Determination of subcellular elemental concentration through ultrahigh resolution electron microprobe analysis.

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REVIEW OF CYTOLOOY VOL. 58

1N"ATlONAL

Determination of Subcellular Elemental Concentration through Ultrahigh Resolution Electron Microprobe Analysis THOMAS E . HUTCHINSON Center for Bioengineering. University of Washington. Seattle. Washington 1. Introduction

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11. Physical Background of Elemental Analysis by Characteristic X-ray

Determination . . . . . . . . . . . . . . . . . . . I11 . Instrumentation Used in Elemental Analysis by Characteristic X-ray Energy Determination . . . . . . . . . . . . . . . . A . Wavelength-Dispersive Analysis . . . . . . . . . . . B . Energy-Dispersive X-ray Spectroscopy . . . . . . . . . C . Eiectron Energy Loss Spectrometry . . . . . . . . . . IV . Critical Reading of the Literature . . . . . . . . . . . . A . State of Tissue Prior to Specimen Preparation . . . . . . 8 . Specimen Preparation . . . . . . . . . . . . . . . C . Conditions of Analysis . . . . . . . . . . . . . . . D . Data Analysis . . . . . . . . . . . . . . . . . . V . Application of Microprobe Analysis to Specific Biological Systems A . Skeletal and Cardiac Muscle . . . . . . . . . . . . B . Smooth Muscle . . . . . . . . . . . . . . . . . C . Lung . . . . . . . . . . . . . . . . . . . . . D . Nerve . . . . . . . . . . . . . . . . . . . . . E . Epithelium . . . . . . . . . . . . . . . . . . . F . Kidney . . . . . . . . . . . . . . . . . . . . G . Calcifiable Tissue . . . . . . . . . . . . . . . . H . Blood . . . . . . . . . . . . . . . . . . . . . 1. Gametes and Developmental Biology . . . . . . . . . J . Chromatin in Mitosis and Aging . . . . . . . . . . . K . Microorganisms . . . . . . . . . . . . . . . . . L . Medical Diagnosis . . . . . . . . . . . . . . . . M . Elements of Particular Interest . . . . . . . . . . . . V I . Methods and Reviews . . . . . . . . . . . . . . . . VII . Conclusions . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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117 120 121 122 125 129 129 130 131 134 134 134

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137 138 139 141 141 142 144 146 146 147

148 150 151 153

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I Introduction Examples of mediation of cell function by elemental concentration gradients on a subcellular level are numerous; one which readily comes to mind is the 115

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Copyright @ 1979 by Acadcmh Press Inc . All rights of reproduction in any form ~ r c w c.d ISBN 0-12--358-9

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THOMAS E. HUTCHINSON

influence of Ca translocation within muscle cells on the contractile process. It seems probable that many other biological phenomena could be better understood if data were available on the position of elements within cells during particular phases of cell activity. In the past a variety of methods has been used, with varying degrees of success, to determine localized elemental concentrations. These range from delicate techniques using ion-selective microelectrode probes to cell disassembly and selective centrifugation as a means of isolating particular cell organelles. Most of these techniques, however, are fraught with problems. Microelectrodesare selective for only one element per electrode, and the diameter of the region probed is in excess of several micrometers, and the microelectrode technique is thus limited in spatial localization. The limit of sensitivity is in general greater than desirable because of the small size of the region probed. One of the most important relationships in biology is that between the distribution and migration of ions in living systems and subsequent cell function. The technique of cell fractionation, however, yields sufficient quantities of individual components and allows rather precise quantitative chemical analysis, but does not permit analysis of the cell in any form resembling the living state. Cell fractionation also has the serious technical problem of not consistently providing clean preparations of individual organelles within the cell and requires larger numbers of cells for analysis than may be convenient to acquire. The requirement of large numbers of cells also implies that variations in elemental concentration among cells cannot be determined; rather, an average concentration is obtained. As a result, biologists have relied upon rather standard techniques of either chemistry or electrochemistry to make concentration analyses. These techniques, while having excellent limits of detectability for a variety of elements, are highly limited in attainable spatial discrimination and cannot be used to make in vivo analyses without disrupting the system. The physical sciences, on the other hand, have long made use of more sophisticated instrumental techniques for determining elemental concentrations. Among the methods developed is electron-induced x-ray emission with subsequent wavelength or energy determination. Until recently, this method was used mainly by metallurgists and geologists in studying inorganic materials. Lately, however, investigators have come to appreciate the effectiveness of the technique as applied to biological systems. The increasing interest in this application is closely coupled with significant advances in electron optical instrumentation. In particular, scanning electron microscopes have reached a new lower limit of spatial resolution. This is of great significance, since the region of electron microprobe analysis depends, to the first order in thin sections, on the diameter of the electron probe beam. Currently, the smallest electron probe used in conventional scanning transmission electron microscopy is about 2.0 nm, and the smallest probe used in sophisticated field emission electron microscopy is less

DETERMINATION OF SUBCELLULAR ELEMENTAL CONCENTRATION

117

than 0.5 nm. This lower limit of spatial resolution, coupled with the modification by instrument manufacturers of general-purpose instruments to make them useful in this type of examination, makes possible the investigation of subcellular regions of cells and tissue through electron microprobe analysis.

11. Physical Background of Elemental Analysis by Characteristic X-ray Determination The physics of x-ray microanalysis is relatively simple, although the instrumentation required to produce quantitative determinations of elemental concentrations by this technique is quite complex. Electrons striking a specimen give rise to several forms of radiation. Backscattered electrons are emitted as a result of elastic interactions of the incident electrons with the atoms of the specimen and have energies which are virtually unchanged from the incident electron energy. Secondary electrons, which have energies much less than that of the incident electron beam-typically 50 eV or less-are also emitted. These electrons are relatively large in number and are used in scanning electron microscopy (SEM)for image formation. Transmitted electrons are available from thin specimens and s 1 ~ eused for imaging in the transmission mode of operation. Several other types of radiation are emitted which are particularly useful in microanalysis. Incident electrons passing through the specimen may ionize atoms by the removal of electrons. When this ionization occurs, the energy levels of the atoms which have been depopulated are available to higher-energy-level electrons which then occupy these vacancies and therefore experience a reduction in energy. The transition of electrons from higher to lower energy states can result in photon production (probability = o, the fluorescence yield). Since these energy levels are quantum mechanically sharp with respect to energy, a transition between levels gives rise to photons of unique energies characteristic of the energy level arrangement in the target atom. The energy and wavelength of the photons are related by Einstein’s relationship: E = hclk

where E is the energy of the photon, h is Planck’s constant, A is the wavelength, and c is the speed of light. Determination of either the energy or the wavelength of the radiation from a particular characteristic transition allows identification of the element from which the radiation was emitted. Low-energy, long-wavelength radiation gives rise to photons in the visible part of the wavelength spectrum. Analysis of the elemental composition in this case is reduced to the determination, using optical instrumentation, of the emitted wavelength known as the electron-induced photoluminescence. A more widely used technique is detection

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FIG. 1 . The energy level configuration of electrons around the nucleus of an atom. Incident electron interaction causes vacancies to occur at lower energy (smaller radius) levels which are filled by higherenergy electrons from outer shells. The difference in energy is compensated for by the production of an x ray characteristic of the transition.

and characterizationof the high-energy, short-wavelength photons classified as x rays. Figure 1 , which considers only the nucleus and surrounding electron shells of an atom of the specimen, illustrates the manner in which characteristic x rays are emitted. When an electron is removed from the K shell by incident electron

Energy of X-ray

FIG.2a.

Phofons In ksV


119

interaction, an electron of the L-shell can reduce its energy by dropping to the K level, giving rise to what is termed K, radiation. Alternatively, M-shell electrons can drop to the K-shell, giving rise to K, radiation. The likelihood of L-shell to K-shell transitions is higher than that of N-shell to K-shell transitions. While K, and KBradiations are seen simultaneously during electron bombardment of large numbers of atoms, the intensity of the K, radiation is higher by roughly a factor of 5 for elements of biological interest irradiated with 20 to 100-keV electrons. In

\

\

\

\

\

NUMBER OF PHOTONS

PI

\

\

X

\

\

PHOTON NUMBER RECORDED BY X-RAY DETECTOR

BREMSSTRAHLUNG

10

2.0

3.0

4.0

5.0

60

Energy of X- roy Photons in keV

10

2.0 30 40 Energy of X- roy Phclons in keV

50

60

FIG. 2. Spectral components from x-ray energy analysis showing characteristic peaks (a), the bremsstrahlung component, (b) and the combined contributions (c).

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addition, since K, photons arise from L-K transitions which are lower in energy than M-K transitions, K, radiation is lower in energy than K, radiation. L, radiation occurs when transitions between the M and L shells take place, that is, when an L-shell electron is ejected from the atom. The difference in energy between the M and L shells is less than that between either the L and K shells or the M and K shells, thus L, radiation is lower in energy than K radiation. A spectral diagram is shown in Fig. 2a. The two characteristic peaks seen in this figure arise when the specimen consists entirely of Na and C1 as in a stoichiometric composition of NaCl. The characteristic K, x ray emissions for Na take place at an energy of 1.07 keV and for C1 at 2.6 keV. The gaussian spread in energy indicated in this diagram is due to instrumental noise rather than energy uncertainty in the characteristic radiation. The area under each of the curves is proportional to the number of atoms excited by the electron beam and thus serves as a basis for quantitative elemental microanalysis using x radiation. The characteristic radiation emitted from the specimen has very sharp characteristic energies. In addition, a continuous radiation, or bremsstrahlung, is emitted as a result of the slowing down of electrons within the specimen. The energy dependence of the bremsstrahlung is shown in Fig. 2b. Since this radiation is not a result of electron transitions within the atoms involved, but is due to a deceleration of the incident electrons themselves, it is continuous in energy. The number of such x-ray photons of all energies is proportional to the incident electron flux and the number of interactions which take place within the specimen. Clearly then, a thin specimen, with which fewer incident electrons interact, yields a lower bremsstrahlung photon number than a thicker specimen and the proportionality between thickness and bremsstrahlung yield can, as suggested by Hall (1968), be used to estimate the thickness of the specimen, which is difficult to measure directly in these studies. The total energy spectrum of x radiation emitted from the specimen is a combination of these two contributions, as shown in Fig. 2c. The sum of the characteristic radiation and the bremsstrahlung comprises the total spectrum of x-ray photons emitted from the specimen.

111, Instrumentation Used in Elemental Analysis by Characteristic X-ray Energy Determination

The instrumentation for ultrahigh resolution elemental microanalysis can be divided into three categories: x-ray wavelength-dispersive spectroscopy, x-ray energy-dispersive spectrometry, and electron energy loss spectroscopy.

DETERMMATION OF SUBCELLULAR ELEMENTAL CONCENTRATION

12 1

A . WAVELENGTH-DISPERSIVE ANALYSIS As noted in the preceding section, the number of x rays having a characteristic energy or wavelength which arise from a specimen is dependent upon both the number of atoms present with the characteristic transitions giving rise to these x-rays and the number of electron-atom interactions which occur. Possibly the oldest form of microanalysis is that which employs high-current, large-diameter electron probes coupled with a wavelength-dispersivespectrometer. A schematic diagram of the wavelength-dispersive system is given in Fig. 3. X rays arising from electron beam interaction with the specimen impinge upon a diffracting crystal. Most of these x-rays are scattered by the diffracting crystal without reinforcement in any particular direction; however, certain x rays which satisfy Bragg’s law:

n h = 2d sin 8 are diffracted with high intensity to the x-ray counter. In Bragg’s equation, A is the wavelength of the characteristic x ray, 8 is the angle between the planes of the diffracting crystal and the incident x ray, and d is the distance between crystal planes in the diffracting crystal. It can thus be seen that, when the angle between the incident x ray and the diffracting crystal, and between the diffracting crystal and the x-ray counter, is set, constructive interference leads to satisfaction of Bragg’s law for selected values of the wavelength. By determining the number of counts entering the x-ray counter as a function of the angle 8 for a known value

m

r-

GAS FLOW X-RAY COUNTER

\

L

ELECTRON BEAM

~

DIFFRACTING CRYSTAL

~

FIG.3. Schematic drawing of a typical wavelengthdispersive x-ray analysis system. X rays having the appropriate wavelength and correct angular acceptance by the diffracting crystal are directed with high intensity to the gas flow x-ray counter which records the presence of the incident x rays. The angle between the x ray incident upon the crystal and the diffracted x ray must be mechanically adjusted to record x rays of differing wavelength.

~

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of d, Bragg’s law can be used to generate a counts-versus-wavelength spectrum. The spectrum thus generated is similar to that shown in Fig. 2c. In addition to this spectrum, a continuous x-ray counter is also included in this system, which allows normalization of the generated data as a function of fluctuating electron beam current and other changing parameters. The advantage of the wavelength-dispersive x-ray system lies primarily in the high resolution which can be obtained in separating closely adjacent spectral peaks. Most peaks of biological interest in closely separated wavelength positions in the spectrum can be resolved by this technique, which is not always the case with the energydispersive system, as will be seen in Section III,B. The second advantage of this technique is that the very low-energy x rays from C, N, and 0 can be detected using special high-sensitivity, windowless systems. This is certainly an advantage in certain biological systems but is not necessarily essential to all studies. There are two disadvantages to this technique. First, a very low efficiency for collection of x rays is inherent in the system. This is due to mechanical difficulties in placing the diffracting crystal in such a position that the solid angle, as seen from the specimen, is large. This drawback is apparently unavoidable, and it seems that little can be done to improve the low collection efficiency. The second disadvantage is related to the fact that the system is basically mechanical and is therefore dependent upon setting a particular angle between the diffracting crystal and the specimen; collection of the spectrum is in essence a sequential process. Because of this, the specimen must be exposed to the electron beam for an excessive length of time in order to collect a complete spectrum for several elements. The large losses of elemental constituents which may occur in biological materials under these conditions further complicate the quantitative analysis of subcellular elements using this technique. B. ENERGY-DISPERSIVE X-RAYSPECTROSCOPY The second technique is energy-dispersive x-ray analysis. This technique depends upon instrumentation which is basically a product of semiconductor technology. X rays of a variety of energies are generated by the specimen and impinge upon a silicon crystal as shown schematically in Fig. 4. This silicon crystal is coated on each side with a thin layer of gold and is isolated from the vacuum of the electron microscope by a thin beryllium window. Upon striking the silicon crystal, the x ray releases its energy in a series of collision events, each of which gives rise to one electron and one positively charged unit known as a hole. A bias voltage of 1000 V exists between the gold layers at the surface of the silicon crystal. As a result, the electrons and holes are attracted to opposite sides of the crystal and collect at the interface, causing a pulse of current to occur. The number of electron-hole pairs generated is directly proportional to the


123

DATA STOR&€ -ELECTRON

SOURCE

CMJDENWR LENS1 ELECTRON E A M CONDENSOR LENSH MINCOMPUTER MTA PROCESSOR

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s,

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DATA INTERRUPTS

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OBJECTIVE LENS SPECIMEN

IMAGING LENSES

FIG.4. Schematic drawing of a modem elemental microanalysis electron microscope. Both the Si-Li energy-dispersive x-ray detector unit and the electron energy loss analysis spectrometer are interfaced with a high-resolution scanning transmission electron microscope to form a complete elemental microanalysis system.

energy of the x ray entering the crystal. Thus the current pulse arising as a result of x-ray absorption within the crystal is proportional to the x-ray energy. The entire crystal is cooled to near liquid N temperature in order to prevent undesirable thermal electron events from occurring which would enter the spectrum as background current. The current pulse is amplified by a field effect transistor, and the resulting signal further amplified by a low-noise solid-state amplifier in an adjacent console. The pulse is converted from a current pulse to a voltage pulse, the height of which is proportional to the energy of the incident x ray. The height of this voltage pulse is then measured by a pulse height analyzer and stored in a preselected memory location of a minicomputer. The particular location selected is determined by the height of the pulse and thus the x-ray energy. The information accumulated in the computer after a significant period of time is a spectrum which has as its ordinate the x-ray energy, and as its abscissa the

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number of pulses proportional to the number of x rays which entered the silicon crystal of this particular energy. The total spectrum is displayed on a television screen, a diagram of which is shown in Fig. 2c. In general, several data manipulations are performed using the software routines of the minicomputer. Software routines are available which, to a greater or lesser extent, remove the bremsstrahlung background from the characteristic x-ray data. This is a rather extensive and difficult procedure, and a satisfactory routine for full removal of the bremsstrahlung has not yet been perfected. A second manipulation which can be performed is Gaussian peak extraction. In this routine a selected peak in the spectrum can be removed in order to reveal adjacent spectral peaks hidden by shoulders of the primary peak. This is extremely valuable in providing full quantitation with the procedure. In addition, a spectral smoothingroutine can be implemented, which uses averaging techniques in order to make small peaks more obvious. This routine is not considered particularly desirable, since repeated smoothings can actually cause pseudopeaks to occur which arise from noise inherent in the background. In order to obtain fully quantitative data from this technique it is necessary to make a significant number of spectral analyses. The technique of spectral analysis has been discussed extensively by Shuman et al. (19761, and the reader is referred to their article for further details. In summary, however, the method consists of obtaining the Gaussian peak area of the characteristic x-ray emissions by a digitizing routine provided with modem systems. The peak area P is the total area of the peak and contains a certain background component, as seen in Fig. 2c. An estimate is made, either by an automatic computer routine or by visual inspection, of the background component of this peak. The background b is then subtracted from P, the peak area, yielding a total Gaussian peak area proportional to the number of atoms giving rise to the peak. The constant of proportionality is based on &heexcitation efficiency of the particular element, the particular electron shell by which the peak was generated, and the efficiency of the detector in recording x-rays of this energy. Each of these factors enters into the equation determining the concentration. In addition, it is obvious that the total peak area is proportional to the electron dose striking the volume of the specimen. Thus the peak area is proportional to the number of electrons per unit area per unit time which strike the specimen, the time of irradiation, and the total volume of material irradiated. A simple way to normalize these data was suggested by Hall (1971), which makes the use of the bremsstrahlung or white radiation arising from noncharacteristic emission of x rays as outlined in Section I. The bremsstrahlung or white radiation, denoted by W ,is proportional to the total electron dose and the thickness of the specimen. Thus the net concentration of any characteristic element C, is

c, - (P - b)/W


125

This equation is valid in the limit of small thicknesses of the specimen (roughly 200 nm) and is known as the thin-film or thin-section approximation. The logic behind this limit is that for thicker specimens x rays arising from emission from higher-atomic number (2)elements can be absorbed by lower-Z elements and give rise to secondary x-ray emissions prior to exit from the specimen. This phenomenon, known as absorption and reemission, yields an anomalously lower than accurate elemental composition for higher-Z elements and correspondingly greater than correct apparent concentrations for lower-Z elements. This effect can be compensated for through the use of a sophisticated theory of x-ray cross section requiring computer analysis (Hall, 1971). With the use of the procedures outlined in this section, it is possible to obtain quantitative concentration data from an energy-dispersive x-ray spectrum through the use of a set of standards. Elemental standards suitable for the quantification of energydispersive x-ray analysis have been fabricated by Chandler (1976) and Spurr (1975), using ionic salts in a polymer matrix. The salt-matrix composite is sectioned into thicknesses within the limit of application of the thin-film approximation. Further studies have been done by Hutchinson and Borek (1977) in which frozen thin-film ionic aqueous solutions were examined in order to calibrate the x-ray spectrum. Each of these standards has particular advantages. The composite method allows examination at room temperature, whereas the method of Hutchinson and Borek requires the use of a cold transfer stage for the examination of standards. The Hutchinson and Borek method more closely approximates frozen hydrated tissue, however. An advantage of the energy-dispersive system over the wavelength-dispersive system is that higher detection efficiencies are possible, since the Si crystal can be moved to within a short distance of the sample, typically on the order of 1 cm. Solid angles of %nhave been attained. A second clear advantage is that the spectrum is obtained simultaneously for elements having atomic numbers greater than that of Na. This much reduces the radiation damage which occurs during the long exposures needed for complete spectra to be obtained from wavelengthdispersive detectors. A major disadvantage is the inability of the system to resolve closely adjacent peaks, however, this has been mitigated to some degree by data manipulation in the computer as outlined above. C. ELECTRON ENERGY Loss SPECTROMETRY

There are two alternative methods for performing elemental analysis of light elements in thin specimens. One is the analysis of Auger electrons emitted from the specimen as a process complementary to x-ray emission. The yield of Auger electrons is just 1 - o per ionization (w = fluorescence yield), so that for low-2 elements there is higher probability of producing an Auger electron than an x ray.

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However, because of the small escape depth of Auger electrons, the technique is limited to analysis near the surface, and quantitation is sometimes difficult. Moreover, the small escape depth poses a further experimental difficulty, since thin layers of contamination formed in medium vacuum systems can completely obliterate the Auger electron signal. The technique is therefore limited to use in high-vacuum systems (pressures less than 10-8 torr). The second alternative method of performing elemental analysis of thin specimens of the type used in transmission electron microscopy is to detect electrons which have lost characteristic amounts of energy in producing inner shell ionizations. This is done by placing a spectrometer beneath the specimen to analyze the energy of the electrons transmitted through the specimen. A typical electron energy loss spectrum for a thin film of biological material is shown in Fig. 5 . This experimentally obtained spectrum shows a large, relatively noncharacteristic energy loss peak due to valence shell excitations of about a 20eV energy loss and also characteristic peaks due to the excitation of N and K levels of C in the molecules present (guanine). Detection of the characteristic energy loss of transmitted electrons holds particular advantage for low-Z materials for two reasons. First, for each inner shell ionization, there exists an electron which has been transmitted through the specimen and lost a characteristic amount of energy in producing this ionization, regardless of the fluorescence yield o.That is, the yield of energy loss electrons to inner shell excitations and ionizations is unity. Second, electrons which have lost energy in the event are scattered through relatively small angles, particularly for l o w 2 elements (and correspondingly low-energy inner shell levels). For

/

NITROGEN K I

I

0

25

50

400

300 ENERGY

LOSS lev)

FIG.5 . A typical electron energy loss spectrum from a biological substance. The spectrum was obtained from a thin (--4M) A), sublimed film of guanine with an incident energy of 25 keV. The spectrum shows characteristic energy loss due to excitation of N and K energy levels, of C, as well as the relative intensity of these peaks compared to the -2O-eV energy loss peak due to valence shell excitations. Specturm courtesy of Dr. D. E. Johnson.


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instance, half of all electrons in the 10-100 keV energy range which have been inelastically scattered, and have lost an amount of energy E in the process, are scattered into angles smaller than about 2EIEo, where Eo is the incident electron energy. Therefore an electron spectrometer with a limited acceptance angle can still collect an appreciable fraction of the transmitted energy loss electrons. As compared to the situation in x-ray techniques, the net increase in collection efficiency due to the above two factors can be translated directly into an increase in elemental sensitivity or, for detection of a given concentration, into an increase in spatial resolution. The improvement in spatial resolution is possible because, with improved collection efficiency, less incident current (and thus smaller probe sizes) can produce the same count rate. A detailed analysis and experimental verification by Isaacson and Johnson (1975) indicates that, for example, over a range of atomic numbers from Z = 6 to Z = 20 the energy loss technique should be able to detect concentrations from 40 to 10 times smaller than the energy-dispersive x-ray technique for a given spatial resolution. And, for example, in the case of a hot-filament electron source where the beam current is a (beam this increase in sensitivity could also be used to increase the spatial resolution by a factor of from 5 to 15 for a fixed concentration. This assumes that the minimum concentration detectable is a (beam current)-1’2 (Isaacson and Johnson, 1975). It should also be pointed out that without special techniques (e.g., windowless detectors) the energy-dispersive technique cannot detect elements below Z = 11 (Na). The energy loss technique, however, becomes more sensitive with lower Z values, and F1, for example, can be detected easily using energy loss electrons. Since Fl is used as a biological marker, its quantitative detection can be useful. In fact, fluorinated serotonin has recently been localized in human platelets by energy loss spectrometry. An example of the use of energy loss electrons to determine the location of S-labeled serotonin in platelets is shown in Fig. 6. An additional and potentially useful aspect of electron energy loss spectroscopy for microanalysis is that very highenergy resolution can be maintained in the energy loss spectrum. One immediate application of such high-energy resolution is the observation of fine structure at the leading edge of inner shell energy loss events. Isaacson and Johnson (1975), with an energy resolution of 0.2-0.5 eV, observed such fine structure in the transmitted energy loss electron spectra at the energy loss peak due to the excitation of K-shell electrons in the C atoms of six nucleic acid bases. The spectra have been correlated by Isaacson and Johnson (1975) with the amount of charge on each C atom and interpreted as transitions from the different bounding states of C in these molecules to single-bond excited states. With the use of this fine structure it may be possible to use the energy loss technique not only to measure elemental concentrations in small volumes but also to identify the bounding states of the elements present.


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IV. Critical Reading of the Literature Critical reading of the literature is of great importance to investigators in any field of endeavor and is a skill which is naturally developed in a particular field. This critical reading skill may not, however, extend to related and useful fields such as elemental microanalysis. Consequently it is appropriate to point out areas of possible misinterpretation and confusion which may arise in the minds of investigators in related fields when reading elemental microanalysis literature. In addition, since the technique of elemental microanalysis now offers great potential benefits to such a wide range of disciplines, it may be used by investigators unfamiliar with its limitations and possible sources of error. Although many of these points are implicit in the preceding and following sections, a more explicit enumeration of these factors is appropriate. Several critical components are required for successful qualitative and quantitative elemental microanalysis of biological tissue. In general terms these can be stated as retention of elemental spatial position prior to and during elemental analysis, attainment of adequate visualization of the micromophology to identify regions in which elemental localization is required, and analysis of the data produced in such a way that maximum information is gained while extraneous and unrelated information is discarded. These three features of successful analysis are not easily separated with respect to experimental procedure, and no effort has been made in this section to attempt this; however, each of these requirements is treated in an explicit form consistent with its appearance in the experimental procedure. A. STATEOF TISSUE PRIORTO SPECIMEN PREPARATION Regardless of the method of specimen preparation selected, the state of the tissue prior to this step is critical in retaining elements in the in vivo positions. The condition of the tissue immediately prior to preparation should be as close as possible to that in vivo. A description should be given by the authors of the steps taken to ensure that in vivo conditions exist prior to preparation for electron microscopy. Clearly preparatory steps which induce cell trauma or high ion motion in the cell counteract efforts to localize elements totally in their in vivo state. This includes such techniques as extraction of the cell mass from tissue or host organisms, whether they involve extraction of muscle in such a way as to FIG.6. Electron energy loss elemental mapping of air-dried blood platelets incubated with a S analog of serotonin. The micrograph was taken using 100-kV electrons in a JEOL lOOb electron microscope. The zero-loss image shows the vacuoles and dense bodies. The image was obtained using S,hence the serotonin as being localized in the dense bodies. Micrograph courtesy of Dr. D. C. Joy, Bell Laboratories and Dr. J. L. Costa, National Institutes of Health.

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change the state of the tissue in an uncontrolled way or extraction of microorganisms into an extracellular medium which may induce osmotic transport. The authors should clearly describe steps taken to ensure that such elemental translocation is unlikely.

B. SPECIMEN PREPARATION Three methods previously outlined for the preparation of cells and tissue for electron microscope examination are (1) conventional fixation and dehydration for electron microscope examination, (2) rapid freezing followed by maintenance of hydration during transfer to the electron microscope and examination, and (3) rapid freezing of cells and tissues followed by freeze-sectioning and drying, followed by examination in the electron microscope at low temperatures. 1. Conventional Preparation Techniques for Electron Microscopy

Numerous examples have been described both in this article and in the literature of techniques which employ conventional preparations for electron microscopy. The central feature of these techniques is fixation with gluteraldehyde or other protein-fixing solutions followed by graded dehydration and subsequent staining of protein elements to provide adequate contrast for electron microscopy. In most cases this procedure is the least desirable of the three methods presented, since it involves the use of solutions for volatile constituents, which may change the ionic distribution within the cells unless they are strongly bound either by natural or artificial means to existing proteins. Thus quantification of elemental concentrations using specimen preparation techniques of the conventional variety is subject to question unless strong binding agents for the element of interest is introduced early in the preparation scheme and it is shown by the authors that there is minimal loss of the element during preparation by this technique. Even then possible redistribution during preparation is difficult to disprove. 2 . Frozen Hydrated Thin-Section Preparaiions Numerous questions may arise concerning the validity of the preparation of frozen hydrated thin sections for elemental microanalysis study. Perhaps the most significant among these concerns the degree to which the rapid-freezing technique translocates ions and elements from in vivo positions to the periphery of ice crystals grown during freezing. Evidence should be given by the authors of the degree of crystallinity (i.e., microcrystal size or amorphous structure) accompaning the freezing step, which may be supplied largely by electron diffraction patterns from which the average size of ice crystals can be inferred. The


13 1

second concern is the degree of hydration, which can be rather easily discerned from an examination of the diffraction pattern and the existence of regions of low scattering and absorption in the bright-field image obtained by either transmission microscopy or scanning transmission microscopy.

3 . Frozen Dried Tissue Many investigators use frozen dried tissue for the examination of elemental distributions within biological material mainly for two reasons: first, ease of preparation, since this method does not require transfer to or maintenance of frozen hydrated material in the electron microscope and, second, the fact that the loss of water and other volatile constituents from the tissue decreases the signalto-noise ratio compared to that for frozen hydrated tissue and allows the detection of wet weight concentrations of elements much smaller than can be realized in frozen hydrated tissue. In addition, greater contrast is frequently attained through the loss of water, making identification of micromorphological features easier. This can be understood by recognizing that about 80% of average biological tissue is aqueous. Care must be taken when reading articles in which frozen dried tissue is used for elemental distribution analysis, since the freeze-drying method is extremely important in determining the degree of translocation prior to analysis. The authors should state the conditions under which freeze-drying was accomplished, which optimally should be as near the critical temperature of water as possible. c . CONDITIONS OF ANALYSIS Although the tissue is brought to the electron microscope in a state in which translocation of elements within it is precluded and the micromorphology of the tissue is revealed, the conditions of analysis can greatly change either or both of these states. The experimental conditions which should be carefully examined in a critical reading of the literature are outlined here. 1, Electron Beam-Induced Loss of Elements during Analysis

The loss of elements during electron microscope examination and analysis has been documented by Hall and Gupta (1974) and Delgado and Hutchinson (1978). The loss of elements from biological tissue during electron beam-induced x-ray examination has been shown to be great during the time required to collect statistically significant information needed for quantitative analysis of biological tissue. These data of course are normalized with respect to electron dose within a particular area of the specimen. The electron beam dose, considered to be the number of electrons striking a particular area of the specimen, should be stated

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by the author and compared to the dose determined by independent studies. To be compensatable for in quantitative analysis, negligible mass loss must occur during this dose period, or the extent of mass loss must be known. These parameters are related to the specimen temperature and the presence or absence of a protective coating on the surface of the specimen, as described by Griffin and Hutchinson (1978). 2. Elemental and Spectral Contamination Two types of contamination can result in nonquantitative results from elemental microanalysis. The appearance of spurious peaks in the spectra may be due to several factors. The first of these is electron-induced x-ray emission from microscope components and specimen constituents, namely, the supporting electron microscope grid, specimen state and carrier, and the imaging lenses of the electron microscope. These may contribute elemental peaks which appear in the resultant spectrum but are not related to the specimen. Authors should state the degree to which these "tramp" peaks are present within the spectrum and the degree to which they have been eliminated from consideration as either constituents of the specimen or contributors to the bremsstrahlung background of the specimen. Each of these conditions can lead to extraneous contributions to the spectrum, which may be wrongly interpreted. A prime example of such an error is the interpretation given by many of the M line of copper interpreted as a concentration of Na, since the region of the spectrum in which they occur is nearly identical. A second type of problem is caused by spurious peaks and contributions to the bremsstrahlung resulting from contamination of the specimen. This contamination may take the form of a buildup of either C or Si at the surfaces of the specimen under investigation, resulting in extraneous Si peaks and more importantly in an inappropriate reading of the bremsstrahlung or white count. An example of the buildup of contaminants is given in Fig. 7. It is clear from this figure that, although the entire contaminant may be C and not appear in the spectrum because of the lower-limit cutoff of the analyzer, its thickness is much greater than the thickness of the specimen and thus it increases the bremsstrahlung radiation, resulting in a white count which makes quantification totally impossible. Authors should state the degree to which contamination occurs and describe experiments which reveal the elemental constituents of the contaminants should they occur. ~

~

~~

~

~

~

~~

FIG.7. Transmission (a) and secondary electron (b) micrographs of contamination formed from residual vapors in the vacuum of an electron microscope. The conical deposit was formed during a lengthy high-intensity exposure of the thin C support film in the microspot mode of operation of the instrument. The diameter of the cone at the base is -100 nm. The transmission micrograph (a) was taken at a 0" tilt, and the secondary electron micrograph (b) at an angle of 42".

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D. DATAANALYSIS Several methods have been used for the analysis of spectral data of x-ray microanalysis once generated. Perhaps the most popular of these and the most useful for high-resolution analysis is the thin-film computer routine devised and employed by T. A. Hall (personal communication, 1977). This routine assumes that the tissue under consideration is thin with respect to secondary scattering events of electrons in the tissue. The authors of articles on microanalysis should state the method used for analysis and the logic for their choice. If the tissue used in analysis is thicker than that which would presuppose secondary electronscattering events, the thin-film routine is not applicable. In any case, a full description of the method of data analysis, with particular emphasis on computer routines used, should be given. Failure to do so should raise the level of skepticism concerning the entire experimental procedure.

V. Application of Microprobe Analysis to Specific Biological Systems The following review of literature reporting applications of electron microprobe analysis to specific biological systems is mainly concerned with its application to mammalian tissue on a subcellular scale. This section also treats, to a lesser degree, application to microorganisms. Further, two elements, Ca and Fe, are deemed to be of sufficient significanceto warrant separate sections. Although this review of the literature is not meant to be exhaustive, examples of application of the microprobe technique are treated for a full range of topics important to physiology and cell biology. A. SKELETAL AND CARDIAC MUSCLE

Skeletal and cardiac muscle are two tissues on which much research using other techniques of elemental localization has been focused. In particular, studies using pyroantimonate, involving efforts to localize Ca and other elements by autoradiography, are extensive. It is therefore quite natural that, when the value of the microprobe technique was recognized, one of the first tissues to receive attention was striated muscle. Of the several methods of specimen preparation used with the microprobe technique, by far the easiest to implement is epoxy embedding followed by thin-sectioning and observation in the microscope. However, the possibility of translocation of elements during the fixation and embedding process has stimulated several studies in order to determine the extent of such translocation. Of particular note is the work of Yarom et al. (1974a,b) on the effect of embedding and other methods of specimen preparation on the


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location of intracellular myocardial Ca. These studies indicate that significant changes occur during fixation with glutaraldehyde, osmium, and pyroantimonate. The pyroantimonate fixation technique produced the highest Ca concentration and the majority of statistically significant concentrations; it has been used successfully by Atsumi and Sugi (1976) and by Myklebust et al. (1975). A combination of osmium tetroxide and potassium pyroantimonate was used by Atsumi and Sugi (1976) to fix skeletal muscle during various states of contraction, while potassium pyroantimonate was used to localize the Ca. Electron probe microanalysis of precipitates showed the presence of Ca in an unambiguous way. In resting muscle, the electron-opaque precipitates were observed on the inner surface of the plasma membrane, the vesicles, and the mitochondria. In muscle fixed at the peak of mechanical response to Ca removal, the precipitates were found to be diffused throughout the myoplasm in the form of a large number of small particles. At the completion of spontaneous relaxation, the precipitate was again seen on the inner surface of the plasma membrane. In experiments to determine the distribution in the catch state, the precipitate was found to reaccumulate in the peripheral structures, with a corresponding decrease in the precipitate in the myoplasm. These workers conclude that the study not only provides evidence for the involvement of Ca-accumulating structures in the contraction-relaxation cycle but also indicates that the transition from active to catch contraction is related to the decrease in myoplasmic free Ca concentration. In a more sophisticated study, Myklebust er al. (1975) reacted potassium pyroantimonate with atrial myocardial tissue and found a pattern of evenly spaced cross-striations of antimonate precipitates along the myofilaments. The spacing was reported to have a periodicity of about 400 A. These investigators suggest that the localization of troponin-bound Ca is demonstrated by the periodicity of the pattern along the thin filaments during contraction. Although the technique of plastic embedding and sectioning with subsequent staining has great advantages in revealing the morphological features of muscle and the use of pyroantimonate can localize Ca redistribution, several investigators have selected the technique of frozen dried, or frozen hydrated thinsection elemental microanalysis in order to minimize the possibility of redistribution during subsequent preparation steps prior to observation. The first of these observations was made by Bacaner er al. (1973), with further details of the work given by Hutchinson et al. (1974). The rudiments of the technique are outlined in the previous section on specimen preparation. In later work, Somlyo et al. (1977), using frozen dried thin sections of skeletal muscle, demonstrated that the electron microprobe technique used for elemental analysis of Na, Mg, P, C1, and K yielded concentrations well within the limits of variability for fibers. They concluded that the electron microprobe analysis of 50-nm- to 2-pm-diameter areas of frozen dried ultrathin sections yielded quantitative results which were in agreement with chemical analysis of whole muscle, and further that, in hypotonically

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treated muscle, the excess Ca was compartmentalized in a manner consistent with ionic communication with the extracellular space. In several excellent studies, Ashraf and Bloor (1976) investigated the mitochondria1 deposits in ischemic myocardium, and the microanalysis results suggest the formation of calcium, magnesium, and phosphate in the mitochondria during ischemia. Further work on ischemia was done by Somlyo et al. (1975). Granules similar to those of Ashraf and Bloor (1976) were observed. It was found that the amounts of Ca in mitochondria containing numerous granules were clearly pathological and represented abnormal accumulations caused by ventricular fibrillation andor ischemia. The Somlyo technique differed from that of Ashraf in that the cells were unfixed and were prepared by cryomicrotomy with freeze-drying and subsequent osmium vapor staining. An extensive investigation of the techniques used to prepare skeletal muscle for transmission electron analytical microscopy of diffusible elements was made by Sjostrom and Thomell (1975). They concluded that brief fixation in glutaraldehyde resulted in gross ionic changes, as did sectioning of frozen material employing liquid trough techniques. Sections cut from unfixed frozen muscle without contact with cryogenic liquids showed numerous spectral peaks indicating the presence of Mg, P, S, Ca, and K. In the various parts of the fibers of frozen dried sections, reproducibIe spectra of these elements were found within different structures. They concluded that the best method for obtaining data on diffusible ions involved rapid freezing of unfixed tissue, followed by dry cutting in the frozen state and freeze-drying. There are, however, questions relating to the alteration of elements during the drying process. These questions have not been treated in sufficient detail in any study as yet, due mainly to the difficulty of obtaining analysis of fully hydrated frozen tissue and the problems associated with determination of full hydration as opposed to partial dehydration in the electron microscope.

B. SMOOTH MUSCLE The field of microanalysis, as well as the determination of elemental distributions by other means within smooth muscle, has been dominated by the work of Andrew and Avril Somlyo. One of the pioneering efforts performed in the Somlyo laboratory involved specimen preparation in which Sr was substituted for Ca prior to induced contraction of smooth muscle. It is suggested by Somlyo and Somlyo (1971) that the translocation of Ca accumulates divalent cations from the sarcoplasmic reticulum in close contact with the surface membranes and is responsible for the action potential triggering contraction in rabbit and guinea pig mesenteric veins. This was pioneering work with respect to localization of electron-dense deposits but did not employ elemental microanalysis to identify these deposits. Later work from the Somlyo laboratory (Somlyo er al., 1974)

DETERMUVATION OF SUBCELLULAR ELEMENTAL CONCENTRATION

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showed that tissues incubated with Sr contained electron-opaque mitochondrial granules and deposits in the sarcoplasmic reticulum. X-ray microanalysis of the mitochondria indicated that the granules contained mainly Sr and Ca. It was further noted that mitochondria, subject to Ba-induced swelling, contained granules which showed characteristic Ba signals. The presence of Ba was in good correlation with that of P. The general conclusion was that the energy-dependent uptake of divalent cations was associated with K and suggested mitochondrial regulation of intercellular divalent cations in smooth muscle. The previously mentioned work involved mainly conventionally fixed preparations of smooth muscle tissue. Somlyo and Somlyo have also used freezing with subsequent freeze-drying in conjunction with an oxalate precipitation method as a Ca ion marker to show by a direct method the significant Ca accumulation inside the sarcoplasmic reticulum of smooth muscle, as had been shown previously in skeletal muscle. In addition, it was shown by Somlyo et al. (1977) that cryosectioned smooth muscle exhibited high Ca concentrations in the mitochondria, frequently associated with high levels of Na and K. Higher than normal net Ca concentrations were seen with C1 of roughly 200 mmoledkg, while K was in the range of 330 mmoleskg. It was concluded from the anomalous non-Donnan distribution in cellular organelles that mitochondria in situ excluded C1 but probably not K from the matrix space. In a study by Garfield and Somlyo (1976), unfixed ultrathin frozen dried sections of cultured smooth muscle cells were subjected to elemental microanalysis. Mitochondria containing electron-opaque granules were identified. In these regions, Ca and P were the dominant elements. In regions outside the mitochondria, the presence of Na, K, S, C1, and Ca was observed. The Ca concentrations in regions other than the mitochondrial granules were deemed to be quite low, but still greatly in excess of that expected in normal cytoplasm. In all these studies, Somlyo and Somlyo went to great lengths to make the best use of the statistical analyses which could be employed with these systems and to obtain the greatest quantification possible with the instrumentation used. C. LUNG Because of the recent increase in interest in the contribution of particles in the lung to cancer initiation, microprobe analysis has been applied rather widely in the study of lung tissue. The literature is extensive, and only two examples of these studies are mentioned here. Ferin et af. (1976) used this technique for the identification of titanium dioxide particles in lung tissue and cells. Conventional fixation techniques were used in these studies, and the particles were identified by electron opacity. Characterization of the particles was aided by their tendency to clump, so that accretions of particles could be Seen in the transmission electron

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microscope and identified by electron probe analysis. The particles were found in the alveolar macrophages and tended to be adjacent to the nucleus. The resolution, however, was not sufficiently high to determine whether the cell organelles or the cytoplasmic membrane was involved in particle encapsulation. Pintar er al. (1976) used the technique to characterize pulmonary silicosis. This study, made on three foundry workers, using lung biopsy specimens, identified a significant amount of Si in fibrous tissues of the septa and pleura and around blood vessels. The amount of Si was sufficient to permit a diagnosis of silicosis in all three patients. All three had severe functional impairment, but it was not clear at the time what factors were responsible for the diffused distribution of Si in their lung tissue.

D. NERVE One example of the application of microprobe analysis to the study of nerve is cited in Section V,M,l. Several other articles are worthy of note, in particular one reporting a study by Oschman et al. (1974) demonstrating the use of microprobe analysis in investigating the squid giant axon. The technique of fixation was similar to that employed with numerous other Ca-containing cells; chloride (5 mM) was added to all solutions used in tissue processing. These workers observed electron-dense deposits along the axonomal plasma membrane, in the mitochondria, and along the basal plasma membranes of the Schwann cells. It was found that these deposits contained mainly Ca and P. They also noted that these elements were not detected in the axoplasm. The findings of this study were supported by the further work of Hillman and Llinas (1974), who examined tissues which were unstained and unosmicated. The findings with respect to Ca and P were identical to those of Oschman et al. (1974). Gambetti et al. (1975) applied electron microprobe analysis to vertebrate glial cells. Again, osrniophilic particles were found in the visceral and cisternal structures. These particles were shown to contain primarily Ca and P. It was further noted that the osmiophilic particles also occurred in astrocytes, and it was suggested that these organelles were the storage sites of Ca. An excellent study was made by Rick ef al. (1976) to determine the distribution of Na, P, C1, and K in different structures of myelinated nerve of Raw escutentur. It was found that the axon showed a typical intracellular distribution pattern of Na, C1, and K, while the interstitial space and the myelin sheath showed a typical extracellular pattern. It was noted that these measurements demonstrated that Na was present in the myelin sheath near the node of Ranvier. Duckett er al. (1977) showed that the localization of Ca and P by scanning electron microprobe analysis provided a morphological outline of normal nerve, which could be used to compare abnormal and normal nerves qualitatively and


1 39

quantitatively. The normal saphenous nerves of eight cadavers and the abnormal saphenous nerves of two cases of diabetic neuropathy were analyzed, and the results were compared. Results were obtained for Ca and P concentrations in the normal and abnormal nerves. In the normal nerve both Ca and P were excluded from the node of Ranvier, while in the abnormal nerve Ca was uniform within the tissue but P was totally excluded from the area of the node of Ranvier to a greater extent than in the normal nerve. These results should be of interest to neurologists, since this method provides an efficient way of detecting abnormalities in peripheral nerves. These investigators are further pursuing studies to refine the technique using air-dried, unfixed nerves, which eliminates the artifactual presence of osmium and lead as stains in these tissues. E. EPITHELIUM The study of epithelial tissue over the past several years by x-ray microprobe analysis has gained increasing importance. Even though epithelial tissue does not lend itself well to studies using frozen hydrated or frozen dried preparations, valuable information concerning elemental distributions in these cells obtained by microanalysis cannot be gained by other techniques. In an early study by Gehring et al. (1972), frozen dried cross sections of frog skin were examined. These studies used freeze-sectioning with subsequent drying in a cryotome for the preparation of tissue. Tissues were examined at room temperature in a scanning electron microscope equipped for x-ray microanalysis. The cytoplasm and the nucleus were regions of particular interest, and the elements Na and K were studied extensively. In the cytoplasm the Na level was 30 meq/kg wet weight, and in the nucleus the Na concentration was identical. The K level was 115 meqkg wet weight in the cytoplasm, and in the nucleus it was 110 meq/kg wet weight. An additional feature of their article is that it outlines a relatively sophisticated quantification technique, although the roughly 5% accuracy of the K concentration determination is not fully justified. Tapp (1975) studied the epithelium of the midgut of the fruit fly. It was known well before microanalysis was used on these tissues that Cu accumulated in the midgut epithelium. This study showed that the Cu was associated with high concentrations of S and that no other elements with an atomic number greater than 9 were present in appreciable concentrations. The Cu was located in granules bound to membranes and morphologically similar to secondary lysosomes. Jessen et al. (1976) studied the amount of S in keratohyalin granules in the interpapillary and papillary lingual epithelium and in the esophageal epithelium of the rat. A quantitative assay of the S concentration of the keratohyalin granules was performed using a suitable S standard. It was demonstrated that the different types of keratohyalin granules had unique compo-

I40

THOMAS

E. HUTCHINSON

sitions. Single granules, present in both nuclei and cytoplasm of the epithelial cells, were rich in S, having a content of roughly 3.6 at 96. Another type, composite granules, contained S-rich components and S-poor components. The S-poor, components contained roughly 0.8-1.4 at 8 S. These investigators suggested that the S-rich keratohyalin granules were involved in deposition of the peripheral envelope protein of cornified cells. Hodson and Marshall (1970) used the electron microprobe technique to study S and K ions in corneal stroma. Frozen sections were cut from the corneal stroma, freeze-dried, and subsequently subjected to x-ray microanalysis. Quantitative analysis determined Na and K concentrations to be on the order of 172 and 22 mM, respectively. Although the limit of resolution of the technique used by Hodson and Marshall was well below the cell size (i.e., 1.O to 0.1 p n ) , the K in the sections appeared to be uniform and not localized in the keratocytes. This strongly indicates that the sections may have rehydrated during transfer, in which case the K may have diffused throughout the tissue. In a paper by Gupta et al. (1976) of the Cambridge University group, extensive studies on the distribution of ions in fluid-transporting epithelium using frozen hydrated sections from the fluid-secreting upper portion of the Malpighian tubule of the insect Rhodnius prolixus were reported. The data presented showed that NayK, and C1 were not uniformly distributed within the cells, that the basal lamina was not entirely a passive layer open to small ions, and that in the particular epithelium studied the stimulation of secretion greatly increased the intracellular Na concentration. These workers stated that their results did not support the standing gradient theory of fluid secretion. The reader is referred to the article for details of the study. One observation of particular significance is that high concentrations of K have been found in other insect tissues as well and cannot be attributed to the limited spatial resolution which only tends to obscure the existing sharp peaks in concentration which do in fact exist. One implication for fluid transport is that the basal lamina, although apparently permeable to quite a large number of neutral molecules, may nevertheless preferentially mtrict the movement of some ions. In a study by Appleton and Newel1 (1977), frozen dried ultrathin sections of regulating epithelium of the snail otala were examined by a x-ray microanalysis technique. This is an especially important cell because of its ability to regulate water and ion flow across the epithelium. Standard techniques of sectioning and freeze-drying were used. The primary results showed that the x-ray microanalysis of frozen dried ultrathin sections was sufficiently sensitive to detect physiologically significant changes in the concentrations of elements at the subcellular level. In addition, the changes in the concentration of Fe and Zn in regulating and control epithelium indicated that translocation of these elements was related to fundamental physiological processes within cells. It was stated by these workers that the increase in the Zn concentration may be related to its role


14 1

as an enzymic cofactor associated with carbonic anhydrase, which is known to be present in mantle epithelium tissue. F. KIDNEY Kidney studies have been made primarily at one laboratory, that of Claude Lechene of Harvard Medical School, and collaborators. This is the result of the extensive facilities and interest in kidney studies at Harvard and the presence there of a Biotechnology Resource for Microprobe Analysis. The primary technique used in these studies is the examination of picoliter samples of fluids extracted by micropuncture techniques from kidney tubules. The studies take advantage of the highly sophisticated automated analysis techniques developed by Lechene and co-workers. The reader is referred to the bibliography for particular results and details of the experimental procedure. G. CALCIFIABLE TISSUE

X-ray microanalysis of calcifiable tissues is an area of investigation which in itself could be the subject of a review article. For this reason only a few articles illustrative of the use of this technique for calcifiable tissue have been selected for discussion. The reason for this extensive literature is that calcifiable tissue presents relatively easily solved problems in specimen preparation and thus has been the subject of extensive study. Perhaps the most extensively studied material using electron probe microanalysis is dental. An example of such a study is that reported by Selvig et af. (1977). Ground sections of human tooth showing early stages of root surface caries were subject to analysis by electron microprobe techniques, and Ca, P, F, S, Mg, Na, Fe, Cu, Zn, Sn, and Ag were observed. The progress of caries in the cementum was followed by sequential analysis, and the pattern of dissolution and precipitation of mineral components seemed to be the same as that seen in dentine caries studied by other methods. A surface layer containing relatively high F content resulted in the development of a distinct zone of recalcification at the surface. Boyde and Reith (1977) examined early stages of the cementum caries of rapidly growing rat incisors which were freeze-fractured, freeze-dried, and subsequently subjected to energy-dispersive x-ray microanalysis. Ca levels were found to be elevated in the distal cell body of odontoblasts where Ca was uniformly low over all parts of the cell body secretory ameloblasts. Results suggested a fundamental difference in the mechanism by which these two types of cells process Ca and that Ca possibly diffused through the secretory ameloblast layer on its way to the enamel.

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In a study by Berkovitz and Heap (1976), the effect of F on Fey Cay and P distribution in rat incisors was studied. It was found that rats subjected to treatment of 25-100 ppm grew incisors with a typical banding pattern of routhly 60 pm. These pigmented bands were found to contain more Fe, Ca, and P than bands exhibiting reduced pigmentation. Diffusion of Sr from desensitizing agents into human dentine was studied by Stazen and Foreman (1977). In this study Sr-containing densensitizing agents were applied to two surfaces and burnished onto another set of diametric surfaces. The variation in concentration of Sr in dentine with depth beneath the surface was determined by x-ray microanalysis. It was noted that burnishing produced deeper Sr penetration which followed Fick 's second law of diffusion. The mineralization of cartilage has been the subject of several investigations. In early research by Hall (1971), calcification was studied in ultrathin sections of costochondraljunctions of 1-month-old guinea pigs. Electron-dense bodies having diameters of 50-200 nm were found. It was concluded from this study that the concentration of Ca in the particles in the early stages of mineralization was not greater than in the surrounding matrix. Observed levels of Caythat is, 1 or 2 ppt, were similar to those in equivalent tissue, although they were much higher than in most soft tissue, suggesting an accumulation of Ca, presumably in an early stage in the process of mineralization. Second, the observed C d P ratio was often much higher than that expected in the phosphate compounds. This suggested that the first step toward nucleation of these globules was the binding of Ca to some moiety other than phosphate. These were regarded as the first sites of apatite nucleation. In a recent study on calcification of elastin, Urry et al. (1976) concluded that the ability of elastin coacervates to initiate calcification was a bulk property of the coacervate and not limited to the serum-coacervate interface, and that calcium phosphate deposits acted to bind the protein units together and slow dissolution and spreading of the coacervate as it floated at the air-water interface. In addition they found there was no inferable involvement of S. Initiation and deposition, it was concluded, were due to neutral sites on the protein which were tightly bound to the phosphate deposits. H. BLOOD Formed elements of the blood such as erythrocytes, lymphocytes, and platelets have become subjects of intense study by x-ray microanalysis, mainly because single cells can be studied by this technique whereas other methods do not offer this advantage. An extensive study of single human red blood cells was carried out by Lechene et al. (1976) at the Biotechnology Resource for Microprobe Analysis at Harvard Medical School. Their report outlines both the techniques of


I43

specimen preparation and the results of microprobe analysis. The method of preparation found to be most reliable was spraying cells onto polished pyrolytic graphite by atomization. It is notable that the mean values of x-ray intensities from the elements studied were linearly related to the content of ions, as determined by optical spectroscopy. The calibration curves were obtained by loading the cells with known amounts of Na and K. The Na and K contents of the cell were measured by flame photometry and the results compared to results from electron microprobe analysis. Calibration curves were thus constructed which allowed quantitative data to be obtained from individual cells. In a study by Yarom et al. (19761,comparative investigations of elemental concentrations in normal and leukemic cells were undertaken with a few subjects using electron microscope x-ray microanalysis. In addition to the normal physiological elements detected, Cu and Zn were found to be above accepted levels in normal cells. The abnormal concentration of these elements appeared to be disease-related. In leukemic lymphocytes, nuclear Zn was significantly lower than that observed in normal lymphocytes, while P was only slightly decreased. This suggested faulty Zn uptake for binding in the leukemic cells. The possible consequences of intracellular Zn deficiency were discussed also by Yarom et ai. (1976). In a study of Ca and P in human platelets, large amounts of these elements were found by electron microprobe analysis of dense bddies of frozen dried human platelets. The Ca and P occurred in a fixed ratio similar to that of dicalcium ATP. Except for C1, no elements other than these were detected. Both dense bodies and membrane-bound particles were absent when platelets were fixed in a Ca-free solution. In a further study by Skaer (1975)and Skaer et al. (1974),mineral elements present in dense bodies of human platelets were detected by the quantitative microprobe analysis technique. Sections of frozendried platelets and also whole mounts of air-dried platelets were used. The only elements detected by the study in the dense bodies were Ca and P. The ratio of Ca to P corresponded approximately to the atomic ratio-just over three P atoms to one Ca atom. When the dried platelets were extracted with liquid solvents, the P/Ca ratio became essentially 1:l. These workers calculated that the amount of Ca within the dense bodies was equal to the total Ca content of the entire platelet as obtained by gross chemical analysis. Microprobe analysis of the platelet cytoplasm substantiated the results of this calculation. It was further noted that the dense bodies dissolved from the platelets in the absence of Ca in the cytoplasm and thus the stability of the dense bodies was dependent upon the presence of Ca. In a further study, Hutchinson (1978) applied ultramicroprobe analysis to frozen hydrated and frozen dried normal and irreversibly sickled erythrocyte ghosts. The presence of membrane-bound particles was evident from the highresolution electron micrographs. Regions of the erythrocyte membrane were found to be devoid of membrane-bound particles, while in other regions they

144


were in a closely packed state. The results of analysis of the concentrations of elements within the membrane-bound particles indicated that they contained largely P and S, although C1, K, Ca, and Fe were also found to be present. With the use of an identical technique irreversibly sickled erythrocytes were examined, and it was found that the major change was an increase in the K/Ca ratio. The technique was also applied to the examination of erythrocytes by Barber (1975). The procedure consisted primarily of tagging the antigen sites with osmium, followed by normal fixation for SEM. The osmium M line was used in the analysis. In this study it was noted that antigen sites were evenly distributed over the surface of the cells.

I. GAMETES AND DEVELOPMENTAL BIOLOGY Very little work has been done on cilia with electron probe microanalysis apart from gametes, although they represent a highly important system much studied by other techniques. Several studies are worthy of note. In the first of these, Tsuchiya (1976) investigated the Ca-binding sites in the cilia of Paramecium. This study revealed the distribution of Ca and other elements within the various components of the cilia using a preparation technique consisting of fixation with glutaraldehyde containing 5 mM calcium chloride, subsequent embedding, and thin-sectioning. Ca and P were present in electron-dense deposits found on the inner side of the ciliary membrane just above the ciliary necklace and less frequently on the outer and central doublet microtubules. This observation suggests that Ca ions may be released from inner ciliary binding sites during excitation of the ciliary membrane and influence ciliary movement. In a supportive study, Fisher ef al. (1976), also investigated Paramecium aurelia and identified granules containing large amounts of Ca, particularly on the cytoplasmic side of the surface membranes in the basal regions of the cilia, in preparations fixed with a high (5 mM) concentration of Ca in the solutions. Certain deposits were also observed on the smooth cytomembranes and within the axonema of the cilia and on the basal bodies. Certain divalent cations, in particular Mg, Mn, Sr, Ni, Ba, and Zn, could be substituted for Ca in the procedure with similar results. Some deposits were larger at the ciliary transverse plates and at the termination plates of the basal body when 5 mM Sr, Ba, or Mn solutions were used in the preparation. Microanalysis showed that Ca and C1 were concentrated within these deposits. It is notable that these deposits were seen only when the ciliates were actively swimming at the time of fixation. These workers also discussed the possibility that the action sites of Ca and other divalent cations were those identified in their study. Stamejohn and Hutchinson (1977) investigated the distribution of ions within bovine spermatozoa using scanning transmissiun electron microscopy and determined the distribution of ions (Na, P, C1, K, s, and Zn) in specimens prepared by


145

ultrarapid freezing and direct observation of the whole-cell preparation in the frozen hydrated state. Large variations were seen in the relative abundance of each of these elements in the various compartments of the cell. In particular, the acrosome, head, midpiece, and tail were studied. The variation in elemental concentration with cell aging for each of these units was determined in semen. Many investigations on mammalian sperm have been carried out in the laboratory of John Chandler at the Tenovus Institute, Cardiff, Wales. He performed x-ray microanalyses on both air-dried and ultrathin frozen dried sections of human sperm and found large variations in elemental distribution and composition among cells in any single ejaculate and an even larger variation among ejaculate samples. The NdK ratios in these preparations were found to be roughly constant. It was concluded by Chandler and Battersby (1976) that airdrying was a valid method of preparation for sperm cells to be subjected to elemental microanalysis. In a study on the effect of Cu on the distribution of elements in human spermatozoa, Maynard et al. (1975) found that incubation with a Cu wire in semen or cervical mucus significantly reduced the levels of both Na and K in the spermatozoa but did not affect the ratio between these two elements. Cu also displaced Zn from the head region, possibly replacing it. They further noted that this may account for the decreased motility of spermatozoa in contact with Cu ions and that the observed toxicity of Cu for human sperm cells lent support to the theory that part of the mode of action of the Cu IUD may be due to alteration of the sperm-fertilizingpotential. Chandler and Battersby (1976) also investigated the use of pyroantimonate in the localization of Ca. Although Na, K, and C1 were all removed during the fixation process, Ca and Zn were found to be present intracellularly in association and independent of the antimonate precipitate. They concluded that there appeared to be a varying degree of binding of these elements subcellularly, precipitation occurring where binding was reduced. In a rather extensive investigation Rosado et af. (1977) studied the elemental composition of subcellular structures of human spermatozoa by the x-ray microanalysis technique. This study determined the elemental composition in the acrosome, head, midpiece, and tail of spermatozoa fixed in the usual manner for electron microscopy. Of particular interest was the finding that Ca, S, and Zn were present in extremely high concentrations in the membrane of the spermatozoa and that sperm heads were richer than tails in Na, Cu, and Zn, while tails had higher concentrations of Ca. This certainly supports the Ca-mediated motility theory of flagellar action. Battersby and Chandler (1977) attempted to correlate the elemental composition with the motility of human spermatozoa. These workers found that x-ray microanalysis data obtained from human sperm cells in donor semen having a range of motility from 0 to 85% indicated that the elemental composition was not strongly correlated with spermatozoa motility. Only the Cu in the midpiece was positively correlated with motility when high- and low-fertility groups were compared. It was noted that aging of cells in semen caused large changes in the

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subcellular elemental concentrations as motility decreased. It was especially clear in the case of uptake of Zn that these changes were not reflected in the range of motilities of clinical samples. In this case the electrolyte balance was measured by NdK ratios which also appeared not to be correlated with motility. It was found that subcellular elemental distributions were not a major factor in determining cell motility in normal human sperm. J . CHROMATIN IN MITOSIS AND AGING A particularly interesting study was made by Cameron et al. (1977), in which the concentration of elements in mitotic chromatin was measured by x-ray microanalysis. These workers used unfixed frozen dried tissue sections of mouse duodenum. The analysis was carried out on carbon planchets and analyzed in scanning electron microscope fitted with energy-dispersive x-ray analysis equipment. The peakkontinuum ratios for S , C1, K, and Ca were measured in mitotic chromatin. It was found that the concentrations of these elements were significantly higher in the chromatin than at other cell sites. These investigators suggested that the redistribution of Ca in mitosis may help explain both chromatin condensation and assembly in the mitotic spindle apparatus. An extremely active group in the area of aging is jointly associated with the Center of Cytology at the Gerontological Research Department in Ancola, Italy, and the Biomedical Institute, Medical University of Hungary. In a report from this group (Pieri et al., 1977) it is noted that they have examined the nucleus and cytoplasm of large brain cortical and liver cells of young and old adults by microanalysis using the preparative method of freeze-drying bulk specimens. It was found that the K and C1 contents per unit dry mass in the nuclei of both tissues were significantly increased between the age of 1 and 24 months, while the Ca content of the hepatocyte nucleus decreased. At the same time some cytoplasm ion concentrations were increased, while others decreased or remained unchanged. It was noted, however, that there was an age-dependent intracellular water loss, the extent of which was not known for the tissues studied, however, in human organs it averages about 10-14% between the ages of 20 and 99 years. The values obtained for the old cell nucleus reached ranges of ionic strength when nonhistonic regulatory proteins were separated from the chromatin in vitro. These results have had a significant impact on recent biochemical observations.

K. MICROORGANISMS Several studies have been made of the distribution of elements in a variety of microorganisms. Coleman et al. (1973) made a quantitative electron microprobe


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analysis of the refractile granules of Tetrahymena pyriformis both individually and in situ. The ratios of several elements were determined in these granules. When Sr was substituted for Ca in the growth medium, all the granules incorporated this element, seemingly at the expense of Ca. The (Ca Mg Sr)/P ratios in the granules were comparable to the (Ca + Mg + Sr)/P ratios in granules from Sr-free media, indicating that the mix of divalent ions in granules may vary but the proportion of divalent ions to P tended to be constant. In a particularly interesting study Murphy et al. (1976) applied electron microanalysis to dormant and germinating Diplodia maydis spores. It was found that the spore population contained large amounts of Si, P, C1, and K, and smaller amounts of S and Ca, with trace amounts of Mg and Al. Analysis of single spores revealed high K and C1 and low P and Mg at one end of the cell, with concomitant low K and C1 and high P and Mg in the central portion and at the other end of the cell. They found that high K and C1 occurred at one end of nongerminating spore cells, whereas germinating cells contained high P and Mg and low K and C1 at the same location. Hutchinson et al. (1977) demonstrated the presence of Ca, K, and P in relative amounts of 1:1:4, respectively, in the viruslike gamma particles found in zoospores of the aquatic fungus Blastocladiella emersonii. Some gamma particles, however, lacked detectable amounts of K. These workers discussed the possible significance of these observations in understanding the triggering of encystment and initiation of wall synthesis mechanisms.

+

+

L. MEDICAL DIAGNOSIS Recently, energy-dispersive x-ray analysis has been applied to medical diagnosis. Two studies may be given as examples of such studies. Paetau and Haltia (1976) examined the sciatic nerve of a patient who died of uremia complicating juvenile diabetes. It was revealed that selective calcification had occurred in the perineurial sheath of the sciatic nerve. Calcium phosphate deposits were found to be limited to outer layers of the perineurium. The end-stage diabetic nephropathy was associated with an extremely high Ca-P ion product known to favor metastatic calcification. The mechanism of selected localization of the P deposit to the outer layer of the perineurium sheath was discussed with reference to the structure and suggested a barrier function of the perineurium in regard to phosphate ions. In a second study, Murphy and Piscopa (1976) investigated subcellular Fe distribution in plasmic anemic human bone marrow. Cells presumed to be proerythroblasts were quantitatively abnormal in their utilization of Fe. It was further suggested that this may provide an index for hematological disorders of this type. These observations revealed the existence of a new class of cells not previously identified by light or electron microscope techniques and identified only by their accumulation of Fe within the cytoplasm and along the plasma

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membrane. This research provides yet another example of the power of the ultramicroprobe technique in cellular investigation. M. ELEMENTS OF PARTICULAR INTEREST

1. Calcium Binding and Transport Much attention has been focused on electron probe studies of Ca transport in mammalian cells. The basic question involved is to what extent electron microprobe analysis can be relied upon to describe the Ca distribution within cells on a scale which is very small in comparison to the cell size. The fundamental biological question is: How is Ca transported through cells which are highly sensitive to Ca levels without the transient Ca interfering with the basic cell function? Coleman and co-workers at the University of Rochester have addressed this question using both glutaraldehyde-fixed and oxalate-stabilized cells followed by routine embedding and sectioning, and by the technique of freeze substitution and freeze-drying. Primarily they investigated Ca movement through cells organized in epithelia, particularly in the two organs of the embryonic chick chorioallantoic membrane and the small intestine of the young rat and chick. Using these techniques coupled with electron microprobe analysis, Coleman and Terepka (1972) identified Ca pools within the cells on a scale of roughly 25 nm. It was clearly established that the Ca in transit through the cells was restricted in location and did not diffuse through the cytoplasm, which was a highly important finding. It was further determined that, of all methods examined, freeze-drying offered the greatest possibility for full quantitation of elemental distributions within cells using the electron microprobe technique. This was established by comparing elemental distribution maps to the values determined by bulk analysis. The close correspondence between the two values for Na and K established for rat small intestine by Nellans and Schultz (1976) strengthened the confidence of the investigators in the electron microprobe technique. Another excellent example of microprobe measurements of Ca binding is found in a study by Routledge et al. (1975), in which the contractile spasmoneme of a vorticellid was examined. The Ca content of isolated contractile organelles from the ciliate zoothamnium was compared in both extended and contracted conditions. Contracted organelles contained a higher Ca content than those in the extended state by about 1.7 g d k g dry mass. It was further noted that this Ca was very strongly bound. These workers concluded from this study that the quantity of Ca bound in this way was in agreement with the theory that the energy of contraction was derived from the chemical potential of Ca ions. They further suggested that stochiometry considerations supported the idea that between 1.4 and 2.1 Ca ions combine per molecule of the sparmanelmaCa-binding protein.


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In a pioneering study, Parducz and Joo’ (1976) demonstrated a method for visualizing stimulated nerve endings by preferential Ca accumulation in mitochondria using the electron microprobe technique. Stimulated and unstimulated nerve endings from cat superior cervical ganglion were used for the study. Standard methods of fixation, embedding, and sectioning were employed. A comparison of x-ray spectrometer signals obtained from mitochondria in different processes within a given section clearly demonstrated that mitochondria with electron-dense granules were significantly higher in Ca than others located in different areas. A nerve stimulated for longer periods resulted in larger areas being occupied by the electron-dense granules in the presynaptic mitochondria. The technique described in this article offers a simple means of identifying stimulated translocation of Ca in relation to the regulation of neurotransmitter release. In addition to the studies described in this section an excellent review of the applicability of microprobe analysis to Ca studies has been compiled by Hall (1975). In this review Hall describes studies ranging from Ca localization in blood to characterization of deposits in squid giant axon. 2. Iron After Ca, Fe is likely the most studied element using the technique of microprobe analysis. Shuman and Somlyo (1976) reported on a remarkable study by electron probe x-ray analysis of single femtin molecules. In this study single molecules and groups of three femtin molecules were subjected to x-ray microprobe analysis. Adequate Fe spectral peaks were recorded during a 100-second x-ray energydispersive analysis, when single fenitin molecules were excited with the electron probe. It was found that there was a linear relationship between the number of femtin molecules analyzed and the count rates. These workers contrasted their experimental results with the theoretically calculated x-ray Fe yields and with the results of Isaacson and Johnson (1975). They concluded that the current state-of-the-art electron probe x-ray analysis could realize the theoretically predicted sensitivity of the method, which is roughly 1 X lo-’’’gm of Fe as a minimal dectable mass. Sprey er al. (1976) studied chloroplasts of young Nicoriana leaves containing electron-dense stroma inclusions. These inclusions may represent a convertible form of the Fe-containing phytofemtin. Considerable amounts of Fe and P were present in the electron-dense inclusions. The mean atomic Fe/P ratio detected by x-ray microanalysis was 2.5. These investigators discuss the gross arrangement of the phytoferritin in the plastic stroma in relation to the different states of chloroplast development. In addition to the direct study of Fe in its naturally occumng form, numerous studies have been made using the fenitin-protein complex in conjunction with concanavalin A (Con A) as an indicator of the binding sites of the latter on cell surfaces. One such example has been given in

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Section V,I. In addition, Amakawa and Barka (1975) studied the distribution of Con A binding sites on the surface of dissociated rat submandibular gland acinar cells. Femtin was also identified in insect vectors of the maize streak disease agent by Kimura et al. (1975). Certainly the use of ferritin in conjunction with Con A as an indicator of sites of binding to cell surface will increase as studies require this technique. Electron microprobe analysis will be used extensively to identify the fenitin positions in these studies.

VI. Methods and Reviews Two very important compilations of articles published in the literature do not lend themselves to comment in the bulk of the text. These are methods and procedures, and reviews by other authors. For this reason, the bibliography will serve as a guide to investigators in selecting articles on the reviews and methods most closely aligned with their research interests. A few comments, however, on methods and procedures are given as a guide to the literature. An excellent set of articles has been published in book form by Academic Press entitled, “Micro probe Analysis as Applied to Cells and Tissues,” edited by T. Hall, P. Echlin,and R. Kaufmann (1974). The book is comprised of the papers given by major contributors to the field of microprobe analysis at a conference sponsored by the Battelle Seattle Research Center in April 1973. The book contains articles which, taken together, can be used as a very substantial introduction to the area of electron microprobe analysis. In addition, Mizuhira (1976) has given a review of the instrumentation techniques used primarily in his laboratory and in addition has referenced the techniques of other investigators. Saubermann and Echlin (1975) have reviewed the particular technique of preparation and examination of frozen hydrated tissue, while the examination of thin sections by scanning transmission electron microscopy has been reviewed by Hutchinson (1977). Bonventre and Lechene (1974). and Roinel (1975) have carefully outlined the methods and procedures for examining picoliter samples of organic compounds. Lechene ’s laboratory has an extremely sophisticated, automated analysis procedure with which routine analysis of picoliter samples can be performed. In addition, there is an excellent general review.of ultramicroanalysis by Lechene and Warner (1977). An article by Marshal (1977) is worthy of note, in that it provides some insight into the procedures and formulas employed in his laboratory for the preparation of frozen hydrated biological specimens used in x-ray microanalysis. This article describes a technique for preparing frozen hydrated bulk specimens for electron probe x-ray microanalysis. This investigator states that the method allows reproducible quantitative analysis of frozen hydrated specimens to be made. The

DETERMlNATION OF SUBCELLULAR ELEMENTAL CONCENTRATION

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article is invaluable in delineating the significant problems in x-ray microanalysis by the electron beam excitation technique, however, many of the problems addressed are presently in a further state of examination. Particularly they concern the effect of coating biological specimens to reduce charging during examination, full qualification of the microanalysis technique, the effect of electron beam heating, and perhaps most importantly the effect on quantification of mass loss during examination.

VII. Conclusions Ultrahigh resolution elemental microanalysis of biological tissue is in a state of infancy as regards full quantification of concentrations within cells and tissues. Further, few studies have been performed which yield highly significant data concerning elemental concentration distributions and gradients within tissue. It is clear, however, that studies which have been performed by careful investigators using advanced techniques and equipment demonstrate the tremendous power ultramicroanalysis brings to the solution of many vital questions relating to physiology, cell biology, and medicine. In summing up the potential of the technique Lechene (1977) states, “In the future, electron probe microanalysis can bring to physiology what electron microscopy brought to anatomy. The discipline of electron probe can help to advance physiology from the realm of the black box approach to describing cellular function.” This is not an overstatement. Clearly a number of very fundamental questions relating to application of the technique to the quantification of elements with ultrahigh spatial resolution on the order of cell membrane thickness have not as yet found satisfactory answers. These questions are, however, being addressed by major laboratories, and there is no basically physical limitation to the analysis required to obtain a solution to all the major problems in ultramicroanalysis. The questions addressed thus far have been mainly in the area of proving the technique a useful biological tool and in application of the technique to biological problems which require only semiquantification results. The intense work presently going on at the several major laboratories attacking various facets of the problem should allow full quantification of the technique within a short time. The areas of investigation which will lead to full quantification are: 1. Complete computer analysis of energy-dispersive x-ray spectra with peak stripping, least-squares analysis, and so on. 2. Quantification of elemental loss during analysis and identification of techniques to minimize this loss.

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3. Fabrication of primary elemental standards having characteristics closely akin to biological tissue. 4. Extension of the technique to include electron energy loss analysis which allows concentration determinations of the light elements, those with an atomic number less than that of Na, not possible with x-ray energy analysis. 5 . Extension of the instrumentation to allow ultrahigh resolution analysis (- 1-10 nm) and full computer analyses of the electron image and control of the electron beam to remove the inefficient unit (i.e., the electron microscope operator) from the analysis scheme.

In addition to the above areas of investigation which are required for full quantification of ultramicroanalysis in the determination of elemental concentrations on the subcellular scale for biological tissues, the entire area of specimen preparation needs serious attention. The two alternative routes in “native state” preparation, frozen-hydrated and frozen-dried, in addition to the conventional embedding process, need careful examination and determination of the degree of loss and transmigration of elements during the preparation technique as well as during analysis. It is likely that the technique of freeze-sectioningand subsequent drying of the tissue prior to examination will be the most widely used method of specimen preparation. Application of this technique, however, requires that full determination be made of the translocation of elements and ions during the drying process. Further, the use of frozen hydrated tissue in analysis is required in every case in which lumen must be examined in addition to the normal cellular concentration. In certain cases compounds which bind selected elements such as pryoantimonate can be used in conventionally prepared electron microscope sections. Care should, however, be taken to ensure that checks of the full binding of these compounds be ensured prior to acceptance of data from the technique. This again requires the use of frozen hydrated tissues. The entire question of morphological destruction during freezing, whether the sample will be subsequently analyzed following freeze-drying or by examination of the frozen hydrated tissue, needs careful treatment. Examination of each of the aforementioned questions is expensive, both in research time and research funds, but it is fully required before meaningful information of a quantitative nature can be obtained from this potentially extremely powerful technique. Prior to the time when these data are available extreme care should be taken that the data obtained by the technique are carefully scrutinized both from their biological implications and their physical significance. The endeavor of subcellular quantitative analysis of biological tissue by ultramicroanalysis requires close collaboration of the biological and physical sciences to a degree seldom required before. This collaboration is occurring and will lead soon to reduction to a relatively simple practice of the extremely powerful technique of ultramicroprobe analysis.


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ACKNOWLEDGMENTS The author gratefully acknowledges the contributions of Drs. Johnson and MacKenzie in the preparation of the manuscript for this article through numerous discussions and extensive editing. I am also particularly grateful to Marie Cantino for assembling much of the reference list and for her critical reading of the manuscript. 1 am also indebted to Keith Monson for his many suggestions and gratefully acknowledge the typing and editing talents of Linda Richter, Judy Bragg, and RenCe Freeman.

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Intracellular elemental concentrations in renal tubular cells. An electron microprobe analysis.

Staining and relative determination of elemental contents by the laser microprobe.

Ultrahigh-resolution scanning electron microscopy of biological materials.

An electron microprobe analysis of osteofluorosis in the rabbit.

Ultrahigh-resolution NMR spectroscopy.

Determination of elemental carbon emission.

Ultrahigh-spatial-resolution chemical and magnetic imaging by laser-based photoemission electron microscopy.

Lack of corrosion of stainless steel instruments in vivo by scanning electron microscope and microprobe analysis.

Elemental mapping in achromatic atomic-resolution energy-filtered transmission electron microscopy.

Electron microprobe investigations into the process of hard tissue formation.

Irradiation effects in the electron microprobe quantitation of mineralized tissues.

Tellurium-induced neuropathy: correlative physiological, morphological and electron microprobe studies.

Quantitative X-ray Elemental Imaging in Plant Materials at the Subcellular Level with a Transmission Electron Microscope: Applications and Limitations.

Laser microprobe mass analysis of organic materials.

Acoustic microscopy: resolution of subcellular detail.

Surface determination through atomically resolved secondary-electron imaging.

Effect of sodium chloride concentration on elemental analysis of brines by laser-induced breakdown spectroscopy (LIBS).

Ultrahigh-resolution imaging of water networks by atomic force microscopy.

Manipulating cell signaling with subcellular spatial resolution.

Transcutical imaging with cellular and subcellular resolution.

Elemental distribution in striated muscle and the effects of hypertonicity. Electron probe analysis of cryo sections.

Cholesterol oxidase: ultrahigh-resolution crystal structure and multipolar atom model-based analysis.

Elemental analysis of occupational and environmental lung diseases by electron probe microanalyzer with wavelength dispersive spectrometer.

Measurement of elemental concentration of aerosols using spark emission spectroscopy.