Concentration evaluation of chromatin in unstained resin-embedded sections by means of low-dose ratio-contrast imaging in STEM

Ultramicroscopy 49 (1993) 235-251 North-Holland

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Concentration evaluation of chromatin in unstained resin-embedded sections by means of low-dose ratio-contrast imaging in STEM B. Bohrmann a, M. Haider b and E. Kellenberger

a

Department of Microbiology, Biozentrum, Universityof Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland b European Molecular Biology Laboratory, Postfach 10.2209, W-6900Heidelberg, Germany a

Received 28 July 1992; at Editorial Office 11 August 1992 Dedicated to Professor Elmar Zeitler on the occasion of his 65th birthday

Quantitative STEM with the imaging mode of ratio-contrast was investigated in order to evaluate the local concentration of DNA in situ for different kinds of DNA plasms in terms of intracellular packing densities (p.d.). The ability of ratio imaging to suppress thickness variations provided the basis to use unstained sections from cryofixed and freeze-substituted material. The DNA p.d. within the nucleoid of E. coli was determined to be about 100 mg ml -I. Quantitative data concerning the p.d. of DNA in condensed eukaryotic chromatin assuming equal amounts of DNA and protein were evaluated for the first time: approximately 400 mg ml-1 chromatin which corresponds to 200 mg ml-x DNA. The p.d. of DNA in chromosomes from the dinoflagellate Amphidinium carterae, a eukaryote devoid of histones and with only small relative amounts of histone-like protein, was also found to be of the order of 200 mg ml-1. The highest p.d. of DNA was measured for the head of the bacteriophage T4 with more than 800 mg ml- 1, in fair agreement with previous calculations. The results provide further support for a condensation mode of low protein chromatins that involves a liquid-crystalline organization of the DNA filaments.

1. Introduction The main advantage of the scanning transmission electron microscope (STEM) is its multi-signal recording capability, which provides the possibility to record the various types of scattered electrons simultaneously without exposure of the specimen each time for every selected imaging mode. After passing, an appropriate electron spectrometer, the electrons can be recorded with respect to their energy losses, and/or regarding their scattering angles by splitting the annular detector into several rings. Z-contrast, as has been suggested for STEM by Crewe [1], is obtained by dividing the simultaneously recorded signal of the annular dark-field detector (Set, the elastic dark-field signal) by the inelastic spectrometer signal (Sin, the inelastic

dark-field signal). For very thin specimens the Z-contrast is independent of the specimen thickness; it should only depend on the scattering cross-sections and thus on the atomic numbers Z. However, in practice this type of imaging is still not completely independent of the thickness of the specimen, and therefore has also been termed ratio-contrast [2]. Concentrations of pure lipids and proteins, if not present as undefined mixtures, should be measureable according to theory

[2-41. The advantage of ratio-contrast as an analytical imaging mode is its ability to suppress influences arising from thickness variations. A particular feature of thin sections is their variation in thickness due to the sectioning process [5]. Therefore, ratio imaging allows the determination of the local concentration or packing density (p.d.,

0304-3991/93/$06.00 © 1993 - Elsevier Science Publishers B.V. All rights reserved

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B. Bohrmann et a L / Concentration evaluation of chromatin by ratio-contrast imaging

according to Woldringh and Nanninga [6]) of macromolecules like nucleic acids within unstained thin sections of resin-embedded biological specimens [7]. An approach using thin sections is an important prerequisite to approach chromatin structure in its native context. The application of cryofixation and freeze-substitution techniques is highly suitable, because especially preparation-induced chromatin aggregation is reduced to minimum [8]. From data describing the volume and mass of particular well characterized D N A containing plasms the p.d. of D N A was calculated with a precision depending largely on the precision of the volume determination. The bacterial nucleoid in exponentially grown E. coli and the replicating and transcribing pool of bacteriophage T4 have p.d. of DNA estimated to be 20-50 mg ml-~ [9]. A similar D N A p.d. was calculated for the nuclei of hepatocytes, although averaged as total D N A concentration assuming an equal distribution within the cell nucleus [9]. In contrast, the highly condensed D N A of bacteriophages was calculated to be 800 mg ml-1 in the case of mature heads of T4 [9]. In T4, DNA is packed most likely in the form of a liquid crystal [10]. While typical eukaryotes exhibit a cyclic decondensation-condensation cycle of their chromosomes, which disappear at interphase, the dinoflagellates always show individualized chromosomes. Therefore, they were claimed to have "permanently condensed chromosomes" and their D N A is proposed to be organized in a liquid-crystalline-like state [11,12]. Since it is generally assumed that any metabolic activity is possible only with decondensed chromatin, this would therefore be different in dinoflagellates. This apparent paradox was challenged by quantitative electron microscopy using ratio-contrast. Ratiocontrast measurements of the DNA p.d. were shown to be consistent with data obtained by quantitative fluorescence light microscopy and morphometrical measurements of A. carterae chromosomes [13]. Intracellularly located proteinous inclusion bodies were used to investigate the contrast of proteins and thus their influence on DNA p.d. evaluations. The limitation of this procedure is

mainly due to the finding that the ratio contrast of proteins is not matched by Epon as initially thought [2]. The non-negligible contrast of protein makes corrections difficult for all chromatins whose protein content is not known. In consequence, only the order of magnitude of the D N A p.d. could be determined. The aim of this study was to measure the local concentration of DNA in various chromatins in situ, using unstained thin sections of resin-embedded cells. Basic information is provided about different degrees of chromatin condensation in selected organisms ranging from prokaryotes to eukaryotes and virions. It could be confirmed that the most highly packed chromatins are not metabolically active.

2. Theory Electrons that are scattered within an object can be used to image this object either with a fixed electron beam (conventional transmission electron microscope, TEM) or with a scanned electron probe (scanning transmission electron microscope, STEM). The scattering processes can be divided into two different types: the elastic and the inelastic scattering processes. Elastically scattered electrons, scattered at the nucleus of an atom, do not lose energy and are scattered mainly in forward directions but into a wider angle than the inelastically scattered electrons. The elastically scattered electrons can be utilized in a STEM with an annular detector to form a dark-field image. STEM dark-field imaging is very efficient when compared with the dark-field imaging mode in TEM. With the annular dark-field detector almost exclusively elastically scattered electrons are recorded and due to the fact that these electrons are well localized, this signal is used as a high-resolution imaging mode in materials science and in biology. In the field of biological research it is mainly used for the localization of small specific heavy-metal labels and for high-resolution mass determination and mass mapping [141. Inelastic electrons, in contrast, are scattered by the electrons of an atom or a molecule and

B. Bohrmann et al. / Concentration evaluation of chromatin by ratio-contrast imaging

lose part of their initial energy. Those electron energy losses where the electrons are scattered at the inner atomic shells contain information about the particular element that is involved in the scattering event. However, most of the inelastically scattered electrons are scattered by the outer shell, i.e. the binding electrons, which are causing an energy loss in the low range (5-80 eV). Almost all (99%) of the inelastically scattered electrons have lost energy in this range. The signal of these electrons is, on one hand, not elementspecific and, on the other hand, the interaction process is not well localized due to the collective excitation of the binding electrons, and thus cause a reduction of spatial resolution. Within biological specimens, that are composed mainly of lowatomic-number elements, this latter type of inelastic scattering occurs more often compared with the amount of electrons which are scattered elastically. The elastic dark-field signal (S~l) as well as the inelastic signal (Sin) are sensitive to mass-density and thickness variations of the specimen. The signal, generated by these scattered electrons, is linearly dependent on the mass density within a certain range of specimen thickness (t) (as long as t < A, A is the mean free path, which is at a primary energy of E = 100 keV, h --- 50 nm). One of the first applications of the multi-signal recording capability of the STEM was the Z-contrast imaging mode in STEM, introduced for imaging single atoms [1]. This imaging mode should be obtained by dividing the elastic signal of the annular dark-field detector by the inelastic spectrometer signal (low energy loss or plasmon loss electrons). The elastic and the inelastic scattering cross-sections ~r~l, ~rin are given by [15]: ¢~, = 1.5 × 10-4Z3/2[1 - 0 . 2 3 Z / ( 1 3 7 ~ ) ] / / 3

2, (la)

gin = 1.5 × lO-4Z 1/2 I n ( 2 / O e ) / / 3 2,

(lb)

with Z the atomic number, /3 the ratio of the electron velocity and the speed of light and 0E = A E / 2 E , the characteristic scattering angle of the

237

inelastically scattered electrons. A E and E are the mean energy loss and the primary energy of the incident beam. The ratio of the cross-sections is obtained by R = O'el/O'in

= Z[1 - 0 . 2 3 Z / ( 1 3 7 / 3 ) ] / I n ( 2 / 0 e ) =

Z/IO,

(2)

which shows, as a good approximation, a linear dependence of the ratio-signal on the atomic number Z. However, the elastic, inelastic, mixed and unscattered signals (Iel, /in, Im and Iun) obtained in a STEM are dependent on the incoming electron beam I o and the scattering cross-sections which are given by I~j =I0(1 - e -'~°'°t) e -'~iaot,

(3a)

e-'~e'ot,

(3b)

/in = I 0 ( ] -- e-~i"Pt)

I m = Io(1 - e-~,°~,)(1 _ e-¢~,pt),

(3c)

Iun = I0 e-°'inpt e-~elPt,

(3d)

with p the atomic density and t the thickness of the object. From eqs. (3a)-(3c), the type of Zcontrast, where the annular dark-field detector signal,

I =lo +Im, is divided by the inelastic signal, is still dependent on the thickness of the specimen. Therefore, this kind of imaging has also been called ratio-contrast [2]. Contrast formation of such a processed signal essentially depends on the atomic composition of the material due to the respective average scattering cross-sections (o-el and ~rin) of the irradiated atoms or molecules and thus, as outlined above, indirectly on their atomic numbers Z. Contrast in ratio images results from local differences in the relative amounts of elastically and inelastically scattered electrons. The higher the atomic number the higher is the relative amount of elastically scattered electrons. Ratio-contrast therefore reflects the atomic composition of the

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238

sample. Considering a sample as a mixture of two different species of molecules, e.g. biological macromolecules and resin, allows for the evaluation of their relative concentrations, presupposing that the atomic composition of these species is sufficiently different. This holds for D N A and resins like Epon and Lowicryl HM20, which are frequently used for the embedding of biological specimens. The phosphorus content of D N A clearly discriminates it from proteins and resins. Unfortunately, R N A cannot be distinguished from DNA by means of ratio-contrast because of a nearly identical atomic composition. Pure Z-contrast has been proposed by several authors [16-18] and it can be obtained by either the combination of more than two signals [16,18] or by means of an energy-filtered dark-field divided by the inelastic signal of a smaller energy window (10-40 eV) [17]. For these pure Z-contrast modes, a thickness-independent image can be obtained. The signal combination of the annular dark-field, the inelastic and the bright-field image as it was proposed by Egerton [16] seems to be the easiest way of obtaining a pure Z-contrast image. The formula for the calculation of the Z-contrast is given by

O'el

In(Io) - l n ( I o - lAD )

O'in

l n ( I o - lAD ) -- l n ( I o -- IAD - - / i n ) ' (4)

with I o =/in + lAD + Iun" However, the combination of just two signals of the ratio-contrast reduces to a certain extent the influence of thickness variations which occur in thin sections [5]. The thickness dependence of ratio-contrast is small as long as multiple scattering does not contribute significantly to the contrast formation. As investigated by Reichelt and Engel [4], Sel and Sin exhibit a linear response between electron scattering intensity and small thicknesses of resin sections. This is guaranteed in the used resin thin sections below a material-specific critical thickness of about 50 nm. The extent of the linear response over the thickness depends also on the detector geometry of the STEM and the applied mode of Z-contrast [17].

3. Materials and methods 3.1. Instrumentation

The experimental work presented here was performed on the Heidelberg cryo-STEM with a liquid-helium-cooled superconducting lens [19] at an accelerating voltage of 100 kV. About ten minutes after the specimen is brought into high vacuum it reaches a temperature of approximately 4 K. The cryo-STEM is equipped with a highly dispersive electron energy-loss spectrometer [20]. In combination with the high-excitation objective lens, the spectrometer has a maximum acceptance angle of @1 = 100 mrad, and the annular dark-field detector collects electrons scattered into an angular interval of 15-130 mrad. The collection angle of the inelastic signal was limited by the annular dark-field detector bore and was therefore @1 = 15 mrad. The energy-loss window was set to 10-50 eV. With this instrument pure Z-contrast can be achieved either with a filtered elastic dark-field signal divided by the inelastic signal or, as is used in the present study, simple ratio-contrast by dividing the conventional annular dark-field signal by an inelastic signal. However, the inelastic signals were recorded with an inelastic detector, whose response depends on the energy loss of the inelastically scattered electrons. In consequence, the signal of each recorded electron is increasing

I.-

0.5 --

0 100

I

I

I

I

I

80

60

40

20

0

)

AE [eV]

Fig. 1. Signal intensity response function of the inelastic detector versus AE. The response is dependent on the energy loss of the inelastically scattered electrons.

B. Bohrmann et aL / Concentration evaluation of chromatin by ratio-contrast imaging

with its energy loss in the low-energy-loss region (fig. 1). Therefore, electrons which have lost a higher amount of their initial energy or have been scattered more than once inelastically are emphasized. According to that response function of the inelastic detector, the thickness dependence of the inelastic signal is about the same as that of the elastic signal. In consequence, the divided signal gives ratio images which appear already by visual inspection independent of thickness variations (figs. 2 - 7 below).

3.2. Digital off-line image processing for ratio-contrast measurements based on STEM images The dark-field and the inelastic image have been recorded simultaneously with the cryoSTEM. A digital data-acquisition system collects the elastic and inelastic signals which are stored in a microcomputer [21], based on a 32-bit microprocessor with 8 Mbytes of memory and 165 Mbytes of local disk storage. Acquired images were transferred via Ethernet to the laboratory central computing facility (a VAX-cluster) for off-line processing. Digital images containing 512 X 512 or 1024 X 1024 picture elements (pixels) with a data depth of 8 bit per pixel were recorded at magnifications of 10 000 X, 20 000 x , 50 000 x and 1 0 0 0 0 0 x and doses of 3-900 e / n m 2 as indicated in the figure legends. For the evaluation of the D N A p.d. of the investigated chromatins only images obtained with recording doses below 100 e / n m 2 were used. The ratio-contrast images have been calculated numerically. The upper threshold of the calculated Z-contrast images were set to 2.0 and the remaining values (0-2.0) are equivalent to 256 gray values. In order to avoid division by zero, 1 has been added to the inelastic image. For image processing the AFS program system was used (written by K. Leonard, EMBL, Heidelberg). Maximum-sized circularly or rectangularly boxed measuring surfaces, entirely covering the particular chromatin of interest, were used. For every image a new background measurement was made. All values for the concentration determination

239

of chromatin were calculated as the normalized ratio R' [3] defined as R' = NSAD/Sin ,

where the normalization factor N = ( S i n / / S A D ) r e s is the ratio of inelastic (Sin) to elastic (SAD) signal of a section area corresponding to pure resin. The normalization factor N depends on t (thickness) and 01 (collection angle) but almost reciprocally on the signal Sin~SAD obtained from section areas with biological matter. Due to this normalization procedure only relative values of the object with respect to the resin are obtained. This is advantageous under normal experimental conditions with predominantly single-scattered electrons, because neither the thickness of a section nor the collection condition is necessarily constant [3]. The normalized ratio signal, e.g., of a particular chromatin is designated as R{~pon and R{jM20 to discriminate between the used resins. The experimentally obtainable values of SAD and Sin are given by the geometries of the annular dark-field detector (AD) and the spectrometer detectors. The contrast setting or offset (e.g. by adjusting amplifier gains) should now be chosen for each microscope session in the same way, such that (i) the object contrast is optical and (ii) the signal normalization is achieved by matching the sensitivity of the detector channels. Hereby contrast intensities of SAD and Sin are adjusted with the help of a line scan displayed on an oscillograph, to rough equality. SAD~Sin is then ~ 1 for the average of a given picture. The local ratio signals S R of particular subcellular structures are then above or below 1. This procedure avoids in particular a contrast reversal of the ratio-contrast if Sin >~>SAD.

3.3. Cell cultures E. coli B cells were grown overnight in M9 minimal medium supplemented with 1% casamino acids at 37°C and permanent aeration and subcultured the following day into fresh liquid medium until cells were clearly visible ( ~ 1.5 h; 2 x 108 ml-1). They are then at the upper limit of exponential growth.

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B. Bohrmann et aL / Concentration evaluation of chromatin by ratio-contrast imaging

E. coli B at (1-2) X 108 cells m l - 1 were treated with chloramphenicol (CAM, stock of 1 mg ml-1 in 100% ethanol) for 60 min to give a final concentration of 3 0 / z g ml - t . E. coli B were infected with T4 at (2-4) x 10 s cells m l - 1, when still in exponential growth with a "multiplicity of infection" (m.o.i.) of 5 and superinfected 8 min later with the same m.o.i. Bacteriophage T4 strains were T4D and T4.31 (amN54). A. carterae was grown in f/2-Si, a silicate-deprived seawater medium for autotrophs. Cultures were maintained at semisynchronized growth conditions that ensure a doubling of the cells every 24 hours at an alternating light (16 h) and dark (8 h) regime (for details see ref. [13]). Euglena W H F L A G was grown in malt seawater medium containing 0.5% peptone.

3.4. Cryofixation and freeze-substitution E. coli cells were harvested from a liquid culture by filtration during 2 min with permanent aeration on a Nuclepore filter (pore size 0.4/zm). Eukaryotic cells were collected on 1.0/zm filters. The cells were p r e p a r e d for cryofixation according to ref. [22], and frozen by slamming them onto a liquid-helium-cooled, polished copper block at 6 K, according to ref. [23]. Samples were stored in liquid nitrogen before the substitution step.

The samples were substituted in acetone containing 3% ( v / v ) glutaraldehyde in the presence of a molecular sieve to ensure complete dehydration (0.4 nm; Perlform, Merck) at 185 K for 90 h. The temperature was then raised at a rate of 6.7°C/h to 228 K and kept there for 2 h. The samples were then embedded either in Lowicryl HM20 or in Epon; in HM20 at 228 K by successive infiltration in the following solutions: 100% acetone for 1 h; H M 2 0 - a c e t o n e (1:1) for 2 h; H M 2 0 - a c e t o n e (2:1) for 2h; HM20 for 2 h and overnight for 16 h. Samples were further infiltrated for 4 h with fresh Lowicryl and placed into closed gelatin capsules. The resin was polymerized by indirect U V irradiation (360 nm) for 24 h at 228 K, followed by further hardening at room t e m p e r a t u r e for 3 days in U V light. For samples embedded in Epon, the procedure was identical up to the temperature rise to 228 K. Freeze-substitution apparatus and further processing steps (temperature rise up to room temperature and Epon embedding) were the same as described by ref. [22]. 3.5. Sectioning and grid treatment

Thin sections were cut with a diamond knife on a LKB ultramicrotome III at a visually judged thickness in the range of 40-60 nm. Sections were transferred with platinum wire loops to 600-mesh hexagonal copper grids without sup-

Fig. 2. STEM images taken at an original magnification of 100000X; elastic dark-field (a), inelastic dark-field (b) and ratio image (c) of an unstained Epon section of E. coli B recorded at 100 keV with a dose of 900 e/nm 2. Bar indicates 200 nm.

B. Bohrmann et al. / Concentration evaluation of chromatin by ratio-contrast imaging

24l

I=1

Fig. 3. STEM images taken at an original magnification of 50000 x; elastic dark-field (a), inelastic dark-field (b) and ratio image (c) of an unstained HM20 section of E. coli B treated with chloramphenicol (30/zg m1-1) for 60 min and recorded at 100 keV with a dose of 270 e/nm ~. Circular measuring area within the nucleoid is indicated in (c). Bar indicates 500 nm.

p o r t i n g film. S e c t i o n s w e r e c o a t e d on b o t h sides with 3 n m carbon.

4. Results 4.1. S T E M imaging o f unstained thin sections

F o r i m a g e - r e c o r d i n g only u n s t a i n e d thin sections o f 4 0 - 5 0 n m thickness w e r e used. It m u s t b e e m p h a s i z e d t h a t d e l i b e r a t e l y no h e a v y - m e t a l atoms were applied during the sample preparation, including f r e e z e - s u b s t i t u t i o n a n d e m b e d ding. F o r o b s e r v a t i o n o f Lowicryl H M 2 0 sections d o u b l e - s i d e d c a r b o n c o a t i n g by e v a p o r a t i o n was

f o u n d i n d i s p e n s a b l e for i m a g i n g t h r o u g h an inc r e a s e o f t h e section stability against e l e c t r o n bombardment. S T E M p r o v i d e s t h e u n i q u e f e a t u r e for simult a n e o u s r e c o r d i n g o f SAD a n d Sin. I m a g i n g in d i f f e r e n t m o d e s is thus possible at t h e s a m e time. In figs. 2 - 7 we p r e s e n t for d i f f e r e n t s p e c i m e n s t h e d a r k - f i e l d i m a g e s o b t a i n e d f r o m SAD a n d Sin , t o g e t h e r with t h a t o b t a i n e d from t h e i r ratio

SAD/Sin. W h e n c o n s i d e r i n g c e l l u l a r d e t a i l s in g e n e r a l t h e elastic d a r k - f i e l d i m a g e p r o v i d e s highest contrast. In b o t h d a r k - f i e l d m o d e s the c o n t r a s t o f the w a t e r - f r e e m a t t e r is d e p e n d e n t on t h e m a s s - d e n sities ( p ) a n d the section thickness (t). A charac-

Fig. 4. STEM images taken at an original magnification of 100000 x; elastic dark-field (a), inelastic dark-field (b) and ratio image (c) of an unstained HM20 section of E. coli KM 4104::pDR 1453 cell with a rec A inclusion body recorded at 100 keV with a dose of 900 e/nm 2. The indicated sector of the inclusion body was exposed to higher electron dose (about 5000 e/nm2). Bar indicates 200 nm.

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B. Bohrmann et a L / Concentration evaluation of chromatin by ratio-contrast imaging

teristic feature of dark-field images obtained from thin sections concerns the observed thickness variations introduced by the sectioning process. Such variations had been estimated to be of the order of 3 nm [5]. Occasionally observed compressions are strongly emphasized in dark-field images. Since water is replaced by the resin, the initial high cellular water content is translated into a corresponding content of resin. For the dark-field imaging mode the p of the resin is of great importance for the resulting contrast. This is reflected by the ribosomes of bacterial cells (figs. 2a and 4a) dependent on the used embedding resin. Epon has a higher density than the hydrogen-rich Lowicryl HM20 and, accordingly, the dark-field contrast of ribosomes was generally high with HM20 (fig. 4a). The clear visibility of ribosomes in Epon (fig. 2a) was only obtained at higher electron doses. At lower magnification of HM20embedded E. coli (fig. 3), the ribosome-containing cytoplasm gave high positive ratio-contrast due to the presence of numerous, but non-resolved ribosomes. The observation of ribosomes at low magnification in Epon embeddings revealed a significant difference. The contrast of ribosomes in the cytoplasm of Epon-embedded E. coli of fig. 5 is far below that observed in HM20 (fig. 2). Lower magnification with STEM is accompanied by a proportional lower resolution, but at the same time, allows for correspondingly lower electron doses. Thus, an increase of quan-

tum noise, which lowers the signal-to-noise ratio, cannot be avoided. The contrast of the cytoplasm is now the result of the mixture of the compact ribosomes, other cytosolic components and approximately 80 vol% of resin. The contrast of ribosomes is particularly high, because their macromolecules are very densely packed and contain practically no water (i.e. no resin) inside. The high density due to the high content of R N A becomes thus fully determinant. The high contrast of ribosomes both in HM20 and in Epon is of some interest for the evaluation of the order of magnitude of their macromolecular p.d. The contrast is due only to RNA and proteins in the respective mass fractions. Unfortunately, for quantitative evaluations ribosomes would need too high doses, because of their small size, thus causing changes of the macromolecular composition provoked by beam-induced mass losses that are known to occur at higher electron doses. A loss of sharpness is observed in inelastic dark-field images concerning subcellular details, e.g. the ribosomal particles are no longer resolved (figs. 2b and 4b). This effect is related to the spatial weakly defined inelastic scattering at the outer electrons of irradiated atoms and molecules. The images of unstained thin sections obtained by ratio-contrast with the cryo-STEM appear by visual criteria to be perfectly thickness independent (figs. 2-7). Compressions of the thin sections, which were visible in elastic and inelastic dark-

Fig. 5. STEM images taken at an original magnificationof 100000×; elastic dark-field (a), inelastic dark-field (b) and ratio image (c) of an unstained Epon section of E. coli with mature T4D bacteriophages60 min postinfectionrecorded at 100 keV with a dose of 3 e/nm 2. Bar indicates 200 nm.

B. Bohrmann et al. / Concentration evaluation of chromatin by ratio-contrast imaging

field images, disappear completely in the corresponding ratio images. The theoretical assumption that proteins and Epon might be nearly similar in contrast due to a comparable atomic composition that provides similar average cross-sections [2] and therefore would not be discernible from each other was experimentally investigated. Pure unstained proteins were observed in the case of intracellularly located inclusion bodies of rec A protein in overproducing E. coli cells, or of intracellular accumulations of geneproduct (gp) 23 from a bacteriophage T4.31 mutant infecting E. coli. The main capsid protein subunits, instead of forming shells, are accumulated in insoluble lumps. For both types of inclusion bodies we found negative ratio signals when embedded in Epon (data not shown). Fig. 4 shows a cell with an inclusion body of rec A protein embedded in HM20. A positive ratio signal was also obtained with inclusion bodies of gp 23 (data not shown). Striations within rec A inclusion bodies which are visible in CTEM as fine, darkly stained lines can also be recognized in cryo-STEM dark-field images (figs. 4a and 4b). Dependent on the plane of sectioning they appear as filamentous or granular structures of higher contrast within the rec A protein lumps when observed by elastic dark-field imaging (fig. 4a). Higher electron doses, which were applied in the indicated peripheral part in fig. 4 during several scans at higher magnification, reveal a

243

strongly amplified filamentous aspect of the rec A protein within the inclusion body in the elastic and inelastic dark-field images. This increase of contrast is most likely the consequence of differential mass losses which are parallelled by an increase of thickness variations on the section surface. With ratio imaging (fig. 4c) no significant differences occurred. This further demonstrates the ability of ratio-contrast to suppress influences arising from thickness variations. Moreover, this observation serves as a demonstration that even 100 times higher doses than the critical dose of 50 e nm -2, as determined for HM20 at room temperature [3], produce no measurable differences in ratio-contrast images obtained with the cryoSTEM. Epon embeddings of the ribosome-free DNAcontaining nucleoid of E. coli showed no significant difference from the resin background (fig. 2c). A measurable contrast difference between nucleoid and resin was observed in HM20 embeddings (fig. 3c). The confinement of nucleoids through the sublethal action of chloramphenicol greatly facilitates the observation of sufficiently large DNA-containing areas within these spherically shaped nucleoids. Very strong positive ratio signals were obtained in the case of mature bacteriophage T4 heads, either in Epon (fig. 5c) or - as expected even more pronounced in HM20 (not shown). This is indicative for an extremely high p.d. of the

Fig. 6. STEM images taken at an original magnificationof 10000x; elastic dark-field(a), inelastic dark-field(b) and ratio image (c) an unstained Epon section of A. carterae chromosomesrecorded at 100 keV with a dose of 20 e/nm 2. Boxed measuring area within the chromosomeis indicated in (c). Bar indicates 1/~m.

of

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B. Bohrmann et al. / Concentration eualuation of chromatin by ratio-contrast imaging

Fig. 7. Cryo-STEM images taken at an original magnification of 10000×; elastic dark-field (a), inelastic dark-field (b) and ratio image (c) of an unstained HM20 section of Euglena spec. with heterochromatin distributed as patches within the nucleus recorded at 100 keV with a dose of 5 e/nm 2. Boxed measuring area within heterochromatin is indicated in (c) Bar indicates 1/~m.

D N A within t h e h e a d o f a m a t u r e T4 b a c t e r i o p h a g e . H o w e v e r , t h e d i m e n s i o n s of t h e p h a g e heads, w h e n c o m p a r e d to t h e values k n o w n f r o m f r o z e n - h y d r a t e d specimens, r e v e a l e d t h a t a cons i d e r a b l e d e g r e e o f s h r i n k a g e m u s t have occurred. C h r o m o s o m e s within t h e cell n u c l e u s o f the d i n o f l a g e l l a t e A. carterae a r e shown in fig. 6. T h e m i c r o g r a p h s o f t h e s e c t i o n e d c h r o m o s o m e s exhibit a p r o n o u n c e d r e l i e f (fig. 6a a n d 6b). T h e i n t e r n a l p e r i o d i c i t y o f t h e l a y e r e d a r r a n g e m e n t of D N A fibrils of t h e p a r t i c u l a r s t r u c t u r e d d i n o c a r y otic c h r o m o s o m e [11] m i g h t c o n t r i b u t e to real

R'

1.5-

a HM20

thickness variations. T h e o b s e r v e d r e l i e f d i s a p p e a r e d c o m p l e t e l y in t h e r a t i o i m a g e (fig. 6c). A distinctly positive ratio signal is o b t a i n e d . Thus, w h e n c o n s i d e r i n g E p o n e m b e d d i n g s , t h e chrom a t i n o f c h r o m o s o m e s f r o m A. carterae a p p e a r s of h i g h e r D N A p.d. c o m p a r e d to t h e n u c l e o i d in E. coil (fig. 2c). U n f o r t u n a t e l y , o u r sections o f c h r o m o s o m e s from H M 2 0 e m b e d d i n g s could not b e u s e d for D N A p.d. m e a s u r e m e n t s , b e c a u s e of e n o r m o u s a c c i d e n t a l thickness variations. Condensed chromatin of histone-containing e u k a r y o t e s as f o u n d in h e t e r o c h r o m a t i n o f Eu glena spec. is shown in fig. 7 as p a t c h e s with

R' ~.5-

b Epon

DNA

1.4-

1.4. Chromatin

1.3,

1.3 • DNA

1.2-

1.2Protein

~

1,1,

1.1

1.0-

1.0

.

Chroma,tin Protein calculated Protein measured

0.9

i

20

-

/

40

-

i

60

-

i

80 vol.

•

i

100 (%)

0.g 0

i 20

.

i 40

•

i 60

m 80 vol.

i 100 (%)

Fig. 8. Numerically evaluated dependence of the normalized ratio R' versus concentration in vol% of embedded DNA, protein and chromatin (assuming a mass ratio of protein to DNA of 1 : 1 for eukaryotic condensed chromatin). (a) Embedding resin HM20; (b) embedding resin Epon. Calculated average protein and extrapolated dependence of measured R' for rec A protein in Epon. The graphs were calculated for 100 kV and collection angles O x= O 2 = 13 mrad, 0 3 = 130 mrad, an illumination angle of 10 mrad and a specimen thickness of 50 nm for Epon and HM20 sections.

B, Bohrmann et al. / Concentration evaluation of chromatin by ratio-contrast imaging

positive contrast. In HM20 both D N A and proteins are more electron scattering than the resin (fig. 8). High positive ratio-contrast is obtained in HM20, because of its high hydrogen content. 4.2. Quantitative evaluation o f D N A p.d. using ratio-contrast in S T E M

We define the packing density (p.d.) or local concentration in vivo, in aqueous environment as the amount in mass (g) of a given macromolecular constituent per unit of volume. It must be emphasized that this value is principally different from the molar concentration. Since the recording dose should not exceed a critical level in order to maintain the atomic composition of the sample during irradiation (e.g. 50 e / n m 2 for the Lowicryl resin HM20 [3]), a significant amount of quantum noise cannot be avoided. As consequence the statistical error of the recorded signal is rather high. This error can be reduced to a large extent by using larger volumes for the measurements. The precision of the concentration determination depends therefore strongly on the considered volume, which is in the range of 0.3 X 10-3-1 X 10 -2 /zm 3 for 50 nm thick sections of the investigated chromatins. In order to achieve a sufficient sensitivity for detection of low amounts of DNA, measuring areas were fixed as circular or rectangular boxes of sizes which guaranteed a location of the measuring area entirely within the particular chromatin (figs. 3, 6 and 7). Besides the correct alignment of the STEM a normalization of the two simultaneously acquired signals Se~ and Sin in terms of intensity is needed, in order to obtain reproducible results for the ratio of Sel/Sin (see section 3). The main problem to be solved is the lack of biological structure of which the p.d. is sufficiently known so that it could be used for calibration of our measurements. We had therefore to proceed by a sort of iteration: the normalized ratio signal ( R ' ) response of D N A and of protein versus the concentration in the used resins was calculated numerically (fig. 8). The calculation was done according to a semianalytical algorithm [17] that takes care of the parameters of the used

245

detector geometry including the angle of the illuminating beam (10 mrad). The atomic composition and the related average scattering cross-sections, which were used for the calculation of the R' signal for the investigated materials, are according to ref. [2]. Response signal curves of R' for D N A were calculated assuming a specific D N A density of 1.6 g m l - l , for dry (water-free) DNA, which was extrapolated from density centrifugation data of different D N A salts according to ref. [24]. Entering the measured R' values into the curves, we could read a p.d. in vol%, or inversely, the original water content. For protein in HM20 (fig. 4) a concentration of 31 vol% was found. This was recalculated into a protein p.d. of about 420 ___80 mg ml -~, assuming a protein density p of 1.35 g ml-1. Data were similar for both types of inclusion bodies. The obtained values for the water content was comparable to that determined for beef liver catalase crystals [7]. This fit meant that the p.d. obtained from the measurements was reasonable. This gave some confidence towards the theoretical values of DNA, where an experimental calibration is more difficult: for bacteriophages the p.d. has been calculated with a fairly good precision [9]. In the thin sections used we observed, however, a substantial shrinkage of the bacteriophage heads. Consequently, ratio measurements led to high values of the internal DNA p.d. (table 1), which prohibited the use of shrunken T4 for a calibration of ratio measurements. In addition, the small size of T4 bacteriophages is such that beam-induced alterations of the atomic composition cannot be excluded. An additional complication has to be envisaged when dealing with naturally occurring DNA plasms as for instance in E. coli. Whereas the DNA content per cell (in eukaryotes per nucleus) is known for many cell types by chemical determinations, these values represent only the average D N A concentration per cell (or per nucleus). The lack of knowledge about the actual spatial distribution of DNA-containing plasms limits the evaluation of DNA p.d. from biochemical and volume data. A combined biochemical and stereological approach might, at least partly, overcome the problem (see dinoflagellate chromatin).

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B. Bohrmann et al. / Concentration evaluation of chromatin by ratio-contrast imaging

Table 1 List of the calculated DNA p.d. by using geometrical dimensions and a biochemically determined amount of DNA in comparison to ratio-contrast measurements of DNA packing densities (p.d.) in thin sections of various chromatins and p,d. of protein and nucleic acids (N is the number of measurements with ratio-contrast) p.d. in mg ml-i Calculated Evaluated by ratio-contrast Epon T4 head E. coli nucleoid a) Dinoflagellate chromosome b) Condensed chromatin c),h) Protein inclusion bodies E. coli protoplasm d),g)

HM20

N

800 e)

1720_+290 1140+250

90

20-50 c)

n.d.

100 -+50

20

_

220-+ 80

n.d.

20

_

320 -+95

430 -+75

10

-

n.d.

420_+ 80

10

100-120 ~ n.d.

190_+70

120

a) Measured in the spherical nucleoid of CAM-treated E. coli. b) Measured in chromosomes of A. carterae. c) Measured in metaphase chromosomes and heterochromatin of Euglena spec. d) Measured in the cytoplasm of E. coli, averaging ribosomal and nucleoid-containing areas. e) According to ref. [9]. f) According to ref. [25]. g) Volume percentages corrected for proteins according to biochemically determined mass fractions of NA = DNA and RNA = 33% and protein = 67% [25]. h) Volume percentages corrected for proteins assuming a mass ratio of proteins to DNA of 1 : 1. n.d.: Not determined.

T h e following results (table 1) can thus b e c o n s i d e r e d c o m p a r a t i v e l y only a n d p r o v i d e o r d e r s of m a g n i t u d e , until a c o r r e c t c a l i b r a t i o n b e c o m e s feasible.

4.3. D N A p.d. in the nucleoid o r E . coli

T h e results of r a t i o - c o n t r a s t m e a s u r e m e n t s of t h e D N A p.d. in t h e s p h e r i c a l n u c l e o i d s of chlor a m p h e n i c o l ( C A M ) - t r e a t e d E. coli (fig. 3) rev e a l e d 7 + 3.5 v o l % D N A within H M 2 0 , which c o r r e s p o n d s to 107 + 53 m g m l - 1 D N A (table 1).

A few m e a s u r e m e n t s in sufficiently large ribos o m e - f r e e a r e a s o f t h e b u l k n u c l e o i d of e x p o n e n tially growing cells gave similar results. N o m e a s u r e a b l e ratio-signal of t h e C A M n u c l e o i d was o b t a i n e d in E p o n (fig. 2). Since in E p o n p r o t e i n s give a negative ratio signal a n d D N A a positive one, t h e final R ' value is a b o u t 1.00. A s s o u r c e s o f n e g a t i v e ratio signals n o t only t h e D N A - b o u n d p r o t e i n s o f c h r o m a t i n s have to b e c o n s i d e r e d b u t also t h e soluble e n z y m e s a n d p r o t e i n s of t h e cellular sap t h a t m i g h t b e p r e s e n t within t h e m e s h w o r k o f D N A filaments. U n f o r t u n a t e l y , very little is k n o w n a b o u t all t h e s e p r o t e i n s in q u a n t i tative terms. O n l y recently, t h e existence o f lowp r o t e i n c h r o m a t i n s was d e m o n s t r a t e d [13]. H o w ever, t h e i r relative p r o t e i n c o n t e n t can only b e e s t i m a t e d as b e i n g b e l o w 0.5 [8]. T h e c o n c e n t r a t i o n of all nucleic acids in a b a c t e r i a l cell as the c o m b i n e d a v e r a g e p.d. o f D N A a n d R N A was also d e t e r m i n e d . F o r t h a t p u r p o s e m e a s u r i n g a r e a s w e r e set in a way t h a t they a v e r a g e t h r o u g h an a r e a o f t h e E. coli p r o t o p l a s m which includes r i b o s o m e - c o n t a i n i n g cytop l a s m a n d n u c l e o i d ( D N A - c o n t a i n i n g plasm); R~pon = 1.02 a n d R~M20 = 1.10. B i o c h e m i c a l analysis of t h e dry mass of cytosolic m a c r o m o l e c u l e s in E. coli [25] gave a c o n t e n t o f a b o u t 33% nucleic acids (4% D N A ) a n d 67% p r o t e i n . T h e R ' v a l u e of t h a t p u r e ( w a t e r - f r e e ) m a c r o m o l e c u l a r m i x t u r e can be c a l c u l a t e d t h e o r e t i c a l l y in a similar way as d o n e for c h r o m a t i n (fig. 8). Such a c o m p o s i t e mixture of p u r e nucleic acids ( N A ) a n d p r o t e i n w i t h o u t H M 2 0 resin s h o u l d give a R~IM20 = 1.25. T h e e x p e r i m e n t a l l y o b t a i n e d R ' value of 1.10 c o r r e s p o n d s to 40 + 14.8 v o l % of a p r o t e i n - N A mixture. This v a l u e is twice as high as t h e 20% o b t a i n e d by M o n c a n y [26] a n d m a n y others. W i t h an a v e r a g e d e n s i t y of 1.43 g m l - 1 for t h e m a c r o m o l e c u l a r mixture, which t a k e s into a c c o u n t the r e s p e c t i v e m a s s fractions o f D N A a n d p r o t e i n , a p.d. of m a c r o m o l e c u l e s of 570 + 210 mg m l - 1 was m e a s u r e d in the p r o t o p l a s m of H M 2 0 - e m b e d d e d E. coli. A c c o r d i n g l y t h e p r o p o r t i o n o f the c o r r e s p o n d i n g p.d. o f 33% N A is a b o u t 190 m g m1-1 in t h e cell interior. H e r e b y , t h e close p a c k i n g of strongly s c a t t e r i n g r i b o s o m e s c o n t r i b u t e s m o s t to t h e m e a s u r e d R ' signal (figs. 2 - 4 ) . T h e p.d. o f D N A c o u l d b e c a l c u l a t e d also

B. Bohrmann et al. / Concentration evaluation of chromatin by ratio-contrast imaging

from these data. Assuming that 4% [25] of the macromolecules are DNA, we calculate a concentration of 23 mg ml-1 when equally distributed throughout the intracellular volume. In vivo the D N A of E. coli is found only in part of the cytoplasm as a dynamic structure. Numerous excrescencies of D N A plasm reach far into the cytoplasm in order to increase the physiological active surface [27]. Therefore, one has to consider a condensation factor of about 3 to 4, thus giving 69 to 92 mg ml -~, which is again comparable to the direct measurements of the D N A p.d. within the E. coli nucleoid. 4.4. DNA p.d. in dinoflagellate chromatin Chromosomes of A. carterae were averaged throughout the chromosomal area. The measured DNA p.d. was 220 + 79 mg ml-1 as revealed for Epon embeddings (table 1). The reliability of ratio-contrast measurements of the D N A p.d. was tested in the case of A. carterae chromosomes by comparison with a combined stereometric and biochemical approach. The D N A content of A. carterae was biochemically determined as 1.8 pg/cell [13]. The stereometrically obtained total volume of the chromosomes was used to calculate the p.d. of D N A to be 285-295 mg ml-1 assuming that the chromosomal D N A is entirely located within the darkly stainable chromosomes [13]. Extrachromosomal D N A which was revealed by immunocytochemistry in the periphery of chromosomes [13] was not detectable by means of ratio-contrast. In order to test the consistency of the results obtained by ratio-contrast and combined biochemical and morphometrical measurements of the D N A p.d., a further control was made. The DNA-specific fluorescence intensity was measured using DNA-specific dyes [13]. For that purpose semithin sections of the same samples as recorded in the STEM were used (data not shown). The D N A content of CAM-induced nucleoids from E. coli was used for relative calibration and set to 1. The relative D N A contents obtained for A. carterae chromosomes were 1.11.4; for details see ref. [13].

247

4.5. DNA p.d. of mitotic chromosomes and heterochroma tin Determinations of the chromatin p.d. using ratio-contrast gave similar results in the investigated types of condensed chromatin. A similar high-chromatin p.d. was evaluated for metaphase chromosomes and heterochromatin (fig. 6) of Euglena spec., a histone-containing eukaryote. R' values were 1.02 and 1.09 in Epon and HM20, respectively. In order to measure the chromatin p.d. of histone-containing eukaryotic heterochromatin equal mass fractions of D N A and protein were assumed. The dependence of the R' response signal of varying concentrations of such a chromatin is shown in fig. 8. The p.d. of condensed eukaryotic chromatin was measured to be 433 _+ 74 mg ml-a as determined in HM20 and 320 _+ 96 mg m1-1 in Epon (table 1). The lower value in Epon can be explained as an underestimate due to the protein influence. The experimentally observed negative ratio-contrast of proteins as found in inclusion bodies was not considered quantitatively, but likely accounts for the observed difference to the chromatin p.d. obtained in HM20. The p.d. of only DNA without proteins accounts for half of the measured value and is thus with about 200 mg ml-~ approximately 2 times higher than that of the E. coli nucleoid. The p.d. of D N A of interphase nuclei present as decondensed chromatin could not be measured by ratio-contrast imaging in STEM. The nuclear matrix appeared homogenously with a slightly positive R' value in HM20. The positive contrast is probably due to contributions from high concentrations of RNA and protein. The R~pon value is 1.0 and equals the resin background. Quantitative data are not obtainable, because the nucleoplasm consists of a quantitatively ill-defined mixture of DNA, proteins and RNA. A considerable amount of phosphorus must be present due to mRNA, rRNA and RNP particles. The presence of D N A in the nucleoplasm could, however, be detected by DNA-immunostaining using CTEM. It is found in the periphery of dinokaryotic chromosomes and also around hete-

248

B. Bohrmann et a L / Concentration evaluation of chromatin by ratio-contrast imaging

rochromatin of Euglena gracilis but not in the more distant nucleoplasm [13].

5. Discussion

5.1. The method of ratio-contrast imaging While the contrast obtained in either of the dark-field modes depends essentially on p and t, this dependence, especially on t, is minor in ratio-contrast, where the atomic composition is the main determinant. As the density p is not linearly and directly related to the atomic composition, the ratio image has different characteristics. They are discussed in detail by Carlemalm et al. [2]. With fig. 8 they showed that the theoretical ratio signal for C, N and H containing organic matter is approximately linearly dependent on the hydrogen content. Obviously for the P-containing nucleic acids the resulting ratio signal is out of this relation. The ratio signal is a consequence of the average atomic composition. Hence, the method of ratio-contrast imaging is suitable to acquire quantitative information about the local concentration or packing density (p.d.) of macromolecules in subcellular structures. Unstained thin sections are thus usable. Evaluation of the p.d. in mg ml-1 was possible, provided that the general category of the involved biological matter was known. The p.d. of mixtures of macromolecules can be measured, if their mass fractions are known. This is realized in eukaryotic chromatin with approximately equal amounts of D N A and protein (histones). This shows that within the experimental limits a chemically non-homogeneous substance can be replaced by a homogeneous one using suitable mean values, like the average scattering cross-sections. Principal limitations for the accuracy of the ratio-contrast measurements are the inability to discriminate between D N A and RNA and the influence of partly unknown amounts of other macromolecules, especially proteins. A direct calibration' using D N A of exactly known concentration was not possible. Water, which is always present in vivo, is replaced by the resin during the embedding procedure. Although the

water content of an E. coli cell has been determined to be 80% [26], we do not know precisely how much the cells shrink during the embedding process. In consequence, we cannot assume that the water is quantitatively replaced by resin. The contribution of proteins to the normalized ratio signal ( R ' ) was indirectly checked by measurements of the ratio signal for proteinous inclusion bodies of E. coli overproducing cells. This was done comparatively for the used resins. Proteins were not completely "matched out" in Epon as was predicted from previous observations with T4 [2]. In the case of identical scattering properties, Epon and the protein would scatter similarly and the protein would not be detectable. Experimental results have shown, however, that this is not entirely correct. Indeed, for Epon only a qualitative atomic composition, but no exact stoichiometry is known. When determining the p.d. of DNA alone, the ratio measurements have to be corrected for the influence of proteins. The problem could be solved by adjusting an embedding resin to the scattering properties of proteins by adding a miscible component with a high H content. This could be done in order to fit the average cross-sections of Epon and protein. Protein inclusion bodies of rec A protein embedded in HM20 were shown to contain about 31 vol% protein and 69 vol% water, which is replaced during the embedding procedure by resin. Protein inclusion bodies were measured to have a high p.d. of 420 + 80 mg ml - i at a R~IM20 value of 1.05. This latter value would be produced by a R~IM20 DNA equivalent of 180 mg m1-1. Chromatins with a relatively low amount of proteins as e.g. with a mass ratio of D N A to protein of 10 : 1 can be estimated with fair accuracy as for instance in E. coli [28]. For example a chromatin of a D N A p.d. of 200 mg m1-1 would accordingly contain about 20 mg m l - l protein. The related R~IM2o value of such a low-protein chromatin is very small ( < 1.01) and can thus be neglected. A property of thin sections is their surface roughness (relief) [5]. Thickness variations are produced by the sectioning process. Differences in the plastic flow properties between resin and biological structure account for the observed surface relief of thin sections [29]. Due to this relief,

B. Bohrmann et al. / Concentration evaluation of chromatin by ratio-contrast imaging

elastic dark-field imaging in STEM, although producing high contrast even with unstained sections, revealed no reproducible results. Quantitative measurements with unstained thin sections using only the elastic signal are thus practically impossible. Ratio-contrast offers the possibility to strongly reduce this limitation by the capacity to largely suppress thickness variations of thin sections. The ability to suppress even compressions of the sections could be demonstrated. The cryo-STEM with the superconducting lens [19], as used in this study, allows one to observe resin-embedded material at a temperature near that of liquid helium, which reduces irradiationinduced mass loss to a minimum [30]. It is known that beam-induced mass losses occur at materialspecific electron doses. STEM measurements at room temperature revealed mass losses already at doses of 50 e / n m 2 for sensitive material like Lowicryl HM20 [3]. Therefore, we employed in ratio imaging for D N A p.d. measurements lowdose imaging conditions with doses not exceeding 100 e / n m 2 which are highly unlikely to introduce mass loss in the cryo-STEM. A limitation of lowdose imaging is given by the small number of electrons collected per pixel of the elastic and inelastic image. As a consequence, a significant quantum noise, also called statistical noise, is introduced which lowers the signal-to-noise ratio also in ratio images. As revealed for rec A protein embedded in HM20, even doses of approximately 5000 e / n m 2 gave no measurable difference for the protein p.d. from that obtained at doses of 100 e / n m 2. Thus, higher electron doses could in principle be applied than those employed in this investigation. This would be advantageous to obtain an improved image resolution due to a reduction of the statistical noise. The exact critical doses have to be determined experimentally for biological materials and resin at the recording temperature of the cryo-STEM. However, at liquid-helium temperature reduction factors of 20-50 [30] appear reasonable. The quality and reproducibility of the sample preparation are crucial for concentration measurements using ratio-contrast. The techniques for specimen preparation must ensure reduction

249

of intracellular rearrangements of molecules and avoid a washout of matter. Considering resin-embedding techniques, cryofixation and freeze-substitution have been shown to minimize aggregation artefacts and ensure superior structural preservation [8]. The ability of cryofixation combined with freeze-substitution to irreversibly cross-link aggregation-sensitive chromatin (low content of protein partners) by physical fixation was demonstrated even in the absence of chemical or heavy-metal fixatives [13]. Therefore, completely unstained specimens can be obtained. Commonly used resins like Epon or the Lowicryls can be applied, which allow conventional thin sectioning. Polymerization-induced shrinkage or swelling might occur. Shrinkage leads obviously to an increase of the measured D N A p.d. as observed in T4 bacteriophage heads. However, we cannot exclude the possibility that shrinkage of some extent might have also occurred in other types of chromatin.

5. 2. Biological relevance Quantitative data about the cellular concentration of biomolecules, especially DNA, are of interest since they are significantly higher than those used in vitro. The knowledge of the local concentration of DNA, defined as its packing density (p.d.), offers a quantitative basis for the description of the structural state of chromatin in relation to its metabolic activity. The whole attempt to know the p.d. of chromatin or of DNA was based on the notion that substrates, enzymes and products are most likely not able to enter or leave a structure of high p.d. Transcriptional activity requires the decondensation of the chromatin in order to make the D N A accessible to gene regulatory proteins or to the R N A polymerase. In condensed chromatin a metabolic activity of the D N A is difficult to envisage. It is therefore generally assumed that decondensation of D N A is needed for making a particular D N A filament accessible to metabolic activity. The data obtained by quantitative ratio-contrast measurements represent orders of magnitude for the D N A p.d. of various chromatins (table 1).

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B. Bohrmann et al. / Concentration evaluation of chromatin by ratio-contrast imaging

The highest degree of D N A condensation is found in mature virions with D N A present in a completely inactive storage form of resting chromatin. Ratio-contrast measurements indeed confirmed their outstanding high D N A p.d. of about 800 mg m1-1 in mature heads of bacteriophage T4 [9]. The D N A content of the mature bacteriophage head forms a nematic liquid crystal as found in bacteriophages lambda and T4 [10]. This high p.d. is incompatible with a metabolic activity of the DNA. The situation of chromatin metabolism in living cells is different. Bacterial chromatin exhibits no changes of the condensation state during the cell cycle in contrast to that observed in most eukaryotes, a finding which was confirmed by ratio-contrast measurements. The bacterial chromatin has a D N A p.d. about 100 mg m1-1 as measured with ratio-contrast in STEM for the nucleoid of E. coli. Although clearly below the D N A p.d. in bacteriophage T4, but still considerable higher than concentrations usually employed in vitro experiments ( ~ 1 mg ml-1), the measured DNA p.d. would allow in principle for the penetration of enzymes into the chromatin bulk as well as for diffusion of the products, e.g. m R N A from the interior of the chromatin to the cytoplasm, if only the mesh sizes of the D N A are considered as the limiting factor which were calculated as function of the p.d. and the fiber diameter [28]. From studies that localize the transcriptional apparatus in E. coli [31] in combination with EM studies of the shape of the E. coli nucleoid [27] a more detailed picture of the metabolic activity emerged. Transcriptional activity appears to reside in a halo of excrescencies of DNA-containing plasm adjacent to the cytoplasm. Eukaryotic condensed chromatin was measured to be about a factor of two more densely packed when compared to condensed chromatin of E. coli. A D N A p,d. of 200 mg m1-1 was found in heterochromatin, mitotic chromosomes and dinoflagellate chromosomes. The measured D N A p.d. of histone-containing eukaryotes was obtained from the total chromatin p.d. of about 400 mg ml-1, which includes the associated structural proteins. We assumed mass fractions of

50% D N A and 50% protein (histones). Similar values were obtained for mitotic chromosomes as well as heterochromatin. A particular situation is found in the chromatin organization in dinoflagellate chromosomes. Dinoflagellates are eukaryotes devoid of histones and with only small relative amounts of histone-like protein [32]. The dinoflagellate chromosome was studied in A. carterae. A D N A p.d. of about 200 mg ml-~ was revealed for the central condensed chromosomal bulk. In contrast, a considerably lower D N A p.d. in a peripheral halo of extrachromosomal D N A was proven by immunocytochemistry [13], which was not measurable by ratio imaging. This might account for the lower value of the D N A p.d. measured by ratiocontrast compared to slightly higher values ( ~ 290 mg ml -~) obtained by morphometrical measurements [13]. Considering the high p.d. of A. carterae chromosomes a real physiological decondensation is needed to make the DNA accessible to metabolic reactions. These findings are in agreement with previous data obtained by autoradiography that demonstrated the existence of a transcriptionally active zone in the periphery of dinoflagellate chromosomes [33]. The measured degree of D N A p.d., which is similar to histonic heterochromatin, suggests a transcriptionally and replicationally inactive state. The chromosome of A. carterae appears to resemble the condensed histonic chromatin of eukaryotes with respect to the observed similar degree in the D N A p.d., but not in the amount of associated proteins. This is in favor of an interpretation that the so-called "permanently condensed chromosomes" are rather patches of heterochromatin which, by definition, are metabolically resting. D N A was found to be able to form spontaneously liquid-crystalline phases in vitro. The concentration of the D N A determines which type of liquid crystal occurs. DNA fragments of 50 nm in the concentration range of approximately 180240 mg ml-~ are arranged as cholesteric liquid crystals [34]. This value fits well with the measured D N A p.d. of 2 2 0 + 7 8 mg m1-1 using ratio-contrast. The data of the DNA p.d. supports the hypothesis that the DNA is packed into

B. Bohrmann et al. / Concentration evaluation of chromatin by ratio-contrast imaging

a twisted nematic (cholesteric) liquid crystal in dinoflagellate chromosomes [11]. 6. Conclusion Ratio-contrast allows for the evaluation of the DNA packing density (p.d.) or local concentrations within various chromatins. Orders of magnitude could be obtained. Limitations arise from uncertainties concerning the amount of protein partners. The construction of a resin that has scattering properties similar to protein would considerably increase the precision of the concentration measurements. The obtained results suggest a correlation of low DNA p.d. below 100 mg ml-1 and transcriptional activity. Further we suggest a universal topological restriction of vegetative chromatin of low DNA p.d., which is localized in a halo at the periphery of a bulk of resting DNA with a high p.d.

Acknowledgements We would like to thank W. Villiger for expert assistance in cryofixation and M. Maeder and M. Zoller for help in preparing the manuscript. References [1] A.V. Crewe, J.P. Langmore and M.S. Isaacson, Resolution and Contrast in the Scanning Transmission Microscope (Wiley, New York, 1975) p. 47. [2] E. Carlemalm, C. Colliex and E. Kellenberger, Adv. Electron. Electron Phys. 63 (1985) 269. [3] R. Reichelt, E. Carlemalm, W. Villiger and A. Engel, Ultramicroscopy 16 (1985) 69. [4] R. Reichelt and A. Engel, Ultramieroscopy 13 (1984) 279. [5] E. Kellenberger, W. Villiger and E. Carlemalm, Micron Microsc. Acta 17 (1986) 331.

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