Digital acquisition and processing of electron micrographs using a scanning transmission electron microscope

Ultramicroscopy 7 (1981) 45-54 North-Holland Publishing Company

45

DIGITAL ACQUISITION AND PROCESSING OF ELECTRON MICROGRAPHS USING A SCANNING TRANSMISSION ELECTRON MICROSCOPE A. ENGEL, F. CtlRISTEN and B. MICHEL Microbiology Department, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland

A digital acquisition system that collects multichannel information from a scanning transmission electron microscope (STEM) and its application are described. The hardware comprises (i) single electron counting detectors, (ii) a digital scan generator, (iii) a digital multi-channel on-line processor, (iv) an interface to a minicomputer, and (v) a display system. Experimental results characterizing these components are presented, and their performance is discussed The software includes assembler coded programs for'dynamic file maintenance and fast acquisition of image data, a display driver, and FORTRAN coded application programs. The usefulness of digitized STEM is illustrated by a variety of biological applications.

1. Introduction More than ten years ago electron microscopists were attracted by the promising results obtained by the first high resolution scanning transmission electron microscope (STEM), as well as its interesting modes of signal collection [ 1]. Experts were excited about the clearness of elastic dark field images due to the lack of interference artifacts [2], the high collection efficiency of the annular dark field detector [3] and the possibility of receiving several images simultaneously and storing them on-line in a digital computer. This led to the initiation of several STEM projects primarily aimed at biological applications. Although these STEMs by now work according to initial specifications, they do not fulfill the expectations of the molecular biologists to visualize proteins Or nucleic acids at atomic resolution. These instruments simply cannot resolve all the problems related to specimen preparation and beam damage that may have been partially disregarded during the early stages of instrumental development. Yet a STEM, once interfaced to a computer, allows quantitative electron microscopy to be performed that is interesting not only for the biologist but can be applied to many fields of research, even if atomic resolution is not achieved. In this paper we describe the use of a digital data acquisition system that inter0304-3991/81/0000-0000/$02.50 © 1981 North-Holland

faces a Vacuum Generators STEM HB-5 to a UNIVAC minicomputer. Many mass determination experiments accomplished in our laboratory [ 4 - 6 ] well illustrate the capability of this system and the usefulness of quantitative electron microscopy in general. Since most of the components of the data acquisition hardware have been described previously [6,7], we discuss their performance in view of several years of routine use rather than presenting individual circuits. Special attention is given to the detectors because there is a lack of experimental data concerning fast pulse counting and because the detection system used (scintillator-photomultiplier) is sufficiently universal to be used in future instruments. The concepts of the softw,are and some application programs are described as far as they are of general interest. Advantages of signal detection with absolute calibration such as single electron counting and the overall performance of the instrumentation are illustrated by a few typical mass-determination results. Finally, an attempt is made to summarize the features of some commercial components of particular interest.

2. Detector system The most important part of any digital data acquisition system suitable for electron microscopy is the

46

A. Engel et al. /Digital acquisition and processing of electron micrographs

detector system. The detection quantum efficiency (DQE), specified as D Q E = (S/N)2out/(S/N)]n,

(1)

should be as close to 1 as possible. Thus, the detectors used should provide single electron detection capability without introducing additional noise. The combination of a scintillating crystal with a photomultiplier has proven to be a convenient solution fulfilling this specification as long as simple detector geometries are used. If, however, the single electron event needs to be precisely localized, the image intensifier-storage system described by Hermann et al. [8] would be the detector of choice. The performance of the scintillator-photomultiplier combination is determined by the properties of the scintillator, the efficiency of the light coupling device and the properties of the photomultiplier. Scintillator specifications are available from the manufacturer (table 1), but there is a lack of information concerning (i) beam sensitivity of various scintillating materials and (ii) afterglow. We have determined the critical dose for beam damage for a plastic, a glass and a CaF2(Eu) scintillator. To this end these scintillators were exposed to a beam of about 0.5/~m diameter and a fixed current in the range of 10 - l ° to 10 - s A for time intervals of variable length. By plotting the diameter of the resulting marks versus exposure time it is possible to specify the dose at which the first discernible damage occurs, i.e., a mark with infinitesimally small diameter (fig. 1). We have also made an attempt to quantify the afterglow of the same scintil-

Table 1 Properties of three solid state scintillators that can be used within a STEM (from the Nuclear Enterprises Scintillator Catalogue)

CaF 2 (Eu) NE 905 NE 160

Light output a)

Decay constant (ns)

Wave length of max. EM (nm)

Meltingor softening point (°C)

110 25 59

1000 18,60 2.3

435 395 423

1418 1200 80-150

a) This is given in % anthracene. The absolute output of anthracene is close to 4% for ~ radiation (Birks [21]).

lators, but the differences between various scintillator types observed within the time interval investigated were very small (fig. 2). This indicated that the background decay shown in fig. 2 may rather be related to the photomultiplier than to scintillator afterglow. Considering that only a small fraction of the primary electron energy is converted to photons, the efficiency of the light coupling device is rather critical. For instance, the coupling efficiency needs to be about 4% if a typical plastic scintillator is expected to produce 10 photoelectrons per incident 100 keV electrons (see tables 1 and 2). While this is easily achieved with simple detector geometries; it is not trivial for the annular detector. Various designs have successfully been applied using either a metal mirror [9] or a quartz light pipe [10]. The annular detector in the STEM HB-5 has previously been described [7]. It consists of an aluminium-coated scintillator tilted at 45 ° to the beam axis and an aluminium-coated glass tube which reflects the light from the scintillator through a vacuum window onto the photocathode. An improvement of the light coupling efficiency of about 20% has been obtained by sandblasting the scintillator side that faces the photomultiplier. Some basic properties of the photomultiplier used in our instrument (RCA 8850) are given in table 2. Of interest is the high secondary-emission ratio (about 30) of the gallium-phosphide first dynode, which makes single photoelectron resolution possible for statistical reasons. In addition, this also leads to a noise pulse-height distribution which sharply peaks at 1 photoelectron equivalent. This permits (i) a simple calibration of the pulse height in photoelectron equivalents, and (i.i) an efficient discrimination of the dark current noise. On first sight, the latter seems to be of little importance, since the dark current amounts

Table 2 Properties of an RCA 8850 photomultiplier (from the specification table provided by the manufacturer) Gain of the first dynode Quantum efficiency Single photoelectron pulse height resolution Anode dark current at 22°C

35 at 600 V 31% at 385 nm 40% FWHM 167 counts/s (typical) 667 counts/s (maximum)

A. Engel et al. /Digital acquisition and processing of electron micrographs

47

C

,,=,

0.5"

:E ~ 0.25"

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CaF2 (Eu)

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NE 110 -10

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,

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:

50

Fig. 1. Plots of the damage mark size versus exposure time allow the critical dose for beam damage of various scintillators to be determined.

typically to about 200 counts/s. However, since the photomultiplier of the annular detector assembly is quite frequently exposed to high light levels, the dark current is considerably increased. In our case, the dark current amounts to 5 × 103 counts/s before switching the beam on and 104 counts/s immediately after switching if off after operating the STEM in imaging mode with a probe current of 10 -12 A for

tot I

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Fig. 2. The count-rate decay after exposure of the detector assembly to a beam current of 2 X 10-12 A and 100 keV for different scintillators.

10 min (see also fig. 2). Considering that we collect about 2 electrons per 40/is when recording a low dose micrograph of unstained protein structures, the dark current noise is thus not negligible any more. In view of the aim to obtain a DQE of about 1 and the need to store images in digital format, we decided to use our detectors in a single electron counting mode and to possibly discriminate any kind of thermal noise. To this end a plastic scintillator (NE 160) and a commercial 100 MHz multichannel discriminator (Le Croy, Model 621 AL) are employed. Although the plastic scintillator is quite sensitive to the electron beam (fig. 1), it is the only one to provide sufficient speed and light output for single electron counting (table 1). In fact, beam damage proved to be a minor problem, since the scintillator life-time is estimated from the critical dose to be at least 1010 S under normal operating conditions. Furthermore, the replacement of the scintillator is a straightforward operation, and scintillators can easily be repaired by polishing the damaged layer (~0.1 mm) off. To set the multiplier gain properly with respect to the minimum discriminator threshold (30 mV for our equipment), the pulse-height distribution needs to be known for all beam energies used. We have determined pulse-height distributions either by differentiating the count-rate versus discriminator threshold function [7], or more simply by scanning the averaged pulse shape with a microdensitometer (fig. 3). A clear separation between signal pulses and single photoelectron events are observed for 80 and 100 kV, thus permitting a clipping of the thermal noise without noticeable signal count losses. The measurement of the count-rate versus probe current confirms single electron counting and illustrates the excellent linear-

48

A. Engel et al. /Digital acquisition and processing of electron micrographs

.J

60kV

uu I--

=UJ

I I•

o.

i

pZ

o

0 Lu '~

PHOTOELECTRON 0

5 nS/U

5

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100 kV

0

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10

0

5

10

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Fig. 3. An illustration of a simple way to determine the pulse height distribution. A time-averaged (5 s)

pulse shape obtained from

a 200 MHz oscilloscope is scanned along the line (arrows) with a microdensitometer. Densitometer traces of pulses due to 60, 80 and 100 keV electrons show the distinct, approximately linear, increase of the averaged pulse height versus beam energy. The predominant single-photon pulses are due to an increased dark current of the relatively strongly excited photomultiplier. ity of this detector arrangement in a count-rate interval of 0.1 to 10 MHz [7]. The sharp cut-offat 12 MHz is due to saturation of the last multiplier dynodes. This maximum count-rate is a function of the multiplier voltage and has been measured for the 6photoelectron pulses produced by a 100 keV beam (fig. 4). As illustrated by this graph, the maximum count-rate can simply be extended by a fast pulse amplifier; in this case, however, dead time losses will become a limiting factor rather than multiplier overloading. The overall-performance of the annular detector is presented in fig. 5. To obtain the count statistics, the detector was exposed to the unscattered beam by shifting the objective aperture off-center. A series of micrographs at different probe current levels were

then recorded and the average and sigma calculated. The DQE was evaluated according to eq. (1), assuming a Poisson distribution of the input signal. In order to illustrate the influence of thermal noise, a series of pictures of a thin carbon film on a holey plastic foil was recorded for several settings of the multiplier gain. Although the thermal background has been efficiently clipped for fig. 5c, a residual background is observed within the hole of the thin carbon rdm which is likely to be due to electrons scattered by the objective aperture. 3. Hardware

The block diagram of the data acquisition hardware shown in fig. 6 gives an overview of the compo-

A. Engel et al. /Digital acquisition and processing of electron micrographs to

uJ

~_ 107 Z

o

X

t o6

1.65 19 2 25 PHOTOMULTIPLIERVOLTAGE(kV)

Fig. 4. Maximum count rate versus multiplier voltage of an RCA 8850 for light pulses producing 6 photoelectrons in average.

nents developed or purchased and their interconnection. The master of the system is the digital scan gen-

1.1

\ °°Et I.O

i

49

erator. It drives the STEM probe along a raster of 512 by 512 or up to 4096 by 4096 picture elements (pixels) and synchronizes the input interface. Pixel times from 5 to 625/as can be selected, thus providing scan times from 1.25 to 104 s per frame. The basic 100 kHz clock from which the pixel times are derived is phase-locked to the line frequency. Subframes of a minimum side-length of 1/8 of the full frame can be selected and conveniently positioned by means of a LED matrix. An interface allows the pertinent parameters concerning the state of the scan generator, the magnification and the beam current to be transmitted before the acquisition of an image. The input interface drives 4 CAMAC scalers (SEN, Geneva, Type 2003), selects 8 out of 12 bit from each of the 8 channels, and packs up to 4 channels in 16 bit words in order to transmit them via direct-memory-access to the minicomputer (UNIVAC-VARIAN, V73). In parallel to the digital data acquisition, a graylevel image is recorded by the high-resolution oscilloscope (Perkin Elmer, ETEC) on 120 trim or on a scan converter (Hughes, MSC 1). The gray-level generator now in use provides 16 gray-levels but is presently adapted to count up to 255 pulses and to convert the result to a gray-level via a 256 by 8 bit RAM look-up table and an 8 bit D/A converter.

O.g

O.1

--

16

~

I

1

10

COUNT RATE (MHz)

b

1

STEM HB5 DETECTORS

U U

HB 5 DISPLAY

F Nhole/Nfilm = 0.11

CRT 1 CRT 2 CRT CAMERA

~

Nhole/Nfitm =0.38

Fig. 5. Distribution from single electron counting (a) and the DQE versus count rate (b). The ratio of counts within the hole (white boxes) to counts on the thin f'llm (black boxes) is 0.11 if single and double photoelectron pulses are discriminated (c) and 0.38 if they are counted (d). Scan speed was 80/~s/pixel and all of the 512 by 512 pixels are shown. The afterglow distinctly visible to the right of the arrow (d) can be related to the slow part of the count-rate decay shown in fig. 2.

U 32K D2,4M R A C KDMA ! SK 9 T TAPE V 73

V 73 DISPLAY ~DI;p~;y~,-.--~

DMA 17 ~

p

"FACE ~

CRTV73

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Fig. 6. The hadrware components of the data acquisition and display system and their interconnection.

50

A. Engel et al. /Digital acquisition and proeessing o f electron m icrographs

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A. Engel et al. /Digital acqu&ition and processing of electron micrographs

In view of the development of a multichannel spectrometer, a digital on-line processor was built that compresses the signals S n from 4 channels into an output of the form: OUT = ]~(1)/~(2)

or

OUT = 7 -2 y(1) y(2),

(2)

where

4 ]~(i) = ~ 0~(~){S n _ ~ n } , 1

The normalization factor (3') required to eliminate tip noise is obtained from the objective aperture current. Processing speed is adapted to the fastest mode of digital data-acquisition (20 #s per pixel) and is achieved due to the application of ROM look-up tables for all divisions. Fig. 7 gives a block diagram of the processor and illustrates the basic functions given in eq. (2), as well as its processing capability. The computer-display system operates completely independently of the STEM image-acquisition hardware. The display interface allows 512 by 512 4 bit gray-level images to be written onto 35 mm film via a second recording oscilloscope or to be stored on a second scan converter. Efficient gray-level plotting is

51

obtained in the line-plot mode, for which only the coordinates of the line starting point and the 4 bit intensity levels for the entire line need to be transferred from the computer. The intensity levels are appropriately adjusted by the pulse lengths of 16 oneshots. Characters and graphs are generated in a pointplot mode, where X - Y coordinates are required for each pixel. A light pen or alternatively a cursor permits to enter X - Y coordinates with a resolution of 8 bit per line. A 96 line opto-coupler is used to connect the microscope as well as the interactive display system with the minicomputer.

4. Software The data acquisition and processing programs developed are based on the features of the real-time operating system (VORTEX) provided by UNIVAC, and the configuration of the V-73 minicomputer (64 kbyte of memory, 1 k of 64 bit writeable control store, a 5 Mbyte moving head disk, and a 9-track magnetic tape drive). The assembler-coded STEM system software includes a tale maintenance program, a VORTEX independent disk-driver for high-speed

Fig. 8. Examples of two mass determination experiments performed to check the calibration of the absolute scale. For Tobacco Mosaic Virus a mass per length of 134 ± 4 kdalton/nm and for glutamine synthetase a mass of 610 -+53 kdalton was obtained, in very good agreement with published data. The scale bar represents 50 nm.

52

A. Engel et al. /Digital acquisition and processing of electron micrographs

STEM-to-disk transfer, and a display-driver which is implemented in the VORTEX nucleus. The File maintenance allows disk as well as tape space to be dynamically allocated, and Fries (i.e. micrographs) to be searched on the basis of identification segments stored in a 120 word File identification block. These segments such as date, specimen, preparation method, etc., are entered at the beginning of a session, and the images subsequently recorded are automatically labelled by this identification. An interrupt signal from the STEM activates the fast disk program, which first acquires data concerning the state of the scan generator, magnification and beam current, subsequently allocates the required disk space and then starts the scan synchronized with the disk. Micrographs are finally saved on tape using the identification block plus microscope parameters as header. The display driver alows graphs, characters of variable size, and gray level images to be plotted. It also received X - Y coordinates from the lightpen-cursor unit. Application programs are written in FORTRAN IV, taking advantage of assembler coded subroutines. They include programs to display pictures from tape or disk, to evaluate the mass of globular particles, Filaments or sheets [4], to calculate the diffraction pattern of up to 128 X 128 pixel fields and to smooth noisy images by convolution with a square box. Computation or transfer intensive programs are optimized with respect to speed as well as memory space. A convolution of a 512 X 512 pixel image with a 4 × 4 pixel box in real space for instance, takes 95 s, or a 128 × 128 pixel FFT 45 s, and is performed in core due to a space-saving 16 bit floating point format [6].

5. Mass measurements

The possibility of quantitative electron microscopy is demonstrated by mass determinations of protein supramolecular structures via electron scattering. This method, long time ago introduced by Zeitler and Bahr [11] and recently reviewed by Wall [12], is of significant interest for the biologist, because it permits mass measurements of globular oligometric particles, friaments or sheet-like large structures that in many cases cannot be measured by any other technique. Concerning precision, it compares favorably with the best method hitherto used: the analytical ultra-

centrifuge. Other attractive advantages are the need of very small amounts of material, and the possibility to investigate mixtures [6]. Furthermore, 2D high-resolution mass maps of supramolecular structures can be obtained, provided that they are well preserved during preparation and are amenable to noise elimination through digital image processing. The calibration of the method can be accomplished by mixing a standard such as Tobacco Mosaic Virus (TMV) with the protein solution to be investigated. Alternatively, the collection efficiency e of the annular detector can be evaluated from scattering theory, if the detector geometry is known. We have determined the geometry of our annular detector using the diffraction pattern of thallium chloride, and calculated e = 0.69 for 80 keV electrons [4]. Since the objective lens is operated at low excitation ( f = 3.5 ram), the collection efficiency is relatively independent of focussing. The average elastic scattering cross-section per atom of a protein as well as the average atomic mass is calculated from the elemental composition of a typical protein; this together with e then allows to determine the calibration factor to be 1910 dalton per electron counted by the annular detector at a recording dose of one hundred 80 keV electrons/nm 2 [4]. Using this calibration we measured for the mass per length of TMV 134 -+ 4 kdalton/nm (published value = 131 kdalton/nm [13]) and for the mass of glutamine synthetase 610 -+ 53 kdalton (published value = 600 kdalton [14]). We also checked the validity of the relative method using TMV as reference. As it turnted out, TMV measured 128 -+ 9 kdalton/nm within a solution of nucleo-protein-DNA complexes, 149 + 8 kdalton/nm together with an oligomeric protein that has a very hydrophobic site, and 248 -+ 11 kdalton/nm with a solubilized membrane protein. Although these values differ quite much, the appearance of TMV was rather similar in all three cases, thus indicating that the use of a reference particle of known mass may not be without problems (see fig. 8).

6. Discussion

Several years of routine operation have given ample occasions to test the data acquisition system described here to its limits. Although in general satisfied with its performance, we have reFmed the initial system [7] in

A. Engel et aL/ Digital acquisition and processing of electron micrographs

many respects. We have found it to be useful (i) to add a recording oscilloscope providing a resolution of 2000 lines per frame to the STEM, (ii) to separate the computer display and image recording facility from the microscope, (iii) to isolate the computer from the STEM via an opto-coupler, and (iv) to employ a cursor rather than a light-pen in order to enter X - Y coordinates. What has proven to be not very satisfactory, but has not been changed as yet, are the two scan converters. Low dose microscopy of unstained air- or freezedried proteins, quantitative studies on beam damage, and mass-determinations on a wide range of protein and protein-DNA complexes have demonstrated the precision and stability of the single electron counting annular detector described extensively in the first section. Dark current pulses clearly contribute to the noise in the image, although this may be neglected in many applications. The elegant charge to pulse ADC described by Zubin and Wiggins [15] is therefore a valuable alternative to a discriminator, in particular for high beam currents. However, the discriminator offers besides the advantages discussed, the further one that it is commercially available. This brings us to a point which seems to be worthwhile to mention here. Over the past decade several powerful STEM data acquisition systems have been developed independently, each having its particular advantages [ 15-18 ]. Many further similar systems have been built for other kinds of digital image acquisition, and all the rather small groups involved in these developments went through similar difficulties. Meanwhile, the electronic industry has produced a wealth of devices that are far more powerful than those available at the time the projects mentioned above were initiated. Thus, a better and more versatile system is commercially available today that can ever be built by a small, individual group. A modular commercial system such as CAMAC furthermore offers the advantage that modules with up-to-date technology can be added at any time. Very attractive frame buffered systems driving a colour TV display, for instance, can be purchased now from most of the bigger CAMAC module manufacturers, including software for convenient data processing. Thus, it seems recommendable to acquire such a commercial system instead of investing the energy to develop another unique system from scratch.

53

A review on possible computer developments has been presented recently [16]. It outlines the processing speed available in the near future, which according to our own experience is crucial to exploit the advantages of the STEM to the fullest extent. Microdose microscopy will remain to be of utmost importance, at least for biological electron microscopy, but it can only be of use if averaged images are at hand immediately. How else should the experimentor be in a position to pursue the rather delicate development of specimen preparation and the extraction of biological significant data with reasonable efficiency? An exciting possibility for on-line processing is the application of look-up-tables. We have demonstrated this by the divider within our processor, and a lookup-table is presently fitted to our gray-level generator that can be loaded with a convenient 7-function from the computer. However, this concept is presently applied with a much higher degree of sophistication for real time processing (100 ms/frame) in conjunction with an IMANCO system equipped with a 720 × 896 pixel frame buffer [19]. This combination of look-uptables with an array processing circuitry allows linear or nonlinear operations to be performed over subframes at a rate of 2 × 108 operations/s. It appears to be the ideal system for real-time cross-correlation averaging, which is now becoming successfully used in off-line processing of electron micrographs.

Acknowledgements The authors acknowledge the continuous interest and support of E. Kellenberger and E. Weibel. The work has been supported by the Swiss National Foundation for Scientific Research through grant No. 3.153.77.

References

[1] A.V. Crewe, Quart. Rev. Biophys. 3 (1970) 137. [2] A. Engel, J.W. Wigginsand D.C. Woodruff, J. Appl. Phys. 45 (1974) 2739. [3] J.P. Langmore, J. Wall and M.S. Isaacson,Optik 38 (1973) 335. [4] A. Engel, Ultramicroscopy 3 (1978) 273. [5] A. Engel, in: Electron Microscopyat Molecular Dimensions, Eds. W. Baumeister and W. Vogell (Springer, Berlin, 1980)pp. 170-178.

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A. Engel et al. / Digital acqu&ition and processing of electron micrographs

[6] A. Engel, J. Microsc: Spectrosc. Electron. 5 (1980) 581. [7] A. Engel, M. Strahm, B. Michel and J. Dubochet, in: Scanning Electron Microscopy, 1977, Vol. I. Ed. O. Johari, pp. 365-370. [8] K.H. Hermann, D. Krahl and H.P. Rust, J. Microsc. Spectrosc. Electron. 5 (1980) 639. [9] J.W. Wiggins, J.A. Zubin and M. Beer, Rev. Sci. Instr. 50 (1979) 403. [10] J.S. Wall, Brookhaven Natl. Lab., USA, personal communication. [11] E. Zeitler and G.F. Bahr, J. Appl. Phys. 33 (1962) 847. [12] J.S. Wall, in: Scanning Electron Microscopy, 1979, Vol. If, Ed. O. Johari, pp. 291-303. [13] J.M. Kaper, in: Molecular Basis of Virology, Ed. tt. Fraenkel-Conrat (Van Nostrand-Reinhold, New York, 1968) pp. 1-133.

[14] R.C. Valentine, B.M. Shapiro and E.R. Stadtman, Biochemistry 7 (1968) 2143. [15] J.A. Zubin and J.W. Wiggins, Rev. Sci. Instr. 51 (1980) 123. [16] A. Jones, J. Microsc. Spectrosc. Electron. 5 (1980) 595. [17] S. Lackovic, M.T. Browne and R.E. Burge, in: Scanning Electron Microscopy, 1979, Vol. I, Ed. O. Johari, pp. 137-144. [18] M. Strahm and J. Butler,.in: 37th Ann. Proc. Electron Microscopy Soc. Am., 1979, Ed. G.W. Bailey, p. 598. [ 19] H.J. Keller, Inst. of Anatomy, Bern, Switzerland, personal communication. [20] O. Saxton and W. Baumeister, submitted. [21] J.B. Birks, The Theory and Practice of Scintillation Counting (Pergamon, Oxford, 1964) p. 241.