Mass Determination by Inelastic Electron Scattering in an Energy-Filtering Transmission Electron Microscope with Slow-Scan CCD Camera

JOURNAL OF STRUCTURAL BIOLOGY ARTICLE NO. SB973861

119, 72–82 (1997)

Mass Determination by Inelastic Electron Scattering in an Energy-Filtering Transmission Electron Microscope with Slow-Scan CCD Camera Bernhard Feja Maurice E. Mu¨ller Institute for Microscopy, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland

Markus Du¨rrenberger Interdepartmental Electron Microscopy, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland

Shirley Mu¨ller Maurice E. Mu¨ller Institute for Microscopy, Biozentrum, University of Basel, Klingelberegstrasse 70, CH-4056 Basel, Switzerland

Rudolf Reichelt Institute for Medical Physics and Biophysics, Westfa¨lische Wilhelms-Universita¨t, Robert-Koch-Strasse 31, D-48149 Mu¨nster, Germany

and Ueli Aebi1 Maurice E. Mu¨ller Institute for Microscopy, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Received December 18, 1996, and in revised form February 11, 1997

approach, a number of typical biological samples were evaluated in the EFTEM and the results obtained were compared with those from STEM. The data presented demonstrate that an EFTEM equipped with a high-performance slow-scan CCD camera is an effective electron optical device for mass determination of biomolecular assemblies with an accuracy and reproducibility comparable to that achieved by STEM. r 1997 Academic Press

One method of determining the mass of biomolecular assemblies takes advantage of the linear relationship between the mass of a thin sample and the scattered fraction of the incident electrons. Mass determination by electron scattering was first performed by scanning transmission electron microscopy (STEM), collecting the elastically scattered electrons by an annular dark-field detector. In the energy-filtering transmission electron microscope (EFTEM), the dark-field images formed by inelastically scattered electrons are recorded with a highly linear and sensitive slow-scan CCD camera. A calibration factor relating the collected fraction of scattered electrons to the mass of a sample can be determined by using a particle of known molecular mass as a reference standard. Processing of the digital dark-field images obtained by the slow-scan CCD camera allows the mass of thin particles, the mass per length of filaments, or the mass per area of planar samples to be determined. To validate this

INTRODUCTION

The mass is an important parameter when evaluating the physical properties of biomolecules or their supramolecular assemblies. Over the years a number of techniques have been developed for mass determination. Most of them either require a significant amount of material (i.e., on the order of 1 mg of purified sample for ultracentrifugation or laser light scattering) or lead to dissociation of the oligomeric complexes or supramolecular assemblies (e.g., SDS gel electrophoresis). Another method for mass determination is based on the electron scattering properties of biological matter (Zeitler and Bahr, 1962;

1 To whom correspondence should be addressed. Fax 141 61 267 2259. E-mail [email protected].

1047-8477/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

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Langmore et al., 1973; Wall et al., 1974). This method was proposed and demonstrated to work in principle many years ago by Zeitler and Bahr (1962) but it was not until the emergence of the scanning transmission electron microscope (STEM) that it became a more practical and effective experimental tool (Lamvik, 1978; Engel, 1978; Wall, 1979). In the STEM the wide-angle elastic dark-field signal is acquired by an annular detector which allows single electron counting of the scattered electrons. Mass determination by STEM is well established and has been in use for over 15 years (for a review, see Engel, 1982; Wall and Hainfield, 1986; Mu¨ller et al., 1992; Engel and Colliex, 1993). In the present paper, we demonstrate that mass measurements by the evaluation of electron scattering can also be done in an energy-filtering transmission electron microscope (EFTEM) to nearly the same precision as by STEM. In the EFTEM the low-angle inelastic dark-field images recorded digitally with a high-performance slow-scan CCD camera are employed. The mass of a number of representative biomolecular specimens was determined with EFTEM/CCD instrumentation, and the results obtained were compared with those from STEM. In addition, Monte Carlo simulations were performed and compared with inelastic scattering data obtained with latex beads of different sizes to determine the maximum specimen thickness for which a linear calibration model is still valid. THEORY

The theory of inelastic electron scattering has been described by Wall et al. (1974). If the thickness t of a specimen is smaller than the mean free path length L of the incident beam electrons, multiple scattering events (i.e., n $ 2) may be neglected. This can be readily assessed by the Poisson distribution of scattering events given as P(n) 5 xn/n! · exp(2x) (Lamvik, 1978), where x 5 t/L. In the case of single electron scattering (i.e., n 5 1), the probability P(1) can be approximated by P(1) < x(1 2 x) < x for x 9 1, i.e., P(1) ~ t. Therefore, the number of electrons inelastically scattered into a given energy interval and scattering angle, Nin, is given by Nin 5 7sin8nN0 /A,

(1)

where 7sin8 is the average partial inelastic scattering cross section of atoms that constitute a protein, n is the total number of atoms within the irradiated area A, and N0 is the total number of incident electrons per area A. Calculating 7sin8 by averaging the inelastic cross sections over the elements comprising a biological specimen does not yield a good approxima-

tion, because both the chemical properties of the sample and the surface and bulk plasmons have to be considered. Instead, the formalism described by Wall et al. (1974) is used to estimate the partial inelastic cross section from the electron energy-loss spectrum (EELS) of an organic sample. With formula [1] the mass M of the proteinaceous specimen is given by M 5 n7Ma85

Nin7Ma8A N07sin8

,

(2)

where 7Ma8 is the average atomic mass of a typical proteinaceous specimen (i.e., with the relative abundance of H:C:N:O being 0.492:0.313:0.094:0.101). Since the sample is adsorbed to a carbon support film, the background scattering B has to be subtracted from the inelastic signal Nin. Introducing the electron dose D 5 N0 /A

(3)

and the calibration factor C 5 7Ma8/7sin8,

(4)

the mass of the specimen can be written as M 5 C(Nin 2 B)/D.

(5)

To estimate the effect of multiple scattering, we have employed a Monte Carlo simulation program described and developed by Reichelt and Engel (1984). INSTRUMENTATION

A LEO EM912 energy-filtering TEM (LEO Electron Microscopy, Oberkochen, Germany) equipped with an LaB6 gun and a Proscan high-speed slowscan CCD camera (Proscan, Penzing, Germany) employing a phosphorus scintillator was used for mass determination (Fig. 1). The CCD camera is interfaced via a Matrox frame grabber to an Intel 486 PC running the LEO ESIVISION image processing software. The acquisition system allows remote control of the microscope functions and monitoring of various microscope parameters via an RS232 interface. The software package also contains a built-in Ccompiler that provides a powerful tool to customize and extend its functionality. One important feature of the EM912 EFTEM is its Ko¨hler illumination system (Benner and Probst, 1994) which produces a parallel and homogeneous illumination of the specimen. The irradiated specimen area is defined by shifting the illumination beam so as to pass through a particular aperture of a

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monitor is used for screening the specimen at low magnification and low electron dose, as well as for focusing. The mass measurements performed in the EM912 EFTEM were critically compared with corresponding measurements made in a Vacuum Generators HB-5 STEM (Vacuum Generators, East Grinstead, UK). Image analysis of both the EFTEM and the STEM data was performed on a DEC Alpha workstation run under VMS employing the modular software package IMPSYS (described by Mu¨ller et al., 1992). METHODS Specimen Preparation FIG. 1. Schematic beam path of the LEO EM912 EFTEM with illumination (dotted) and image (solid) beam paths. The AIS aperture defines the illuminated area, and the objective aperture fixes the acceptance angle to 25.4 mrad.

five-hole diaphragm (aperture sizes: 37.5, 75, 150, 300, and 600 µm). The aperture defining the illuminated area may be selected either automatically depending on the magnification (Automatic Illumination Selection, AIS) or manually by the user (Manual Illumination Selection, MIS). The imaging energy filter allows a particular range of the electron energy-loss spectrum to be selected by the insertion of an adjustable slit aperture in the energy-dispersive plane. A liquid-nitrogencooled anticontamination device protects the sample from contamination by residual hydrocarbons etc. An electron collector mounted in the final image plane allows the beam current, and thus the electron dose incident on the sample, to be measured. For this purpose, the collector current corresponding to a homogeneous beam with all apertures removed from the beam path and in the absence of a sample is measured. The actual electron dose in the specimen plane is calculated from the collector current, the magnification, and a calibration factor which includes the collector area and is also corrected for the fraction of backscattered electrons. The slow-scan CCD camera system consists of a glass fiber plate coated with P43 phosphorus coupled by another fiber optic plate to a Peltier-cooled 1024 3 1024 pixel CCD chip with 19 mm side length. The readout frequency is 1 MHz, and the information depth is 14 bit. A so-called flat-field correction eliminates the dark current contributions of the CCD chip (black-level pattern) and the nonuniform contributions of the scintillator (fixed pattern) to the raw image (Liu, 1992). A side-mounted TV camera in a low-magnification position with integrated image buffer and separate

For mass determination specimens were adsorbed to a thin (2–5 nm) carbon film which was supported by a thick fenestrated carbon layer deposited onto a 200 mesh/inch gold-coated copper grid (Engel, 1978, 1982; Fukami and Adachi, 1965). To facilitate focusing and astigmatism correction, small gold particles had been deposited onto the thick fenestrated carbon layer. To achieve more effective and reproducible adsorption of the sample to the thin carbon film, the grids were subjected to glow discharge in a reduced atmosphere of air for 15 sec. (Aebi and Pollard, 1987). The specimens were directly adsorbed to the grids from stock solutions. Adsorption was followed by several washing steps using quartz bidistilled water. When required, freeze-drying was performed at 280°C in the EM912 microscope column at a pressure of 2 · 1027 hPa for 3 hr, using an Oxford cryostage (Oxford Instruments, Oxon, UK). In the STEM, freeze-drying was performed in a specimen pretreatment chamber directly attached to the microscope column (Engel et al., 1982). Instrumental Parameters To facilitate direct comparison, all mass measurements performed by both EFTEM and STEM were carried out at an acceleration voltage of 80 kV. The energy-loss interval in the EFTEM used for recording the inelastic dark-field images was set to 8–48 eV. This range, including the low-energy-loss peak with its maximum at approximately 20 eV, was found to be a suitable compromise considering the instrumental limitations of the mechanical slit of the EM912, possible drift of the spectrometer, and the maximum signal efficiency. The lower energy-loss edge (8 eV) is a minimum in the electron energy loss spectrum (EELS) of a typical proteinaceous specimen between the zero-loss peak and the plasmon-loss peak. The maximum slit width and an EELS intensity comparable to that at the lower edge were decisive for the selection of the upper edge (48 eV). Thereby, a small energy shift ,0.5 eV would not influence the image intensity measurably. A 180-µm objective lens aperture restricted the collection halfangle a to 25.4 mrad. The images were typically recorded at a microscope magnification between 32 000 and 50 000 3, corresponding to an image size (i.e., as defined by the CCD chip) in the specimen plane between 537 and 338 nm. Suitable electron doses were selected between 200 and 500 e2/nm2. Doses lower than 200 e2/nm2 lead to an increase of the statistical noise of the images, and doses higher than 500 e2/nm2 lead to significant beam damage and mass loss of the specimen. The CCD camera was operated in the 2 3 2 pixel grouping mode, thus producing 512 3 512 pixel images. The conversion rate of the scintillator–CCD system, which is defined as the ratio of the gray value and the number of incident electrons per pixel, is an important parameter when attempting to count single electrons. It can be calculated from the mean gray value of a homogeneously

EFTEM MASS DETERMINATION illuminated empty image area, the incident electron dose, and the image size in the specimen plane. Image Acquisition Because of beam-induced mass loss, both minimal specimen preirradiation and low recording doses are indispensable. To achieve this, three operation modes are defined by the software: search, focusing, and acquisition. Magnification, brightness, illumination aperture, and camera type (TV or CCD) can be set for each of the three modes. Keyboard shortcuts allow fast switching between the modes. This setup assures minimal irradiation of the specimen. An EFTEM mass measurement session typically starts with the alignment of the microscope after a 60-min warming-up phase once the filament and the high tension have been switched on. Next the dose for the requested illumination brightness and exposure time is measured. The slit aperture is then adjusted to the desired energy-loss range, and the objective diaphragm is centered onto the optical axis. The specimen grid is moved into the focus plane, which is fixed by a calibration procedure of the objective lens current so that defined values for the magnifications and collection half-angle are obtained. In the search mode, the TV camera is used for screening the specimen at dose rates ,10 e2/(nm2 · sec) and magnifications of 8000 to 10 0003. For focusing, the gold particles deposited on the thick carbon film lying near the desired specimen site are used by activating the microdose focusing function (MDF) of the microscope at the magnification desired for image acquisition. This function performs a shift of the illumination and the image, so that the sample area of interest is not irradiated during focusing. Afterward, the CCD camera is exposed with the predefined parameters (typically: magnification, 40 0003; corresponding image size, 423 nm; dose, 200–500 e2/nm2 ). Usually, a series of 10 to 50 images from different specimen areas is recorded for a mass determination, depending on the sample to be measured, its distribution on the grid, and the required statistical precision. Image Analysis Each recorded image has a corresponding header file containing all of the parameters required for the quantitative off-line data processing. The images together with their header files are stored on magneto-optical discs. The data processing is performed in three steps. First, regions of interest (ROIs) are defined interactively by the user on each image, and a file containing all ROI coordinates for a given image set is created. Next, the processing routine calculating the mass for every ROI is activated. Finally, the data set can be analyzed by creating mass distribution histograms, by Gaussian fits, and by calculating means and standard deviations or other statistical parameters (Mu¨ller et al., 1992). The measured mass values can be corrected for beam-induced mass loss. By determining the mass of the sample at different electron doses, a sample-specific relationship between mass loss and electron dose may be determined. From this relationship, the mass at zero dose can be extrapolated.

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warming-up time of 60 to 90 min, the emission current of the gun remains stable, and the measured dose does not drop by more than 2.5% per hour (Fig. 2). The linearity of the high-performance slow-scan CCD system was tested by plotting the mean gray values of homogeneously illuminated images (i.e., without a specimen in place) versus the number of incident electrons for the low-dose range (i.e., up to 40 e2/pixel; Fig. 3). The slope of this plot yields the conversion rate of the CCD camera which is 7.60 6 0.04 digits/e2 for 80 kV when using 2 3 2 pixel grouping. The measured error of 0.5% for the conversion rate also contains any inaccuracies in the dose measurements performed with the electron collector. Additional experiments were performed to estimate the effect of two factors which could also influence the precision of mass determination in the EFTEM. The first factor is due to the circumstance that in a real imaging spectrometer the energy selection is not isochromatic: the energy range selected by the energy selection slit is not the same for each object element, it depends on the off-axial position of the object element (Rose and Krahl, 1995). In the EM912 the energy difference over the area imaged on the CCD chip is #1.5 eV. As the width of the energy-loss window is 40 eV and the intensity of the energy-loss spectrum of a typical thin biological specimen is minimal at the edges of this window (i.e., at 8 and 48 eV), an energy shift of 1.5 eV should produce a minimal contribution to the width of the mass distribution histogram. To demonstrate this experimentally, 12 images from a thin carbon film specimen were recorded under the experimental conditions employed for mass determination (i.e., 80 kV; DE: 8–48 eV; a 5 25.4 mrad; magnification, 40 0003; 512 3 512 pixel images; see Fig. 4a) at different grid locations and averaged to compen-

RESULTS

Calibration and Limits of Accuracy It is a prerequisite to work with stable and reproducible microscope and camera parameters during a mass determination session. The magnification was calibrated by using catalase crystals as a standard (Wrigley, 1968). Its deviation from the calibrated value is ,0.5% after a magnification change. After a

FIG. 2. Relative beam intensity (scaled to the maximum) versus time starting with the turn-on of the filament and the high tension. After a warming-up time of 60 min the intensity drop-off is smaller than 2.5% per hour.

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FIG. 3. Linearity of the Proscan high-speed slow-scan CCD camera in 2 3 2 pixel grouping mode. The mean gray values together with their standard deviation error bars of homogeneously illuminated images are plotted versus the number of incident electrons (per image pixel).

sate for possible thickness gradients. The center of symmetry of the isochromacy pattern usually matches the optical axis and the center of the CCD camera; this depends on the height alignment of the slit aperture. Although the alignment is generally stable, the effect of misalignment was considered by positioning the center of symmetry of the isochromacy pattern at the upper left corner of the CCD camera. As illustrated in Fig. 4a, 50-pixel-wide averaged line scans in the x and y direction were computed and least-squares-fitted by a linear curve (Fig. 4b). The deviation from the mean gray value along these averaged line scans was found to be 60.5% for the vertical and 60.25% for the horizontal direction.

In this context, it should be mentioned that the adjustment procedure for the width and lateral position of the slit aperture is very accurate. The error of reproducibility is ,0.5 eV. A second factor which may affect the precision of the mass measurement is the defocus at which the images have been recorded. To assess this, a focus series (i.e., from 21.5 to 11.5 µm defocus in steps of 250 nm at a magnification of 40 0003) of a thin carbon film was recorded. The mean gray values of four ROIs (80 3 80 pixels each) per image were plotted versus the defocus (Fig. 5). The intensity varied less than 0.8% over the whole 3-µm defocus range. For precise absolute mass determination, the calibration factor C (see Eq. [4]) was estimated from the known mass of a standard specimen, which is more convenient than using the calculated partial inelastic scattering cross sections. For this purpose, preparations of the rod-shaped tobacco mosaic virus (TMV) are a good choice, as this is a very stable specimen with a theoretical mass per length (MPL) of 131 kDa/nm (Namba and Stubbs, 1986). A series of TMV grids were examined in STEM and the results compared with the intensity distribution measured in EFTEM from grids prepared using the same stock. The experiments were always performed in parallel. This yielded a mean calibration factor C of 135 kDa/nm2 (80 kV; 25.4 mrad; 8–48 eV). Raw data images and mass distribution histograms of one EFTEM/STEM pair of unstained air-dried TMV samples are displayed in Fig. 6. The MPL measured by EFTEM was 125.1 6 5.1 kDa/nm (Nsegments 5 77; dose, 385 e2/nm2; Figs. 6a and 6c), whereas STEM

FIG. 4. Non-isochromatic filtering. (a) Average from 12 images (80 kV; DE: 8–48 eV; a 5 25.4 mrad, magnification, 40 0003; 512 3 512 pixels) of thin carbon film with horizontal and vertical 50 3 512 pixel ROIs being marked; (b) the relative deviation from the mean gray value is plotted versus the pixel coordinate.

EFTEM MASS DETERMINATION

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the radial intensity profile about the center of the beads was calculated from their inelastic dark-field images (Fig. 8a), and the distance from the center of each bead was converted into corresponding thickness values assuming a spherical bead shape. After normalizing the intensity values to the incident electron dose, the fraction of inelastically scattered electrons was plotted versus bead thickness (Fig. 8b). Employing a Monte Carlo simulation, a curve was fitted to the data points by a least-squares procedure. Within a thickness range of 0 to 35 nm, the fitted curve deviates by less than 10% from linearity. Applications FIG. 5. Influence of focus on scattering. Mean gray values from four 120 3 120 ROIs versus defocus (images recorded at 80 kV; DE: 8–48 eV; a 5 25.4 mrad; magnification, 40 0003; 512 3 512 pixels).

gave a MPL of to 127.6 6 5.5 kDa/nm (Nsegments 5 59; dose, 295 e2/nm2; Figs. 6b and 6d), where Nsegments represents the number of segments for each histogram. The latter value became 130.6 6 5.6 kDa/nm after correction for beam induced mass loss (Mu¨ller et al., 1992; see below). The mass-loss behavior of TMV in EFTEM was also examined. For this purpose, image series from the same TMV particles were recorded at doses of 180–200 e2/nm2 per image, and the resulting MPL values were plotted versus the cumulative dose. As in STEM experiments, for TMV kept at ambient temperature (25°C) the relationship was linear up to a cumulative dose of 2000 e2/nm2, allowing extrapolation to yield the zero-dose mass. It is convenient to express the data in terms of the relative residual mass m/m0 (in %) which depends on the total dose d (in e2/nm2 ) as m/m0 5 100 2 c · d (Fig. 7). The fit parameter c was found to be 1.1 3 1022 (Mu¨ller et al., 1992: 6.5 3 1023 for STEM). Correction of the mass histogram accordingly yields a statistically meaningful zero-dose mass in agreement with the STEM result. Application of the same correction factor to other specimens allows an approximate mass-loss correction. As mentioned above, calculating the mass using a linear calibration function assumes single inelastic scattering. However, with increasing specimen thickness the effect of multiple scattering, in particular the inelastic–elastic scattering signal, cannot be neglected. To experimentally estimate the upper limit of specimen thickness, for which a linear calibration can be employed without introducing an unacceptable error, the scattering profiles of polystyrene beads having different average diameters (i.e., 91, 234, and 312 nm) were evaluated. Specifically,

To evaluate the practicability and faithfulness of mass determination by EFTEM, three typical samples, a particle, a filament, and a planar sheet, were examined. In each case the results were compared with corresponding STEM data and, as far as available, the effective mass was calculated from the masses of the constituent proteins. The first specimen was glutamine synthetase, a key enzyme in nitrogen metabolism consisting of 12 identical 51 772-Da subunits arranged in two hexameric rings which are associated back to back (Almassy et al., 1986). The mass of freeze-dried enzyme particles isolated from Escherichia coli (Fig. 9a: raw data image) was measured as 604 6 103 kDa (N 5 173) in the EFTEM (Fig. 9b). For comparison, in a parallel STEM experiment the mass of the enzyme particles was found to be 625 6 90 kDa (N 5 193). In both cases the total electron dose given to the sample was about 300 e2/nm2, and the mass values presented were not corrected for beaminduced mass loss. The calculated mass of glutamine synthetase is 621 kDa. It should be noted that the mass per area (MPA) of a glutamine synthetase particle is about 3.5 kDa/nm2, i.e., in the range of the MPA of the thin carbon support film, therefore yielding a relatively poor signal-to-noise ratio and hence a relatively large statistical error. The second specimen was synthetic actin filaments assembled from purified rabbit muscle actin (e.g., Bremer et al., 1991, 1994). Freeze-dried, phalloidin-stabilized actin filaments (Fig. 10a: raw data image) yielded a MPL of 15.1 6 1.8 kDa/nm (N 5 105; electron dose, 322 e2/nm2 ) in the EFTEM (Fig. 10b) compared to the MPL of 15.3 6 1.4 kDa/nm (N 5 653; electron dose, 325 e2/nm2 ) determined by STEM. As with glutamine synthetase, these MPL values were not corrected for beam-induced mass loss. Applying the dose-induced TMV mass-loss relationships to the MPL data yielded MPLs extrapolated to zero electron dose of 15.7 kDa/nm for the EFTEM data and 15.6 kDa/nm for the STEM data. For comparison,

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FIG. 6. Raw data images of air-dried TMV recorded on a LEO EM912 EFTEM (a) and a Vacuum Generators HB-5 STEM (b). MPL histograms: (c) EFTEM (MPL, 125.1 6 5.1 kDa/nm; N 5 77; electron dose, 385 e2/nm2 ); (d) STEM (MPL, 127.6 6 5.5 kDa/nm; N 5 55; electron dose, 295 e2/nm2 ). The mass data have not been corrected for beam-induced mass loss.

FIG. 7. Mass-loss behavior for TMV. The relative residual mass (in %) is plotted versus the electron dose [e2/nm2]. The data points ,2000 e2/nm2 were fitted by a linear regression. The relative residual mass (in %) is 100 2 (1.1 3 1022 · d), where d represents the electron dose in e2/nm2.

the calculated MPL for phalloidin-stabilized actin filaments is 15.7 kDa/nm. The third specimen, the hexagonally packed intermediate (HPI) layer is a 2-D crystalline protein layer from the cell envelope of Deinococcus radiodurans (Engel et al., 1982; Rachel et al., 1983). The MPA of air-dried, glutaraldehyde-fixed HPI layer (Fig. 11a: raw data image) was measured as 2.50 6 0.11 kDa/nm2 (N 5 79) in the EFTEM (Fig. 11b) and 2.44 6 0.15 kDa/nm2 (N 5 171) in the STEM (Mu¨ller et al., 1992). From the primary sequence of the HPI layer protein the MPA of the unfixed specimen is calculated as 2.29 kDa/nm2. Considering the 6% mass increase caused by glutaraldehyde fixation (i.e., determined experimentally by Mu¨ller et al., 1992) yields a calculated MPA of 2.43 kDa/nm2 for the glutaraldehydefixed HPI layer, a value in excellent agreement with both the EFTEM and STEM mass measurements.

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FIG. 8. (a) EFTEM image of latex beads with the radial intensity profile superimposed onto one of the beads (MC). (b) Fraction of inelastically scattered electrons (a 5 25.4 mrad; DE: 8–48 eV) plotted versus thickness. The data are from latex beads of three different mean diameters, i.e., 91 nm (S), 234 nm (N), and 312 nm (X), and they were fitted by a curve calculated with a Monte Carlo (MC) simulation. Between 0 and 35 nm thickness the deviation of the linear plot from the MC fit is ,10%.

DISCUSSION AND CONCLUSIONS

The results presented in this communication demonstrate that an EFTEM equipped with a highperformance slow-scan CCD camera is a powerful instrument to determine the mass of biomacromolecules and their supramolecular assemblies. The selection of individual particles after visual inspection of their characteristic structural features (i.e., their overall size and shape) together with the need of only nano- to micromolar amounts of material to determine their masses or related parameters, i.e. MPL or MPA, are two significant advantages of

electron scattering over the more conventional methods of mass determination (i.e., analytical ultracentrifugation or laser light scattering) involving bulk measurements and requiring micro- to millimolar amounts of material. State-of-the-art slow-scan CCD cameras are linear and highly sensitive detector devices achieving singleelectron counting, a prerequisite for EFTEM to be employed for mass determination via quantitating the inelastically scattered low-energy-loss electrons. In this context it is worth noting that the LEO EM912 is a commercial EFTEM which does not

FIG. 9. EFTEM raw data image (a) and mass histogram (b) of freeze-dried glutamine synthetase from E. coli. Mass, 604 6 103 kDa; N 5 173; electron dose, 282 e2/nm2.

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FIG. 10. EFTEM raw data image (a) and MPL histogram (b) of phalloidin-stabilized and freeze-dried synthetic actin filaments. MPL, 15.1 6 1.8 kDa/nm; N 5 105; electron dose, 323 e2/nm2.

require any instrumental modification in order to be used for mass determination. Therefore, operation of the fully digitally controlled EM912 EFTEM is significantly more user-friendly than that of a dedicated STEM such as the Vacuum Generators HB-5. Nevertheless, to convincingly document the faithfulness and reliability of mass determination by a commercial EFTEM, it was important to rigorously compare the results, both the average mass values and their standard deviations, with those obtained by a dedicated STEM. The experiments have shown that the instrumental errors related to the imaging spectrometer or to the detection of the low-energy-loss (e.g., DE: 8–48 eV) electrons do not affect the precision of mass

measurements significantly. Moreover, the nonlinearity of the camera system and the effect of nonisochromatic filtering on the accuracy of mass determination amount to no more than 0.5%. Defocusing, which deviates usually by no more than 80 nm from the Gaussian focus, seems to have no systematic influence. Nevertheless, a limitation of using the inelastically scattered electrons for mass determination concerns the nonnegligible effects of multiple scattering for specimens thicker than about 35 nm at 80 kV. This effect is mainly caused by filtering out the multiple inelastically scattered electrons with energy losses .48 eV and by eliminating the elastically– inelastically scattered electrons by the objective

FIG. 11. EFTEM raw data image (a) and MPA histogram (b) of glutaraldehyde-fixed, air-dried HPI layer. MPA, 2.50 6 0.11 kDa/nm2; N 5 79; electron dose, 322 e2/nm2.

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aperture. This limiting thickness might be extended by a factor of 2 to 3 by using a nonlinear calibration function. One possible approach to generating a nonlinear calibration function is by a Monte Carlo simulation. Unfortunately, in the course of exploiting this approach it became evident to us that the known models for calculating differential inelastic scattering cross sections (Wall et al., 1974) do not correspond closely to the experimental data. A more practical approach might be to experimentally determine an actual calibration curve with a couple of standard samples of known density and mass thickness, for example, carbon films of different thickness evaporated onto mica and floated onto EM grids or multiple lipid layers. Anyway, the practical limit of sample thickness is reached with the contrast inversion which occurs at about 120 nm for 80-keV electrons, the given energy-loss range and acceptance angle. Yet another, and probably the easiest, way of extending the thickness limit is to increase the acceleration voltage of the primary electrons. Whereas with increasing primary energy of the beam electrons their mean free path length increases, their scattering cross sections decrease. So in future we will perform our mass measurements at an acceleration voltage of 120 kV, which will extend the practical specimen thickness before contrast inversion occurs by approximately 30%. On going from 80 to 120 kV the actual decrease of scattering cross sections is acceptable and will basically be compensated by a higher conversion rate of the phosphorus scintillator at 120 kV. Rigorous comparison of the mass measurements for glutamine synthetase, actin filaments, and the HPI layer performed by EFTEM with those obtained by STEM has clearly documented that there are no significant differences neither in the absolute mass values (i.e., as judged by the means calculated from the mass histograms) nor in their relative accuracy (i.e., as judged by the respective standard deviations). As the electron collector in the EM912 which is used for the dose measurements has a limited accuracy, the precision of absolute mass determination could be improved by normalizing the inelastic darkfield images with homogeneously illuminated images obtained by recording the direct beam intensity without a specimen in the beam, under specimen imaging conditions. By multiplying these normalized images with the calibration factor C, i.e., as defined by Eq. (4), absolute mass maps can be obtained. The knowledge of the conversion rate would become unnecessary, and the dose measurement would only be important for mass-loss correction. For this approach to be used more routinely

some major changes in the IMPSYS software would become necessary. To this end, one future goal is to integrate the entire image and data analysis, which is presently performed by using the IMPSYS software, into the CCD control package. In further research, the effect of cooling the sample during data collection on the precision of the mass measurements will also be investigated. Under these conditions beam-induced mass loss should become negligible. Mass determination by electron scattering was first introduced on the STEM by evaluating the elastically scattered electrons. Over the years it has become an extremely valuable analytical tool. In the present investigation, we have implemented this methodology on a commercial EFTEM equipped with a high-performance slow-scan CCD camera system by evaluating the inelastically scattered lowenergy-loss electrons. This development now opens the possibility of making mass determinations by electron scattering available to a wider circle of users in cell and molecular biology. We are very grateful to LEO Electron Microscopy, Oberkochen, Germany, for putting the EM912 to our disposal and to Dr. W. Probst for his continued support concerning technical questions and problems. We also thank Mrs. B. Fedtke for acquiring the STEM images and Prof. Dr. A. Engel for many useful suggestions and constructive criticism. This work was supported by the Canton Basel-Stadt, a research grant by the Swiss National Science Foundation, and by the M. E. Mu¨ller Foundation of Switzerland. REFERENCES Aebi, U., and Pollard, T. D. (1987) A glow discharge unit to render electron microscope grids and other surfaces hydrophilic, J. Electron Microsc. Tech. 7, 29–33. Almassy, R. J., Janson, C. A., Hamlin, R., Xuong, N.-H., and Eisenberg, D. (1986) Novel subunit-subunit interactions in the structure of glutamine synthetase, Nature 323, 304–309. Benner, G., and Probst, W. (1994) Ko¨hler illumination in the TEM: Fundamentals and advantages, J. Microsc. 174, 133–142. Bremer, A., Millonig, R. C., Su¨tterlin, R., Engel, A., Pollard, T. D., and Aebi, U. (1991) The structural basis for the intrinsic disorder of the actin filament: The ‘‘lateral slipping’’ model, J. Cell Biol. 115, 689–703. Bremer, A., Henn, C., Goldie, K. N., Engel, A., Smith, P. R., and Aebi, U. (1994) Towards atomic interpretation of f-actin filament three-dimensional reconstructions, J. Mol. Biol. 742, 683–700. Engel, A. (1978) Molecular weight determination by scanning transmission electron microscopy, Ultramicroscopy 3, 273–281. Engel, A. (1982) Mass determination by electron scattering, Micron 13(4), 425–436. Engel, A., Baumeister, W., and Saxton, W. O. (1982) Mass Mapping of a Protein Complex with the Scanning Transmission Electron Microscope, Proc. Natl. Acad. Sci. USA 79, 4050–4054. Engel, A., and Colliex, C. (1993) Application of scanning transmission electron microscopy to the study of biological structure, Curr. Opin. Biotechnol. 4, 403–411.

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