Journal of Structural Biology 129, 17–29 (2000)doi:10.1006/jsbi.1999.4203, available online at http://www.idealibrary.com on

Complementary Visualization of Mitotic Barley Chromatin byField-Emission Scanning Electron Microscopy

and Scanning Force Microscopy

A. Schaper,*,1 M. Roßle,† H. Formanek,† T. M. Jovin,* and G. Wanner†,2

*Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, 37070 Gottingen, Germany;and †Botanical Institute of LMU–Munich, Menzingerstrasse 68, 80638 Munich, Germany

Received June 26, 1999, and in revised form November 12, 1999

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The surface structure of mitotic barley chromatinas studied by field-emission scanning electron mi-

roscopy (FESEM) and scanning force microscopySFM). Different stages of the cell cycle were acces-ible after a cell suspension was dropped onto alass surface, chemical fixed, and critically pointried. Imaging was carried out with metal-coatedpecimen or uncoated specimen (only for SFM). Thepatial contour of the chromatin could be resolvedy SFM correlating to FESEM data. The experimen-ally determined volume of the residue chromatinuring mitosis was within the range of 65–85 mm3. Aomparison with the theoretically calculated vol-me indicated a contribution of about 40% of inter-al cavities. Decondensation of chromosomes byroteinase K led to a drastic decrease in the chromo-ome volume, and a 3-D netlike architecture of theesidue nucleoprotein material, similar to that inhe intact chromosome, was obvious. Incubation ofetaphase chromosomes in citrate buffer permitted

ccess to different levels of chromatin packing. Wemaged intact chromosomes in liquid by SFM with-ut any intermediate drying step. A granular sur-ace was obvious but with an appreciably loweresolution. Under similar imaging conditions pro-einase K-treated chromosomes exhibited low topo-raphic contrast but were susceptible to plasticeformations. r 2000 Academic Press

Key Words: AFM; cell cycle; plant chromosome;NA; scanning electron microscopy; scanning forceicroscopy; SFM.

INTRODUCTION

The structural integrity and functional repertoiref genomic DNA in vivo are maintained by the

1 Present address: Stephanusstrasse 2, D-30449 Hannover,ermany.2

wTo whom correspondence should be addressed.

17

ontrolled condensation of DNA–protein assemblieshroughout the cell cycle. Many efforts have beenndertaken to resolve the structural details at theifferent levels of chromatin condensation usinglectron microscopy (EM), crystallography, spectros-opy, and molecular biological techniques (for aeview see van Holde, 1988). Although the higherrder structural hierarchy of chromatin organiza-ion, in terms of the folding of the elementary fibrilnucleosomal chain) into fibrous structures of dis-inct sizes, has received much attention over the pastwo decades, the different levels of chromatin organi-ation have hardly begun to be elucidated (for reviewee Cook, 1995). Several preparation techniquesave been elaborated for rendering the chromatinccessible to structural investigations by high-esolution microscopy (for a review see Zentgraf etl., 1987). Scanning electron microscopy (SEM) canrovide valuable data about the three-dimensional3-D) organization of the chromatin and differentevels of condensation (Harrison et al., 1982, 1987;

ullinger and Johnson, 1987; Rattner and Lin,987; Sumner, 1991; Wanner et al., 1991; Pelling andllen, 1993; Takayama and Hiramatsu, 1993; Rizzoli

t al., 1994). Since FESEM is limited to the investiga-ion of dried samples, it is crucial that the samplereparation procedure preserve the native structuref the biomaterials. Martin et al. (1994, 1996) haventroduced the drop preparation technique thatielded well-preserved structures of mitotic, meiotic,r interphase chromatin suitable for high-resolutionEM. Scanning force microscopy (SFM) permits thetructural resolution of the topography of biologicalaterial adsorbed at air–solid or liquid–solid inter-

aces with up to subnanometer resolution. The advan-age of the SFM for biologists is that it can visualizeonconductive materials in a nonvacuous (i.e., air or

iquid) environment. In effect, fixation, contrast en-ancement, and labeling are not required. The SFM

as successfully applied in the investigation of the

1047-8477/00 $35.00Copyright r 2000 by Academic Press

All rights of reproduction in any form reserved.

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18 SCHAPER ET AL.

ucleosomal chain morphology (Allen et al., 1993;esenka et al., 1992; Fritzsche et al., 1994a; Leuba etl., 1994), of chromatin fibers in air and in liquidFritzsche et al., 1994b, Fritsche and Henderson,996), and of the overall structure of air-dried andehydrated metaphase chromosomes (De Grooth andutman, 1992; De Grooth et al., 1992; Putman et al.,993; Rasch et al., 1993; McMaster et al., 1994).In this study we have investigated different states

f barley chromatin condensation during mitosis byeans of FESEM and SFM in air–vacuum and in

iquid. We show SFM images of different condensa-ion states of the chromatin, indicating that the dropechnique is of general use for the preparation ofhromatin free of any surface contaminations and forhromosome decondensation assays.

MATERIALS AND METHODS

All reagents were of analytical grade and were from ServaHeidelberg, FRG), if not otherwise stated. We used double-istilled (quartz glass distillery) and ultrafiltered (Amicon) watern the preparation of all buffer/salt solutions. Glass slides withaser engraved locator grids (Laser Marking, Fischen, FRG) weresed after they were cleaned in chromic acid.Preparation of chromatin samples. Seedings of barley (Hor-

eum vulgare, Steffi) were germinated on moist filter paper in aet chamber for 3 days at 4°C in the dark and subsequently

ncubated for 8 h at 20°C. For synchronization of the meristematicells in the S-phase of the cell cycle the roots were incubated for 18

in 1.25 M hydroxyurea. For arresting the cell cycle in meta-hase, roots were incubated in 4 µM amiprophosmethyl (Bayer,everkusen, FRG) for 4 h at 20°C and washed 33 with water

Dolezel et al., 1992; Busch et al., 1994). Root tips were chopped,xed in ethanol/acetic acid (fixative I, 3/1 (v/v)), and stored at20°C. The meristematic tissue of the root tips was digested in aixture of 10% cellulase ‘‘Onozuka’’ R10 and 10% pectolyase Y-23

Kikkoman, Dusseldorf, FRG) in 75 mM KCl, 7.5 mM EDTA, pH.5, for 90 min at 30°C. The cell suspension was filtered through aylon mesh (80-µm mesh size) and treated with hypotonic KCl (75M) for 5 min at 20°C. The nuclei were spun down for 7 min at

0g, resuspended in fixative I, and washed and pelleted 33 inxative I. Finally, nuclei were stored in fixative I at 220°C.hromosomes were prepared according to the drop technique ofartin et al. (1996). Briefly, an aliquot of the nuclei suspensionas dropped on ice-cold glass slides. After a drop of 45% acetic acidas added and just before the drop was evaporated the area was

overed with a coverslip, gently squashed, and incubated for 30in on dry ice. Finally, the coverslip was removed and the glass

lide was incubated for 45 min in fixative II (2.5% glutaraldehyden 75 mM cacodylate (Merck, Darmstadt, FRG), 2 mM MgCl2, pH). For FESEM and SFM of dried specimens, samples wereehydrated in ascending concentrations of acetone (20, 40, 60, 80,00% (v/v)) and critical point dried (CPD) (Critical Point Dryer,alzers, Liechtenstein) using CO2 as the transition fluid. For SFM

maging in liquid the glass slide was transferred to water andounted to the BioScope. For mounting samples to the Nano-cope the glass slides were cut into pieces #15 mm in diameter.Controlled decondensation of metaphase chromosomes. Decon-

ensation assays were performed with metaphase chromosomeamples prepared by the drop technique. Enzymatic degradationf chromosomes was carried out with proteinase K. Glass slidesere fixed in 2.5% glutaraldehyde/75 mM cacodylate buffer, pH 7,

or 1 h, washed 33 for 15 min in water, and subsequently

ncubated with proteinase K (0.1 mg/ml), 10 mM Tris–HCl, pH 7, s

or 2 h at 37°C. After proteinase treatment glass slides wereashed 33 (15-min incubation for each step) in water. Samplesere incubated in fixative II for 2 h and again washed 33 followedy CPD. For imaging in liquid the CPD step was omitted. Forhemical decondensation the metaphase chromosomes were incu-ated in 60 mM sodium citrate, pH 7.2, for 1 h at 20°C according to

procedure of Marsden and Laemmli (1979). Samples wereashed twice with water prior to fixation in 2.5% glutaraldehyde

n 10 mM Tris–HCl buffer, pH 7.2. Again samples were washedwice in water and were critical point dried. Light microscopyphase and differential interference contrast) was applied for thereselection of chromatin-covered surface regions facilitated byhe locator grid engraved in the glass slide (cf. Martin et al., 1996).he latter also enabled localization of the same region in theESEM and in the SFM.Field emission scanning electron microscopy. For FESEM we

sed a Hitachi S-4100 equipped with an Autrata YAG-typeetector for back-scattered electrons (BSE). Samples were coatedith 2–3 nm Au/Pd (80/20) with a magnetron sputter-coater (050CD, Balzers, LIE) and examined at various voltages.Scanning Force Microscopy. SFM measurements of CPD dried

pecimen were performed with a NanoScope IIIa-multimode SPMDigital Instruments (DI), Santa Barbara, CA). In some cases aticky tape was applied to the dry sample surface prior to SFMmaging (Pietrasanta et al., 1994, 1996). The SFM was operatednder ambient conditions at 18–27°C and at a relative humidity

n the range of 20–45%. Scanning was carried out with a J-canner with a 135 3 135 3 5 (x, y, z) µm3 scan range. All imagesere taken at a line scan rate of ,4 Hz. Investigations with theanoScope in liquid were conveyed with a liquid measuring

hamber (DI). Alternatively, for imaging in liquid we used aioScope (DI). Surface profiling with the NanoScope and theioScope was in permanent contact mode and in tapping mode

only for imaging in liquid with the NanoScope equipped with anltralever). We used different types of microfabricated scanning

ips integrated into triangular cantilevers with spring constantsf ,0.2 N/m: (i) microfabricated pyramidal shaped Si3N4- tips (DI)ith a typical tip radius of 20–50 nm, (ii) conical shaped Si-tips

Ultralever, Park Scientific Instruments, Sunnyvale, CA) with aypical tip radius of 3–5 nm, and (iii) electron beam depositedEBD) tips of ,1.5 µm in length and with a tip radius in the rangef 5–10 nm as specified by the supplier (Hart Probe, Materialsnalytical Services, Raleigh, NC). EBD tips were deposited on topf a pyramidal shaped Si3N4 tip from DI. The standard planefitorrection (first-order fits) of the NanoScope software was appliedo the data. The various images were combined and processed forresentation with the programs Photoshop (Adobe Systems) andanvas (Deneba Systems) for the Apple Macintosh computer.ross-sectional analyses were performed with the NanoScopeoftware. The reported lateral dimension of a surface feature ishe full width at half-maximum height. For image processing wesed ImageSXM software (Barrett, 1997). For volume determina-ions (see also Fritzsche and Henderson, 1996; Allen et al., 1996) aean background value was subtracted from the image data.tructural features were selected by applying a binary maskenerated from thresholding the surface corrugations. Residueurface contaminations were erased from the binary mask. Theask was overlaid on the original image and used to extract those

ata points that comprised the chromatin. The chromatin volumeas calculated by collecting the volumes of the data points

voxels) within the mask. For the determination of the meaneight of condensed chromatin a similar procedure was applied tohe data. The upper 104 pixel values within the masked chromo-ome domain were collected for calculation of the mean height and

tandard deviation.

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19VISUALIZATION OF MITOTIC BARLEY CHROMATIN

RESULTS

he Apparent Volume of the Residue Chromatin IsConstant during Mitosis

Figures 1A–1F present SFM images of the chroma-in of the cell cycle separated by digital imagerocessing, i.e., background subtraction, threshold-ng, and binary filtering. The chromatin in all phasesppeared well distinguished from the basal planend free from any particle contaminations. Differentorphologies that were typical for the distinct phases

f mitosis were obvious, including the amorphousass of interphase chromatin (Fig. 1A), the con-

ensed states from prophase to metaphase (Figs. 1C,nd 1D) comprising 14 chromosomes in barley, andhe redistribution of the chromatin between mothernd daughter cells in anaphase and telophase (Figs.E and 1F). For comparison, the correspondingESEM micrographs of the anaphase and telophasere shown in Figs. 1H and 1J. The chromatin inetaphase (Fig. 1D) is more condensed by about one

rder of magnitude compared to its interphase ap-earance (Fig. 1A) as calculated from the chromatinovered surface area per unit height. The latter ishe mean height of the chromatin domain. In pro-hase the condensation process started from theentromeric region presumably by aggregation/ransformation of the granular interphase chroma-in into chromomers, which then condensed to form aylindrical body. In this state the chromatids wereften discernible from a topographic point of viewven in the uncondensed telomeric region duringrometaphase (Martin et al., 1996). Parallel arrange-ent of fibers, which is also a characteristic feature

redominantly seen in prophase chromatin by FE-EM (Martin et al., 1996), was not resolved by SFM.rophase chromosomes separated into pairs of chro-atids in late prophase or early metaphase. During

ubsequent chromatin condensation in prometa-hase and metaphase, the limitations of instrumen-al resolution caused by finite SFM tip size werebvious and multiple images of the tip face at steephromosome edges were perceptible (data not shown,ee Discussion). In anaphase (Fig. 1E) decondensa-ion proceeded from the telomeric region and anmorphous chromatin structure was visible in telo-hase (Fig. 1F). The total volume of the resident partf each phase during mitosis was determined byeans of image processing. From the diagram in Fig.

G it is evident that the apparent volume of theesidue chromatin during mitosis is within the rangef 65–85 µm3. The slightly higher volume in ana-hase is mainly due to the overlap of chromosomes,hereby forming gaps not accessible to the SFM tip,nd thus led to an additional contribution to the

pparent volume. Thus we can conceive that the d

olume is nearly constant within the range of errorsndependent of the cell and of the mitosis phase.

In Fig. 2 representative SFM images of the ultra-tructure of chromatin are presented that exhibithe most prominent morphological changes of thehromatin during the cell cycle on the nanometercale. In interphase (Figs. 2A, and 2B) a globularorphology was the common structural motif with

n apparent globule diameter of about 100–300 nm.n prometaphase the chromosomal surface wasensely packed with granules and condensation ofhromatin was accompanied by a drastic increase inheir size (Figs. 2C and 2D). A granular chromosomeurface morphology (tens of nanometer corruga-ions) was typically observed, presumably a conse-uence of the condensation process of the nucleopro-ein fiber during mitosis. In prometaphase the largestorrugation amplitude was visible, whereas in meta-hase the chromosome surface appeared smoothlyontoured (Figs. 2E and 2F). At higher magnificationhe most prominent structural features of meta-hase chromosomes were the chromatids and theentromere. The latter is composed of parallel bundlesf fibrous elements (Wanner et al., 1991; Martin etl., 1996). During condensation the clusters of chro-atin fibers may assemble as intermediates in the

onstruction of an axial structure, which is furtherompacted in the fully condensed (metaphase) chro-osome.

Complex Chromatin Network Remainedafter Controlled Decondensationof Metaphase Chromosomes

For revealing structural details of the chromosomeubstructure, samples of metaphase chromosomesere treated with proteinase K and citrate buffer. Inig. 3 representative SFM images of chromosomesfter proteinase K digestion are shown at variouscan sizes. As a prominent consequence of deconden-ation, chromosomes were flattened on the glassurface. For removal of scan distortions from sampleontaminations during SFM imaging (e.g., such asndicated by the arrows in Fig. 3A), a sticky tape waspplied to the sample surface without any detectableoss of the chromatin fine structure (Fig. 3B). Theticky tape removed an upper layer of contamina-ions and was generally applicable for removingeakly adsorbed contaminations of dried samplesxposed to air. For comparison, the mean height ofpro)metaphase chromosomes was extracted fromFM images obtained under various experimentalonditions and summarized in Table 1. Within theange of error similar mean heights were obtainedor CPD prometaphase and metaphase chromosomesTable 1, first and second rows). In contrast, decon-

ensed chromosomes appeared to be appreciable

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20 SCHAPER ET AL.

FIG. 1. Different stages of barley mitosis in the SFM after background subtraction and structural filtering of the residue chromatintructure. Normalized lateral dimensions. Sputtered with Au/Pd (coated) and uncoated. (A) Interphase, uncoated. (B) Prophase, coated. (C)rometaphase, coated. (D) Metaphase, uncoated. (E) Anaphase, coated. (F) Telophase, coated. (G) Apparent volume of the chromatintructures in A–F. The volume calculation based on an image processing algorithm (for details see text). (H) FESEM micrograph ofnaphase and (J) of telophase shown in E and F, respectively.

fE

FIG. 2. SFM images of selected regions of different states ofeatures are discernible down to 50 nm in diameter. (C,D) Prometa

condensation; uncoated hardware zooms. (A,B) Interphase. Structuralphase with corrugated surface. (E,F) Metaphase with smooth surface. In

a region with a chromatid gap is shown and in F the centromeric region is visualized.

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22 SCHAPER ET AL.

ower by a factor of 4–5 (Table 1, third row). Thusrom Table 1 it is evident that after decondensationy proteinase K treatment the mean height of thehromosomes is drastically reduced compared to theondensed (undigested) structures. In Figs. 3C and

FIG. 3. SFM imaging of proteinase K-treated chromosomes inample instabilities typical scan distortions (indicated by the arroas applied. (C) Hardware zoom of an individual chromosome. (D)

learly discriminated from the structure of the basal plane is obvio

D a porous, netlike surface morphology is obvious n

ith a mesh width in the range of 50–100 nm. Atigh resolution it was revealed that the poroustructure was homogeneously distributed over thentire chromosome domain. In a former publicatione have shown chromosome structures after impreg-

Surface area prior to applying a sticky tape to the surface. Due tos) are obvious. (B) Same surface area as in A after the sticky tapeare zoom of the ultrastructure. A netlike morphology that can be

air. (A)wheadHardw

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23VISUALIZATION OF MITOTIC BARLEY CHROMATIN

um blue) for specifically staining DNA (Wanner andormanek, 1995). In the BSE micrographs of suchhromosomes, a netlike DNA pattern is perceptibleith morphology similar to that in Fig. 3D. Thus it is

onceivable that part of the protein can be removedy proteinase K digestion without changing theverall appearance of the DNA distribution withinhe chromosome domain.

Chemical decondensation of metaphase chromo-omes with citrate buffer was performed in order tonvestigate the different states of decondensation ofhe nucleoprotein fiber. In Fig. 4A a metaphase plates presented after decondensation in the FESEMrior to SFM and in Fig. 4B after SFM imaging. Theorresponding SFM surface relief is shown in Fig.E. Drastic changes in the FESEM image contrastfter SFM are obvious. Since the sample surface isovered by a thin Au/Pd layer we must conclude thathe SFM tip alters the layer properties during scan-ing. Consequently, it must be considered that thehysicochemical surface properties are modified byip–sample interaction (e.g., reconstruction and/orechanical or chemical aging of the metal layer) in

n unknown manner. In Figs. 4C and 4D a detailediew of the fine structure in the FESEM before andfter SFM imaging, respectively, are shown. Afterareful inspection of the FESEM and SFM imagese conclude that the fine structure of the surface

elief is not changed during SFM and we could notetect any loss of surface material during SFMmaging.

Decondensation was initially associated with aradual elongation and loosening of the chromosomexis. Strikingly, decondensation did not affect thehromosome structure isotropically but seemed toroceed in a bipolar manner starting from the telo-eric regions. As loosening progressed, higher order

tructures (aggregates) became visible, i.e., clustersf nucleoprotein material and fibrous structures ofistinct size. The arrangement of the aggregateshowed much variation between different meta-

TABLE IMean Height of Prometaphase and Metaphase

Chromosomes under Various Conditions

Mean height (µm) SD (µm)

rometaphase, CPD 0.61 0.05etaphase, CPD 0.57 0.07etaphase, proteinase K, CPD 0.13 0.02etaphase, in liquid 1.20 0.12etaphase, proteinase K, in liquid 0.37 0.07

Note. Data were collected from samples comprising 7 to 14hromosomes. For consistency, from each sample the upper 104

ixels were used for the height statistics including mean andtandard deviation (SD).

hase plates and samples. In the final stages of e

econdensation, aggregates separate and individualhromosomes were no longer recognizable. FiguresF–4K show details of the decondensed chromatinalo at higher resolution in the SFM. The chromatin

s dispersed radially from the chromosome domainnd is more decondensed in the outer regions of thealo. Inspection of different frames at higher struc-ural resolution revealed a mixture of fibers ofarious diameters and of granules and voluminousggregates probably from partially condensed chro-atin. Despite the heterogeneous morphology of the

econdensed chromatin, selected regions exhibitinglamentous structures were amenable for SFM me-rology. Taking into account the broadening of theateral dimensions by the SFM tip, the apparentidth of the various fibers averaged in the size

anges expected for DNA, for the nucleosomal chain,nd for higher order chromatin structures such ashe 30-nm fiber and 100- to 300-nm fibrous elements.fiber substructure was not resolvable by SFM. The

nvestigation of the nanometer topography of decon-ensed chromatin clearly shows the limitations ofhe applied sample preparation technology includingurface contaminations (nanometer particles) andisruption of the chromatin fine structure (disassem-ly and dissociation of nucleosomes and other DNA-ssociated proteins).

hromosomes Prepared by the Drop Technique AreSuitable for SFM Imaging in Liquid

We imaged chromosomes in the SFM by omittingny intermediate drying step in the drop prepara-ion procedure. The combination of an inverted lighticroscope with a SFM imaging tool in the BioScope

nabled localization and positioning of the chroma-in within the scan range of the SFM. The isolatedhromosomes, which were not allowed to dry duringreparation, retain a 3-D appearance. In general,maging was delicate since the chromosomes werefficiently removed from the glass support by sharpips, as was the case with Ultralevers and Hartrobes (tip radius 3–5 and ,10 nm, respectively)ven under minimum tip load. The detachmentrocess could be followed in the light microscope andsually appeared immediately during the first con-act (scan line) of the tip with an individual chromo-ome, which was then removed as a whole from theurface. Imaging with pyramidal shaped Si3N4 tipstip radius 30–50 nm) yielded more stable imagingonditions but was accompanied by a decrease intructural resolution. Figure 5A shows a metaphaselate in an aqueous environment in the SFMquipped with a pyramidal shaped tip. The latterxhibited characteristic SFM imaging artifacts. Fromig. 5B at higher resolution it is obvious that the

dge profiles of the chromosomes are images of the

24 SCHAPER ET AL.

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25VISUALIZATION OF MITOTIC BARLEY CHROMATIN

ip profile, thus creating sidewall images of theyramid at steep surface profiles. Inspection of theurface morphology of the chromosome surface ex-osed to the top of the pyramid revealed a granularurface structure in qualitative agreement with thePD dried structure. According to Table 1, the meaneight of hydrated chromosomes is about twice thePD structure, indicating that the native hydrated

hree-dimensional structure could not be fully pre-erved during dehydration and/or CPD. We mustmphasize that this discrepancy is very mild com-ared to results of the swelling behavior of rehy-rated (former air-dried) chromosomes of humanymphocytes. For those, a factor of 5–7 was typicallybserved (De Grooth and Putman, 1992; Fritzsche etl., 1994b), indicating a severely collapsed chromo-ome structure.In Figs. 5C–5F SFM images of proteinase K-

reated chromosomes in liquid are presented (cf. Fig.). The contours of the chromosomes are clearlyisible but details of their fine structure were notesolvable (Fig. 5D), probably due to the convolutionf topography and elastic response. The apparentean height of the hydrated chromosome was en-

arged two- to threefold, compared with the CPDtructure (Table 1), which should be due mainly tohe contribution of the hydration behavior of thehromatin. Operating the SFM in tapping mode atinimum tip load was essential for nondestructive

maging of the fragile residue chromosome struc-ure, while permanent contact mode failed due tolastic deformation. The contribution of plasticitynd elasticity in the image contrast could be trig-ered by switching between permanent contact andapping mode during tip tracking. In Fig. 5E part ofhe structure was removed in a controlled manner byardware zooming (see region indicated by the arrow

n Fig. 5E) in permanent contact mode prior tomaging in tapping mode. After positioning an appro-riate SFM imaging frame (in tapping mode) evenn entire chromosome could be easily removed liney line from the glass surface by switching to theermanent contact mode (cf. Figs. 5C and 5F, theegion indicated by the arrows). These proteinase-treated samples should be attractive for chromo-

omal microdissection essays; the latter were estab-ished by Thalhammer et al. (1997). The behavior ofroteinase K-treated chromosomes during SFM imag-ng in liquid indicates that non-DNA material cru-

FIG. 4. Controlled decondensation of glutaraldehyde-fixed mncubation in citrate buffer, fixation in glutaraldehyde, and CPD.maging and (B) the same area after SFM imaging. (C,D) Zoom of turface relief of the structure shown in A. (F–K) SFM hardware z

esolution a complex surface pattern of granules and fibers was obvious.

ially contributes to the stiffness of the intact con-ensed structure in Fig. 5A.

DISCUSSION

By using unsynchronized barley root tips, snap-hots of different states of mitosis were obtained.hanges in the morphology of chromosomes during

he different stages of mitosis have been examinedy FESEM and SFM. For all chromatin samplesnvestigated by SFM, stable imaging was achievedithout any measurable changes of the topography.ur results show that the drop preparation tech-ique originally adapted for FESEM is widely appli-able in microscopic investigations of the chromatintructure during mitosis and is amenable for struc-ural investigations/manipulations by SFM in airnd in liquid. Prior to sample preparation for micros-opy, chemical fixation with ethanol–acetic acid andlutaraldehyde was essential in order to preserveetails of the native chromatin structure. For SFMmaging in liquid, chromosomal structures wereemarkably stable without any intermediate dryingtep. In former SFM studies of chromosome struc-ure an intermediate drying step was always essen-ial during sample preparation in order to attainufficient sample stability and fixation of the rehy-rated specimen on the solid support. The investiga-ion of the different states of condensation of thehromatin during mitosis shows that higher resolu-ion by SFM is achievable on flat and dry surfaces,.e., on chromosome surfaces facing the tip apex (Fig.), exhibiting nanometer corrugations, and on decon-ensed chromatin that spread out on the glassurface (Fig. 4). For the former, the topographicesolution was down to chromomer dimensions andor the latter it was down to the level of the nucleo-omal chain and of other fiber classes in chromatinrganization.

imits of Structural Resolution in FESEM andSFM of the Chromosome Structure

Although much information about chromatin ap-earance and behavior within the cell has beenbtained using light microscopy, greater resolution iseeded for a thorough understanding of the chromo-ome organization. Compared to the instrumentalesolution of modern light microscopes of about halfhe wavelength of the incident light (typically ,250

ase chromosomes. The drop technique was applied, followed bymetaphase plate after decondensation in the FESEM prior SFMges shown in A and B before and after SFM, respectively. (E) SFMof selected regions of the decondensed chromatin halo. At higher

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26 SCHAPER ET AL.

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27VISUALIZATION OF MITOTIC BARLEY CHROMATIN

m), the resolution power of the FESEM is about 1.5m. However, due to sputter coating and restrictions

n the exposure time of chromosomes toward theigh-energy electron beam, the experimental limit ofesolution is typically in the range of 5–10 nm. Byeans of SFM we could not detect any significant

tructural alterations introduced by sputter coatingf CPD dried samples. In SFM, surface profiling iserformed with a stylus of finite sharpness and theesolution capability of the SFM is directly related tohe tip geometry. The structural resolution on flaturfaces (with corrugation amplitudes within the tipiameter) is basically limited by the point detectionapability of a SFM tip as given by the diameter of itspex. At present, long tips grown in a FESEM fromontaminations of the vacuum chamber (EBD tips)re the most suitable tips for imaging steep struc-ures. The batch of Hart Probe tips we used inhromosomal imaging had lengths of about 1.5 µmith a tip diameter of roughly 15–20 nm. At steep

idewalls the contact between tip and sample is notestricted to the very top of the tip but also involvests face. From inspection of SFM images of meta-hase chromosomes (e.g., in Fig. 1D) there was anbvious structural dilation of ,50 nm at side wallshile surmounting a mean step height of about 0.6m from the glass surface to the chromosome top.ssuming a conical tip shape, this is consistent withn apex angle of about 5°, which is an excellent valueor these EBD tips and should yield only a minorontribution to the volume calculation.As a disadvan-age, imaging in tapping mode with these long EBDips was not achievable due to large scan instabili-ies, probably induced by intrinsic bending motionsf the tip and amplified by the feedback electronics ofhe SFM. One reason for the sample instability ofhromosomes in liquid using sharp tips (Figs. 5A andB) is the elasticity of the nucleoprotein structure.harper tips exert a higher local pressure than bluntnd tips (same force constant and cantilever bend-ng). In a former study rehydrated chromosomesxhibited a rubber-like consistency (Fritzsche et al.,994b). Due to this cohesion of the condensed chroma-in, the SFM tip penetrates and sweeps the entirehromosome from the glass surface. Our data indi-ate that imaging intact chromosomes is a delicate

FIG. 5. SFM imaging of chromosomes in liquid prepared withetaphase plate immersed in water and imaged with a pyrami

canning. (B) Hardware zoom of prometaphase chromosomes withrometaphase plate. Si3N4 tip. Inset frame size is 33 3 33 µm2.rrows), i.e., apparent structural broadening by the tip–sample con liquid. Tapping mode. Ultralever. (C) Overview of a selected surs decreased probably due to elastic contributions of the residuehromatin by triggering the tip load. As indicated by the arrow, permanent contact mode prior imaging in tapping mode at minim

n C.

ompromise between structural resolution and sam-le stability.

tructural Hierarchies in ChromosomeOrganization

Chromosomes are formed from chromatin in itsost condensed state, which is abundant in equal

mounts of DNA, histones, and nonhistone proteins.he amount of DNA in the diploid genome of barley

s Nbp 5 10.7 3 109 bp (Bennet and Smith, 1976,991). The chromosome structure is dominated byhe nucleosome (8 histones 1 140 bp) particles, each5.5 nm in width (a-axis) and ,11 nm in diameter

b-axis) and linked by ,60 bp (nli) DNA on averageRichmond et al., 1984; Kornberg and Klug, 1981).he nucleosome is the repeating unit of the elemen-

ary fibril (nucleosomal chain) and is responsible foracking and organizing the DNA in the cell nucleus.he number of nucleosomes per barley genome isnuc 5 5.35 3 107 (Nbp/200). Thus for a chain of nu-

leosomes with their short (a) axis oriented eitherarallel or normal to the surface, the contour lengthf the barley genome is within the range of.3(Nnuca) 2 0.6(Nnucb). That is, for the infinitelyispersed chromatin, i.e., a surface (mono)layer ofucleosomes similar to the chromatin appearanceresented by Fritzsche et al. (1994a; Fig. 1), a surfacerea within ,3000 µm2 (a b Nnuc) and ,5000 µm2 (pb/2)2 Nnuc) should be covered by nucleosomes. Withhis approach the contributions of the linker DNAnd nonhistone proteins were ignored. In Fig. 1A thehromatin covered surface area is ,490 µm2, whichs only 1/7 to 1/10 the area occupied by a nucleosomalmono)layer. That is, the interphase chromatin inig. 1A is not fully dispersed on the level of theucleosomal chain, which is probably due to incom-lete spreading during sample preparation and/orue to the chromatin appearance in a state of partialondensation.An interesting question is the biological implica-

ion of the absolute volume values calculated fromhe topography of the CPD samples (Fig. 1G). Assum-ng a cylindrical symmetry of the DNA strand withadius r 5 1 nm and with a rise per basepair for theanonical B-form h 5 0.34 nm, the total DNA volumep r2 h Nbp) is 11.4 µm3 for the diploid (mitotic) set.

y intermediate drying step during drop sample preparation. (A)aped Si3N4 tip. The arrow points to sample instabilities duringegion indicated by the square in the inset. (Inset) Overview of theM image exhibits characteristic tip artifacts at steep edges (seeion. (C–F) Metaphase chromosomes after proteinase K treatmentea. (D) Hardware zoom of a region in C. The topographic contrastosome structure. (E) Imaging and manipulation of the hydratedthe residue chromatin was removed by the tip during imaging inad. (F) Selective removal of the structure indicated by the errors

out andal shin the rThe SFnvolutface archromart ofum lo

TNtnictoF(nitcs1atc1ss,prctsdsciossccmioiphimilibmrhadCa

wobeicdepttdbwsacrtpdcp

fMG

A

A

B

B

B

B

C

D

D

D

28 SCHAPER ET AL.

he total volume of the nucleosome core (p (b/2)2 anuc) is 28.0 µm3 and it is 3.4 µm3 (p r2 h nli Nnuc) for

he linker DNA. Including an equal amount ofonhistone proteins (assuming the nucleosome core

s 20.0 µm3) the total dry volume of a densely packedhromosome plate should be ,51 µm3, which is inhe range of 60–70% of the volume extracted fromur experimental data on the CPD specimen. FromESEM we know that the chromatin appears porous

due to internal cavities) during mitosis, most promi-ently in prometaphase (Martin et al., 1996). Accord-

ng to our SFM measurements these internal cavi-ies contribute about 30–40% to the apparenthromosome volume. Inspection of the lateral dimen-ions of the dry and hydrated chromosomes (cf. Figs.D and 5) led us to conclude that the glass surfacerea covered by the chromosomes differed no morehan 10–20%, although the height of the hydratedhromosomes was enlarged by a factor of ,2 (Table). That is, the hydrated chromosome plate volumehould be in the range of 150–200 µm3. In compari-on, the radius (rn) of the barley nucleus is typically5 µm, corresponding to a volume of ,520 µm3 (4/3rn

3), which is about 2.5–3.5 times the volumeequired for harvesting the hydrated diploid barleyhromatin. According to the present knowledge ofhe chromosome architecture it is believed that aister chromatid is organized by a series of loopedomains of a single fiber of densely packed nucleo-omes. Decondensation of chromosomes either byitrate buffer or by proteinase K treatment as shownn Figs. 4 and 5C–5F, respectively, led to eliminationf the structural integrity (unfolding) of the chromo-ome. After incubation with citrate buffer, fibrousubstructures became obvious; they are believed toonstitute intermediate structures during chromatinondensation in nearly all of the chromatin packingodels (discussed in Cook, 1995). The citrate-

nduced decondensation process was on a time scalef minutes, and the amount of chromatin dispersedn the halo varied from sample to sample. Mostrobably, the latter observation is related to sampleistory. In this context, one must keep in mind that

n contrast to the preparation of the ‘‘snapshots’’ ofitosis (Fig. 1) the chromosome decondensation is

nduced under the constraint of the surface immobi-ization. In the initial step of decondensation, thentramolecular interactions are decreased followedy chromatin dispersion in the telomeric and centro-eric regions (Wanner and Formanek, unpublished

esults). Such a behavior correlates with a structuralierarchy of chromatin organization. Since we wereble to prepare chromatin of different levels ofecondensation, we conclude that dehydration andPD influence only the appearance of the dissociated

mount of chromatin, i.e., the chromatin halo.

It is not clear whether the chromatin halo in Fig. 4as generated by diffusional processes (driven bysmotic pressure) followed by adsorption or inducedy the interfacial forces during fluid exchange. How-ver, the partially disrupted chromatin topographyn Fig. 4 indicates the importance of an appropriateontrol of the kinetics and of the binding affinitiesuring sample preparation. Our results on the influ-nce of the CPD, hydration, citrate buffer, androteinase K treatment on chromosome manifesta-ion demonstrate the importance of adequate chroma-in preparation and of the necessity of combiningifferent microscopic techniques in revealing theiofunctionality of these structures. In this context,e have shown that the drop method for chromatin

preading provides specimens suitable for FESEMnd for SFM imaging in air and in liquid. Morphologi-al features of the isolated mitotic chromatin areetained, enabling the investigation and manipula-ion of its structure. Further refinement of thereparation technique should provide specimens forirect in vitro SFM observations of the structuralhanges induced by chemical and/or enzymatic com-ounds under physiological conditions.

We thank W. Fritzsche for valuable discussions and S. Steineror excellent technical assistance. This work was supported by the

ax Planck Society and the German Research Council (DFGrants Jo 105/9 and WA 603/2) to T.M.J. and G.W.

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