t a/., 1964). Wedeen and Jernow (1968) advocate the use of a liquid adhesive for attaching dried sections to the emulsion before the storage period begins. It must be assumed that contact between the sections and a liquid adhesive is likely to cause the translocation of some solutes within the sections, thus limiting the usefulness of this technique for general application to all diffusible substances. For some applications, however, the use of an adhesive has not introduced any obvious undesirable effects (Wedeen, 1969).

3. Low- Temperature Storage of Radioautographs Theoretically, diffusion of soluble radioactive sources will be minimized during radioautographic exposure if dry sections are attached to dry emulsion without using any adhesive. This is especially true when molecular movement is restricted by storing the preparation at - z o T or lower. In the technique developed by Appleton (1964, 1966), wet, stripping-film emulsion (Kodak AR-10) is coated on microscope coverslips in such a way that the radiation-sensitive emulsion layer (4 p thick) faces away from the cover slip and the inert gelatin layer (10 p thick) is pressed against the glass surface. After thorough drying the coated coverslips are cooled to - 5 "C. Cryostat sections of rapidly frozen and radioactively labeled tissue are prepared at - z > O C . Immediately sections have been cut, a cold, coated cover slip is touched to them. On contact the sections adhere to the emulsion surface without the need for any adhesive material. The radioautographs are stored throughout the exposure period at --2j°C. At the end of the exposure period, the slides are brought to room temperature when the ice presumably melts. The dried sections may be fixed by immersion in 5 yo acetic acid (or buffered formaldehyde, Rogers, I 967) before the radioautographic image is developed in the usual fashion. This technique has been used successfully in several laboratories for observing diffusible ions and molecules (Pelc and Appleton, 1965; Gahan and Rajan, 1965; Kinter and Wilson, 1965; Rogers e t al., 1966; Waser e t a/., 1965)). The resolving power, sensitivity, and latent image fading inherent in this technique have all been measured (Appleton, 1966; Pelc and Welton, 1968; Welton, 1969). The conclusions from these studies were that there is no significant diffusion of soluble compounds during exposure at -zj"C, but that the emulsion sensitivity is reduced by about 24% at this temperature when compared with the sensitivity at +4"C. This is a small loss

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in sensitivity when considered in relation to the advantages of the technique for localizing soluble substances. Further reduction of storage temperature as proposed by Rogers (1967) results in considerable loss of emulsion sensitivity (64 yo)without any apparent change in resolution (Appleton, 1966).

4. Latent Image Fading at Low Temperatures The sensitivity loss at -25°C becomes insignificant in the light of studies on latent image fading. When emulsions are exposed to a specimen, radioactive disintegrations in the specimen induce changes in silver halide crystals in the emulsion which cause the formation of a latent image. It is this latent image that is developed during immersion in photographic developer solution to produce a permanent silver grain. During the prolonged exposure periods common in radioautography, some latent images can fade so that a permanent image no longer develops from such halide crystals. Latent image fading can significantly reduce the efficiency of radioautography during exposure periods of several weeks or months at 4°C or warmer (Ray and Stevens, 1953; Baserga and Nemeroff, 1962; Lord, 1963). When radioautographs were exposed at -23°C for up to zoo days, no fading of the latent image was detected (Pelc and Welton, 1968; Welton, 1969). This finding has since been confirmed for exposures up to 3 years (Pelc, personal communication). Thus the loss in emulsion sensitivity at -23" to -zj0C is more than offset by the reduction in latent image fading apparent at this temperature. 5 , The Use of Freere-Dried Sections

A different technique for attaching dry sections to dry emulsion was developed by Stumpf and Roth (Stumpf, 1964; Stumpf and Roth, 1965a). In this procedure radioactively labeled tissue that has been rapidly frozen to -180°C in liquid propane is sectioned in a cryostat at -30" to -6oOC. Temperatures below -55°C appear to favor the cutting of ~-p-thicksections (Stumpf and Roth, 1965b). The frozen sections are then dried at -68°C in a cryosorption pump at better than 10-5 mm H g for 24 hours (Stumpf and Roth, 1967). Microscope slides are separately coated with fluid emulsion (Kodak NTB-3) and dried over a dessicant. The dried sections are then brought to room temperature and transferred to pieces of Teflon. An emulsion-coated slide is placed over the sections, and the Teflon and slide are pressed together with the thumb and forefinger. The Teflon is allowed to fall away, leaving the sections adhering to the emulsion layer (Fig. 4). The dry-mounting procedure is carried out at room temperature at low relative humidity (20-40 yo). Subsequently, the radioautographs are stored in dry boxes at - I 5 "C. After storage the boxes are warmed to room temperature before opening. At this stage it is recommended that the preparations be briefly

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(A)

FIG. 4. The dry-mounting radioautography technique for diffusible substances. Sections containing radioactive material, 0 , and control sections, 0,are placed on Teflon. Under a safelight, a dry, emulsion-coated slide is placed over the sections (A) and the Teflon and slide are pressed together (B). (From Stumpf, 1970.)

moistened, by exhaling on the slide, and then dried to ensure adhesion of sections to the emulsion during development. Moistening the sections at the end of the exposure period is not expected to affect materially the distribution of the latent image immediately prior to its development. Although there is no precise measure of the resolving power obtainable with this procedure, several published applications indicate that normally diffusible substances can be localized with high precision. In a comparison of five other radioautographic methods with their own, Stumpf and Roth (1966) showed that any method using frozen sections that involved thawing or contact with organic solvents resulted in varying degrees of diffusion. When the distribution of e~tradiol-~H in various tissues was examined in detail using the dry-mounted, freeze-dried section procedure, a distinct pattern of cellular and subcellular radioactivity distribution was observed in target tissues for the hormone. In the nucleus ventromedialis of the rat diencephalon, for example (Fig. 5 ) , radioactivity was concentrated in the nuclei of neurons

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FIG. 5 . Dry-mount radioautograph of the pars lateralis of the nucleus ventromedialis tuberis cinerei of a mature ovariectomized female rat prepared z hours after subcutaneous injection of 0.4 pg of 17/3-estradiol-6,7-8H. Radioactivity is concentrated in nuclei of neurons. x zoo. (From Stumpf, 1970.)

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(Stumpf, 1968). The further successful application of this technique for localizing soluble hormones within individual cells has been recently reviewed (Stumpf, 1970). A simple method for detecting diffusible compounds in single cells was developed in which air-dried or freeze-dried cells on a microscope slide are covered with a layer of emulsion formed in a wire loop (Miller e t al., 1964). To ensure adherence of the emulsion to the specimen slide, it was necessary to moisten the emulsion by breathing on it. While this procedure may cause diffusion in some situations, this was not apparent in a study of nucleotide pools in Tetrabymena pyriformis.

D. DIFFUSIBLE SUBSTANCE RADIOAUTOGRAPHY AND ELECTRON MICROSCOPY Many problems must be solved before a routine method for making electron microscope radioautographs of soluble compounds can be developed that has universal application. Advances have been made recently in such areas as the design of special equipment for cutting ultrathin frozen sections (Appleton, 1969; Christensen, 1969), but there are many problems concerning the adhesion of sections to emulsion layers at low temperatures, and the effects of high vacuum and an electron beam on unfixed, nonembedded tissue sections. It may be possible to circumvent some of the problems inherent in handling ultrathin frozen sections by employing a different approach similar to that used in a recent study by Eckert (1969). A discussion of this subject has been postponed until the end of the section on electron microscope radioautography.

111. Electron Microscope Radioautography A.

GENERALCONSIDERATIONS

The most important requirement for preparing reproducible radioautographs is uniformity in specimen thickness and in the distribution and thickness of the radiation-sensitive emulsion layer. This is especially true for electron microscope radioautography which requires the use of ultrathin specimens and emulsion layers. Several methods described for preparing electron microscope radioautographs (Caro and van Tubergen, 1962; Hay and Revel, 1963) are often used for semiquantitative studies in which the relative concentration of radioactivity in different regions of labeled cells is to be compared (Revel and Hay, 1961; Car0 and Palade, 1964; Jamieson and Palade, 1966; Lane e t a/., 1964; Neutra and Leblond, 1966). In these methods sections of radioactively labeled tissue are first placed on electron microscope grids and then covered with a layer of emulsion. Attention has been drawn to the possibility that the

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spreading of fluid emulsion can be influenced by the crossbars of a grid such that sections become unevenly coated (Pelc e t a/., 1961; Budd and Pelc, 1964; Salpeter and Bachmann, 1964). As an aid in obtaining more uniformly spread emulsion over a section supported on a grid, emulsion layers formed in a wire loop may be dried before application to the sections (Caro and van Tubergen, 1962; Hay and Revel, 1963). This method is satisfactory for some emulsions but is not applicable to the very fine-grain emulsions with a high ratio of silver halide to gelatin. For these emulsions other methods must be used. To ensure the uniform spreading of fluid emulsion over ultrathin specimens, two methods have been developed in which the radioactive specimen, usually a section of epoxy-embedded tissue of known thickness supported on a flat surface, is covered with a layer of emulsion of known thickness (Fig. 6). After storage and processing, the specimen with its radioautograph is transferred to a standard specimen grid for observation in an electron microscope (Pelc e t al., 1961; Budd and Pelc, 1964; Salpeter and Bachmann, 1964). The glass slide technique of Salpeter and Bachmann (1964) was recently used in detailed studies of sensitivity and radioautographic resolution (Salpeter and Bachmann, 1961; Bachmann and Salpeter, 1967; Salpeter e t al., 1969). The results obtained in these and other studies (Kopriwa, 1967; Vrensen, 1970) demonstrated that with proper regard for specimen and emulsion thickness and emulsion distribution it is possible to use electron microscope radioautography for quantitation of radioactivity within defined regions of a specimen. That this is true for experimental situations has been demonstrated in recent cytological applications (Israel e t a/., 1968; Budd and Salpeter, 1969).

B.

THEMEASUREMENT OF EFFICIENCY

It is important to know the efficiency of a radioautographic technique before it can be used for determining the amount of radioactive material in a biological specimen. Efficiency depends mainly on the type and energy of the radiation, the thickness of the specimen and emulsion, the thickness of any intervening layer, and on the type of emulsion and the method of processing. In electron microscope radioautography it is possible to control all these factors so that accurate estimates of efficiency for a given set of conditions can be determined (Bachmann and Salpeter, 1965, 1967; Kopriwa, 1967; Vrensen, 1970). Efficiency has been measured in a number of ways. In order to measure the response of L4 emulsion (Ilford, Ltd.) and NTE emulsion (Eastman Kodak) to electrons, Salpeter and Bachmann (1961) irradiated emulsion layers in the form of close-packed silver halide crystals with 10-keV electrons to obtain a measure of emulsion sensitivity or grain yield (grains/electrons hitting the emulsion). A grain yield of 1 / 1 2 was observed for L4 emulsion developed with hlicrodol X.

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(a

Add emulsion

Drain onto filler poper and dr)

I

*

Storage period

Remove from slide

( 4 ) ond attach grid

/

Process

Invert slide and (40) attach g r i d

cG

FIG. 6. Diagram of two techniques for electron microscope radioautography that employ a smooth substrate (see text for detailed account). (I) A microscope slide coated with collodion. (Ia) In one technique (Budd and Pelc, 1964) the collodion is removed from the slide onto water. (Ib) The collodion is then picked up to form a membrane over a hole in another slide. (2) In the other technique thin sections are transferred directly onto the collodion-coated slide (Salpeter and Bachmann, 1964). (28) Sections are transferred to the membrane with a wire loop. (3) and (3a) After staining the sections and coating them with evaporated carbon, fluid emulsion is dropped onto the section and the excess is removed. (4) After storage and processing, the collodion, sections, and radioautograph can be removed from the slide onto water and a grid is attached. (4a) Alternatively, the grid is attached to the sections and radioautograph which are on a membrane. ( 5 ) The grid and attached preparation is removed from the water with a suction device. (la) Alternatively, the grid and attached preparation are cut out of the membrane. (6) The complete preparation composed of collodion (M), sections (S), carbon (C), and processed emulsion (E) attached to a grid is observed in an electron microscope.

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For accurate quantitation the relationship between radioactive disintegrations in a specimen and the number of silver grains in the developed emulsion should be determined. This relationship was measured under controlled conditions using a calibration specimen composed of a thin layer of dry gelatin containing a known amount of a radioactive substance which formed a layer of known thickness on a glass microscope slide (Bachmann and Salpeter, 1967). Such a calibration specimen was closely apposed to a test emulsion layer on a collodion-coated slide for a predetermined time to irradiate the emulsion. After removing and processing the emulsion, the number of grains per unit area of the emulsion was determined from electron micrographs and could be related to the number of disintegrations in the region of the calibration specimen that was in contact with the measured area of emulsion. By using this method the sensitivity of a layer of L4 emulsion equivalent in thickness to a single layer of close-packed silver halide crystals (1400- to ~ j o o - A ) ,after development in Microdol X (3 minutes, 24OC), was 1/10 ( I grain per 10 disintegrations) for tritium and 1 / 2 1 for sulfur-) 5 . Bachmann and Salpeter obtained a range of sensitivity values for L4 emulsion monolayers and NTE emulsion layers after varying the development conditions. When comparing the grain yield for L4 emulsion exposed to 10-keV electrons with the index of grains per disintegrations in the later experiment, it should be realized that only about half the radiations emitted in the specimen hit the emulsion. For comparative purposes the grain yield value (Salpeter and Bachmann, 1965) should therefore be halved (giving a value of 1/24). The value obtained is much lower than that in the later experiment of Bachmann and Salpeter (1967). These investigators explained that tritium electrons have a lower mean energy ( 5 . 5 keV) than the ro-keV electrons and a greater average path length in the emulsion. Thus the higher sensitivity under conditions of actual radioautography is, in principle, consistent with theoretical expectations. Bachmann and Salpeter (1967) have discussed the influence of specimen thickness on efficiency and conclude that as long as the specimen is not thicker than 1000 A differences in section thickness have no practical influence on sensitivity to tritium. They did, however, observe an average deviation in mean sensitivity values of z o x , on different occasions when radioactive sources of standard thickness were used. The deviations were in part attributed to errors in judging emulsion thickness and variations in developing conditions. Using a different approach, Kopriwa studied the influence of development conditions on the relative sensitivity, expressed as reaction intensity (or number of grains per unit area in exposed regions of the emulsion), of three different emulsions: Ilford L4, Gevaert NUC-307, and Kodak N T E (Kopriwa, 1967). The test specimen in the case of each emulsion-developer combination consisted of thin sections (light-gold interference color) from a polymerized block of methacrylate homogeneously labeled with tritium. These sections

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were assumed to be of constant thickness for all the tests. By using a specially designed emulsion-coating apparatus (Kopriwa, I 967), test specimens supported on a microscope slide were coated with a monolayer of one of the three emulsions. After an appropriate exposure period, the emulsion was processed with one of a series of developers used in the investigation. Many different combinations of emulsion, developer, development time, and processing temperature were used with separate test sections in each case. Some examples of the reaction intensities observed are illustrated in Table I. It was observed that for all three emulsions D19b developer consistently gave a higher reaction intensity than the other developers tested. Salpeter (personal communication) also measured efficiency using 1000-A sections of metha~rylate-~H.For comparable conditions of processing and emulsion thickness, the efficiencies observed were within 20% of those obtained with slides coated with a thin radioactive layer (Bachmann and Salpeter, I 967). Recently, Vrensen (1970) used an approach similar to that of Kopriwa (1967) to study both the effects on efficiency of varying the type of emulsion, exposure time, and development procedure and, also, the effect of section thickness on efficiency. Efficiency was expressed as: Number of grains over the source Efficiency

=

x

I00

Number of disintegrations in the source Values for efficiency in relation to sections with a gray interference color (mean thickness 430 A) for L4 and NUC-307 emulsions, after processing in Dr 9b or Elon-ascorbic acid preceded by gold latensification (GEA), are included in Table I. When sections with differing interference color (and therefore varying thickness) were coated with L4 emulsion and developed in D19b, it was observed that an inverse relationship between section thickness and efficiency exists. Increasing the section thickness from about 400 to around 1600 A resulted in a change in efficiency from 3 5.7 ( I grain per 2.8 disintegrations) to 19.5 ( I grain per 5.2 disintegrations). Increasing the section thickness still further to about I p resulted in a further efficiency change to I 1.0 ( I grain per 9.1 disintegrations). Within the range from about 400 to 1010 A (equivalent to sections with gray to light-gold interference color, according to the data of Salpeter and Bachmann, 1964), the efficiency changed from 35.7 to about 25.6 ( 1 / 2 . 8 to 1/3,9) that is, an absolute efficiency change of about IO%, or a relative reduction in efficiency of 2 8 yo. These observations are not consistent with the conclusion of Bachmann and Salpeter (1967) that section thickness has no influence on efficiency provided the sections are not thicker than 1000 A. In Vrensen’s (1970) study the standard deviation from the mean efficiency was high, especially for sections in the 400- to 500-A range. The deviations were large enough (*8 to hz5y0 of the mean values) to make it uncertain

TABLE I

EFFICIENCY OF ELECTRON MICROSCOPE RADIOAUTOGRAPHY ~

Emulsion

Undeveloped grain size (A)

Developer

Developed grain size (A)

Monolayer sensitivity (grains/disintegrations)”

Combined results expressed as Reaction Efficiency efficiency intensity [(grains/disinte[(grains/disinte(yoof D19b)~ grations) x I O O ] ~ grations) x I O O ] ~

~~

I!ford L4

Gevaert NUC-307

Kodak N T E

a

Microdol X Microdol X D19b Dr9b GEA

2000-4000 2740-3790 2190-3360

Microdol X D19b D19b D19b

1030-1830 1030-1680

Microdol X D19b Dektol

5 20-1 I 70 930-1430

Dektol

800-ljOO

GEA

400-600

Bachmann and Salpeter (1967). Kopriwa (1967). Vrensen (1970).

-

4 (see ref. c) 9.’ 36.5 32.7

100.0

-

-

-

-

0.8

8.0 100.0

-

-

-

-

800-1 5 00

I0

43.5

(700-A monolayer) 1/12 (1400-A layer)

1/23

I/8

-

14.6 (in air) 16.7 (in nitrogen)

10.3 14.6 16.7

4.6

0.2

100.0

3.7 4.4

-

-

12.5

G. C. BUDD

40

whether or not the apparent change of 10% in absolute efficiency in the 400to 1010-A-thickness range is as significant as it appears at first sight. Clearly, further studies are needed to resolve this question. The conclusion from recent studies is that for sections that are all of one color the radioautographic efficiency from section to section will probably vary by not more than zj yo and is likely to be less variable than this for sections with a silver or gold interference color. Such a conclusion is consistent with the observations of Williams and Meek (1965). It is difficult to compare the efficiency data obtained in various studies because the radiation sources and developing procedures vary. The existence of substantial self-absorption in thin sections and uncertainties as to the emulsion distribution add to the difficulties. It is possible, however, to show where differences occur and to indicate possible reasons for the discrepancies. The efficiencies observed by Vrensen for L4 emulsion developed with D19b were 3 to 4 times greatcr than those observed by Kopriwa using the same developer. By using a rather roundabout argument, it was suggested (Vrensen, 1970) that the discrepancy may be partly attributable to the use of an overlapping or double layer of silver halide crystals in Vrensen’s emulsion layer, while Kopriwa (1967) used monolayers of emulsion. Bachmann and Salpeter (1967) observed that there is a 60% increase in emulsion sensitivity when an L4 monolayer is increased to a double layer. In addition, it was suggested that increased self-absorption in Kopriwa’s sections (gold interference color -900-1 j o o A) as compared to Vrensen’s (gray interference color ~ 4 0 0 - - ~ 0A)0 and possible overexposure of the radioautographs could have contributed to the observed differences. The conclusions from these studies are that electron microscope radioautography can be applied for absolute quantitation provided the efficiency of the radioautography procedure is measured for each experiment. The factors that are most important in influencing efficiency when the radioautograph is prepared with sections supported on a flat surface are section thickness (Vrensen, 1970), emulsion type, distribution and thickness (Bachmann and Salpeter, 1967), and conditions of processing (Bachmann and Salpeter, 1967; Kopriwa, 1967). C. I.

RESOLUTION MEASUREMENT

Definition and Limits

In addition to knowing the efficiency of the radioautography technique, the position of radioactive sources within labeled biological specimens must be accurately defined. The accuracy with which the position of a source can be determined, or resolution, is dependent om the individual features of each radioautographic technique. The general factors that affect the resolution

DEVELOPMENTS I N RADIOAUTOGRAPHY

41

of electron microscope radioautography techniques are the same as those that influence radioautographic efficiency, namely, type of radiation, specimen and emulsion thickness, and proximity and conditions of processing in the development of latent images. Resolution may be defined numerically as the horizontal distance from the source to the point at which the probability of finding a developed grain is half that over the source (Doniach and Pelc, 1950). Using this definition, Car0 (I 962) determined for electron microscope radioautographs that a resolution of ~ o o o Ais theoretically possible when a close-packed monolayer of L4 emulsion is in contact with a section j 00 A thick. Experimental histograms of grain distribution around tritium-labeled virus particles and bacteria were in good agreement with this prediction. After considering more fully the photographic factors that limit resolution, Bachmann and Salpeter ( I 96 y ) calculated the resolution for two situations, one in which 3 yo-A sections were covered with a monolayer of N T E emulsion, the other in which rooo-A sections were covered with an L4 emulsion monolayer. They used a novel definition in which resolution was defined as the radius of a circle around a point source within which half the total grains produced from the source are found to occur. The predicted values for the two situations using NTE and L4 emulsion were, respectively, 770 and 18jo A. Theoretical predictions of the probable limiting resolution in electron microscope radioautography have been made based on the energy relationships of /3 particles of various energies passing through matter (Pelc e t al., 1961; Pelc, 1963). The best resolution that can be expected with photographic emulsions as the recording layer is about IOO A (definition of Doniach and Pelc, 1950). There is thus still some possibility for improvement of existing techniques. 2.

Experimental Determination of Resoltition

Recently, a method was devised for experimentally determining the distribution of grains in relation to an extended linear source lying parallel to the plane of an emulsion layer (Fig. 7) (Salpeter e t al., 1969). By using the radioactive line as a calibration source, histograms of the distribution of developed grains perpendicular to the center of the thin radioactive line were determined experimentally for various section thicknesses using L4 and N T E emulsions. For each histogram the distance from the center of the line, within which 10% of the total developed grains fell, was defined as the “half-distance’’ (HD). By normalizing the grain distribution in terms of units defined as HD divided by distance, it was observed that a universal curve of grain density distribution in relation to a linear source could be constructed. Experimental values for HD in relation to a line source are reproduced in Table 11. The error attributable

42

G . C . BUDD

FIG. 7. Electron microscope radioautographs of polystyrene-*H “hot line” sources sectioned at 500 A (gray interference color). (a) Coated with a monolayer of Kodak NTE and developed with Dektol(2 minutes). (b) Coated with monolayer of Ilford L4 and developed withp-phenylenediamine ( I minute). (c) Coated with a monolayer of Ilford L4 and developed with Microdol X (3 minutes). x 35,000. (From Salpeter ef a/., 1969.)

DEVELOPMENTS I N RADIOAUTOGRAPHY

43

TABLE I1 RESOLUTION IN RELATION TO

A

LINE SOURCE" Resolution, HD (A)

bmulsion Ilford L4 monolayer (1400 A)

Undeveloped grain size (A) 1000-1600

Kodak NTE double layer (1400 A)

3 00-5

Kodak monolayer (700 A)

300-1 5 0

a

jo

Developer

Gray section, 500 A

Gold section, 1200 A

Microdol X p-Phenylene diamine

'450 I 300

1650 I450

Dektol

I000

1250

Dektol

800

I000

Data from Bachmann and Salpeter (1967) and Salpeter ~ta/. (1969).

*IOO A or less. The HD for a section with a medium-gold interference color, coated with a monolayer of L4 emulsion (purple interference color) and developed in Microdol X, was measured to be 1670 A. The best resolution observed in this study, which is the highest resolution observed in any radioautographic technique, was obtained when 100-A (gray interference color) sections were coated with a monolayer of N T E emulsion which was developed in Dektol. The observed HD was 800 A. These observed values agree very well with the predictions (Bachmann and Salpeter, 1965). Recent observations using a 14C-labeled line source indicate that for similar conditions of emulsion distribution and section thickness the HD values are poorer than those for tritium by a factor of 1.5-2.0 (Salpeter, personal communication). Using these experimental results as a basis for further theoretical consideration, Salpeter and co-workers went on to determine the calculated grain distribution over and around hypothetical sources which resembled in size and shape potential sources commonly encountered in cytological material. These included circular sources and band sources which where either uniformly labeled (i.e., solid disc source or solid band source) or preferentially labeled at the periphery (hollow circular source or hollow band source). From these theoretical data it is possible to generate new curves for other special types of sources (Salpeter e t al., 1969). These theoretically determined curves, having experimental data as their base, are likely to be of great potential value in quantitative experiments. I n those studies in which theoretically derived

to statistical fluctuations was estimated to be

44

G. C . BUDD

curves have been used for comparison with experimental determinations of grain density distribution in relation to suspected sources, it has been demonstrated that labeled sources can be defined with greater precision than was previously the case (Israel e t a]., 1968; Budd and Salpeter, 1969) (Fig. 8). The use of such resolution curves offers new possibilities for increased precision in localizing radioactive sources with electron microscope radioautography. The same principle is also applicable to light microscope radioautoPPhY.

D. ABSOLUTEQUANTITATION The value of electron microscope radioautography as a cytological tool has been clearly demonstrated in many studies published within the last 10 years. Most of these studies have given information on the localization of a labeled end product of biochemical activity, for example, newly synthesized D N A (Revel and Hay, 1961), or have provided semiquantitative data on the relative distribution of radioactivity among the organelles and specific granules in secretory cells (see Figs. 9 and TO) (Caro and Palade, 1964; Jamieson and Palade, 1966; Lane e t al., 1764; Budd, 1964, 1966; Neutra and Leblond, 1966). More recently, data on the efficiency of various emulsion and developer combinations (Bachmann and Salpeter, 1967; Kopriwa, 1967; Vrensen, 1970) and resolution measurements (Salpeter et a/., 1969) paved the way for determining, from the density of developed grains in an radioautograph, the absolute amount of radioactive material within a labeled specimen. Before quantitation was possible, however, further factors had to be considered. One of these is latent image fading. During a prolonged exposure of a layer of emulsion to radiations from a labeled specimen, a latent image formed early in the storage period may fade because of slow reoxidation (Ray and Stevens, 1953). It is therefore essential to detect and control this process if accurate quantitation is to be achieved. Some data are available concerning latent image fading in the emulsions used for electron microscope radioautography. When layers of NTE emulsion, previously irradiated with known doses of 5 - and Io-keV electrons, were stored in air for periods up to 2 months, a 60% reduction in (Dektol) developed grains was observed (Salpeter and Bachmann, 1964). The effect could be eliminated by storing the emulsion in gaseous helium. No similar latent image fading was observed with L 4 emulsion (Microdol X-developed). Recently, Vrensen ( I 970) confirmed that there is no latent image fading when L4 emulsion is stored at 4°C in air for periods up to 40 days. Under the same conditions latent image fading was observed in NUC-307 emulsion, although the effect was not as marked as that observed by Salpeter and Bachrnann with N T E . The fading effect in the NUC-307 emulsion was eliminated by storage in a nitrogen atmosphere. Another factor that must be considered before quantitation can be achieved

FIG. 8. Electron microscope radioautographs of adrenergic nerve terminals in the pineal body of mice injected intravenously with n~repinephrine-~FI.The radioautographs were prepared at 8 minutes (A) and 30 minutes (B) after injection. Most developed grains overlie granular synaptic vesicles at both labeling intervals, as confirmed by quantitative measurements of grain distribution. x 63,000. (From Budd and Salpeter, 1969, and unpublished observations.)

FIG. 9. Electron microscope radioautographs of goblet cells in the intestine of mice prepared I hour after injection of sodium ~ulfate-’~S.Radioactivity is distributed throughout all mucigen granules in cells near the base of an intestinal crypt (A, x >zoo) but is concentrated in mucigen granules close to the Golgi cisternae in cells near to the outlet of a crypt into the intestinal lumen (B, x 8000).

FIG. 10. Electron microscope radioautographs of cells in early gastrulas of sea urchins (Lyfechinus pictus) labeled with ~ r i d i n e - ~ HSections . were exposed to L4 emulsion. After development in D19b (A) grains form long randomly coiled filaments. p-Phenylenediamine developer (B)produces short thick filamentous or punctate grains. x I 5,000. (From Claybrook and Budd, unpublished observations.)

48

G. C . BUDD

is the possible chemical or physical interaction between the specimen and emulsion. Positive chemographic effects tend to give a false increase in grain yield in an emulsion layer directly over the specimen. Negative chemography results in reduced grain yield over the specimen. These effects are discussed at length by Rogers (1967). A very strong negative chemographic effect was observed when NTE emulsion was in direct contact with histological sections (Salpeter and Bachmann, 1964). A small effect was also seen when L4 emulsion was developed in p-phenylenediamine. In both cases the effect was abolished by inserting a 30- to 60-A carbon layer between the specimen and emulsion. Such a layer is likely to have a small eAect in reducing resolution. With the knowledge obtained in these studies, it was possible to determine the absolute number of acetylcholinesterase molecules at the skeletal neuromuscular junction (Rogers et al., 1966; Salpeter, 1968). Subsequently, the total number of diisopropylphosphorofluoridate (DFP)-sensitive sites (some of which were identified as being in acetylcholinesterase) at the neuromuscular junction were determined (Salpeter, 1968). The use of DFP-3H in these and other radioautographic studies has been reviewed recently (Barnard, I 970; Budd, 1970). For accurate quantitation the resolution of the radioautographic technique should be known (for discussion, see Section 111,B). When the resolution is known, it is possible to compute for the total grains produced by a given source the fraction that will occur over the source and the fraction that will be scattered beyond its boundary. This fraction is small when large structures (for example, whole nuclei) are considered but increases markedly for structures approaching point sources. Salpeter and her associates ( I 969) have published integrated curves of grain distribution from which it is possible to see the percentage of total grains expected to fall over a radioactive structure and the percentage that falls outside.

IV. Light Microscope Radioautography with Thin Sections and Emulsion Layers It is common practice in many electron microscopy laboratories to section epoxy- or methacrylate-embedded tissue at I p for light microscope studies. Sections in the thickness range of approximately 0.25-1 p are usually visualized after staining with toluidine blue or other stains. Alternatively (or in addition), such sections can also be observed with phase-contrast optics. Sections in the 0.25-I p range may also be used in radioautographic studies where they offer several advantages over thicker sections obtained from frozen or paraffin-embedded tissue. An important advantage is the increased optical resolution of cytological detail that results because there is less superimposition of cells or cell components than is usually the case when thicker sections are

DEVELOPMENTS I N RADIOAUTOGRAPHY

49

used. The presence of transparent embedding medium, which need not be removed, helps to support cell components in a correct spatial relationship to each other. When section thickness is reduced, it is to be expected that radioautographic resolution will improve (Pelc, 1962). Reduction in thickness of the emulsion layer also has the effect of improving radioautographic resolution for most 8-emitting radioactive isotopes except tritium. In the case of tritium, the average energy of radiation is such (7.5 keV) that most 4, particles hitting the emulsion lose their energy within the first one or two layers of halide crystals, and only a slight change in resolution is likely to be observed when a multilayered emulsion is compared with a monolayer. That this is true in practice has been recently observed in experimental measurements of resolution in relation to radioactive line sources labcled with tritium (Fig. 11) and carbon-14 (Budd

FIG. I I . Light microscope radioautograph of a linear source of poly~tyrene-~H. Section thickness 0.5 p. AR-10 emulsion developed in D19. x stoo.

et a]., 1971). When 0.5-p sections were used, the HD (Salpeter e t a/., 1969) for a tritium line was 0 . 4 ~ for a multilayered emulsion (Kodak AR-10) and a monolayer emulsion (L4) of comparable undeveloped grain size. Maximum resolution in light microscope radioautography, limited only by the optical resolving power of the microscope, is therefore achievable when sections of about 0 . 2 5 - to 0.5-p thickness are covered with a close-packed monolayer of fine grain emulsion (when the isotope is tritium, thicker emulsion layers, e.g., Kodak AR-10, can be tolerated).

V.

Electron Microscope Radioautography of Diffusible Substances

It was inevitable that attempts would eventually be made to develop a method for visualizing diffusible radioactive substances with the electron

50

G . C . BUDD

microscope. One logical way to do this might be to modify existing diffusible compound radioautography techniques that have been developed for use with the light microscope. The changes that must be made include the preparation of very thin specimens in which the diffusible materials under investigation have been immobilized. Attempts have been made in several laboratories to cut ultrathin sections of frozen tissue for electron microscopy with variable success (Bernhard and Nancy, 1964; Fernhndez-Morin, 1966; Bernhard and Leduc, 1967; Christensen, 1969; Appleton, 1969). Using an ultramicrotome in a cryostat at -3 5 "C, Bernhard and associates (Bernhard and Nancy, 1964; Bernhard, 1965; Bernhard and Leduc, 1967) sectioned tissue that had been fixed and embedded in gelatin. The sections were of good quality and were useful in ultrastructural cytochemical studies (Leduc e t a/., 1967) but are likely to have limited use for diffusible substance radioautography. Recently, Appleton (1969) described a technique for cutting ultrathin sections of unfixed liver and kidney, using a specially modified thermal feed ultramicrotome in a cryostat at a temperature within the range -60" to -80°C. Fairly thin sections could be cut without much difficulty, but their transfer to an electron microscope grid was more of a problem. Section transfer was eventually accomplished by touching a cold stainless steel grid, previously coated with Formvar and L4 emulsion, against a ribbon of frozen sections. After transfer the sections were freeze-dried in a cryosorption pump (Stumpf and Roth, 1967) prior to viewing. Although this procedure represents a first step in the direction of a radioautographic method, Appleton did not carry out the sequence of steps under safelight conditions but instead fixed the emulsion in sodium thiosulfate before picking up the sections. It was observed that sections prepared in this way can withstand electron irradiation under vacuum without tending to disintegrate. Cellular detail was visible in electron micrographs of the fixed liver, and structures resembling sectioned mitochondria were observed in unfixed kidney sections (Fig. 12). Christensen (1969) modified an ultramicrotome in a different way to obtain thin sections of frozen, fresh tissue. A standard ultramicrotome was used at room temperature after making the following modifications. The chuck used for holding frozen tissue was made from an ebony bar which extended vertically down into a bowl-shaped, insulated flask containing a knife holder for a diamond or glass knife. The inside of the bowl was cooled with cold nitrogen gas at a controlled temperature between -40" and -1zo"C. During operation a temperature of -75°C was used. As was observed also by Appleton (1969), using a similar temperature, ribbons of sections were readily obtained. It was found that sections could be transferred with a fine wire probe to a grid kept at the same temperature. Flattening and adherence of the sections to each grid was accomplished by pressing them with the end of a copper rod. Freezedrying was achieved with a jet of anhydrous nitrogen gas at -75OC for

DEVELOPMENTS I N RADIOAUTOGRAPHY

FIG. 1 2 . Electron micrograph of part of a hepatic cell in a section of rapid frozen, glutaraldehyde-fixed but unembedded mouse liver. A portion of the nucleus (upper left) and cytoplasm including granular endoplasmic reticulum can be seen. Light areas may represent the position of mitochondria or ice crystal damage. x 50,ooo. (From Appleton, 1969.)

52

G . C. BUDD

30-60 minutes. The cellular detail observed in sections of rat liver very closely resembled that seen in A[Jpleton’s (1967) study. So far, no results of attempts to localize diffusible substances by combining this technique with radioautography have been published, although there have been plans to localize steroids in this way (Christensen, 1967). It is to bc hoped that these techniques can be developed before long into a useful method for localizing diffusible substances with greater resolution than is possible with the available light microscope techniques. When this has been done, the resolution curves for circular and band-shaped structures developed by Salpeter e t a/. (1969) will permit the resolution of diffusible substances to be checked. Only if the values resemble those obtained with nondiffusible sources will it be possible to confidently state that diffusion has not occurred. In a novel approach to the problem of localizing diffusible compounds, Eckert (1968, 1769) modified the apposition technique in the following manner. Frozen sections cut on a cryostat microtome were placed on a thin polyethylene sheet supported on a slide. The sections were then pressed against an emulsion-coated slide at --20°C and stored between -25’ and -30°C during exposure. After exposure the polyethylene sheet was removed, the sections remaining attached to the emulsion. After brief immersion in 5 % glutaraldehyde solution at low pH, the emulsion was processed in photographic developer and fixer. A more prolonged immersion in glutaraldehyde followed, succeeded by dehydration and embedding of the whole radioautographic preparation in Epon. After separating the glass slide from the embedded preparation, thin sections were cut on an ultramicrotome in a direction normal to the plane of the emulsion layer. Developed silver grains were observed in the secfioned emulsion layer, adjacent to the tissue. A problem inherent in this approach is that there is a probability that because of radiation spread any observed silver grain in the sectioned emulsion layer may result from radioactive disintegrations in a region of the tissue specimen not included in the section. When tritium is the isotope and monolayers of L4 emulsion are used, however, the radioautographic resolution is such that on the average -75 yo of developed grains can be expected to occur within 2900 A of a point source situated within 1000A perpendicular to the emulsion layer, and 5 0 yo of the grains will be within 1450A of the source (calculated from data of Salpeter e t d.,1969). From this it may be inferred that when sections with a pale-gold interference color are selected (-1000-A thickness) most of the grains produced by radiations from a point source close to the emulsion layer will occur within a series of three or four adjacent sections. Many radioactive sources within tissues are larger than point sources. It follows that such a source will produce a similar silver grain image in two or more adjacent thin sections. By observing a series of such sections it should therefore be possible to accurately locate immobilized diffusible and nondiffusible sources

DEVELOPMENTS I N RADIOAUTOGRAPHY

53

in double-sectioned frozen tissue. At the present time, this method appears to offer the best chance for success in locating diffusible substances with high resolution.

VI.

Summary

Advances have been made recently in several areas of radioautographic technique. I n addition, a more complete knowledge of the resolution limits and efficiency of the available methods has evolved. Several methods have been developed which allow diffusible materials to be located in whole cells or frozen tissue sections. The success of these methods in the area of light microscope radioautography has stimulated renewed interest in ultra-thin frozen sectioning for electron microscopy. These studies are preparatory to the development of high resolution radioautographic methods for diffusible materials. The use of standardized tritium and carbon-14 sources which resemble sections of biological tissue has enabled the limits of resolution and sensitivity of electron microscope radioautographs to be defined precisely. Using similar sources the resolution of light microscope radioautographs with specimens 0.25-1 p thick has also been determined. It is likely that present and future work will continue to lead to improved radioautographic efficiency and increased knowledge of the location of diffusible and nondiffusible substances in cells. The current trend, therefore, is to continue to develop radioautography as a qualitative and quantitative method for visualizing biochemical reactions within undisrupted cells and tissues. ACKNOWLEDGMENTS The author gratefully acknowledges the donation of recent material from: Drs. T. C. Appleton, H. Eckert, R. Claybrook, S. R. Pelc, G. Rowden, M. M. Salpeter, and W. E. Stumpf. Some of this material is included in the review with the author’s permission. Thanks are also due t o Dr. M. M. Salpeter for collaboration in several studies discussed in the text. The technical help of Mrs. Sharon Mattimoe is also acknowledged. The author also acknowledges the use of a Phillips E. M. 300 electron microscope awarded to Dr. Leonard Nelson under NIH grant number ROI-5-HD-03266. REFERENCES Appleton, T. C. (1964). J . Roy. Microsc. Sac. 83, 2 7 7 . Appleton, T. C. (1966). J . Histochem. Cyfocbem. 14, 414. Appleton, T. C. (1967). J . Roy. il4icrorc. SOC.87, 489. Appleton, T. C. (1969). Its “Autoradiography of Diffusible Substances” (L. J. Roth and W. E. Stumpf, eds.), p. 304. Academic Press, New York.

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