3 Electron Microscopy of Biomolecules - Wiley-VCH...3 Electron Microscopy of Biomolecules 109 Tab. 1...

103

3Electron Microscopy ofBiomolecules

Claus-Thomas Bock1, Susanne Franz2, Hanswalter Zentgraf 2,and John Sommerville3

1Department of Molecular Pathology, Institute of Pathology,University of T

..ubingen, Germany

2German Cancer Research Center, Applied Tumor Virology,Electronmicroscopy, Heidelberg, Germany3School of Biology, University of St. Andrews, St. Andrews, United Kingdom

1 Principles 105

2 Techniques 1062.1 General 1062.2 Nucleic Acids 1062.3 Chromatin 1092.4 Proteins 1132.5 Macromolecular Assemblies 117

3 Applications 1193.1 Use of Antibodies 1193.2 Detection of Radiolabeled Molecules 1213.3 Electron Spectroscopic Imaging (ESI) 1233.4 Image Reconstruction 124

Bibliography 124Books and Reviews 124Primary Literature 124DNA Spreading 124Long DNA Molecules 124DNA–DNA Heteroduplex 124DNA–RNA Heteroduplex 125Small Chromatin Units 125Large Chromatin Units (transcriptionally inactive chromatin) 125Large Chromatin Units (transcriptionally active chromatin) 126

Proteins. Edited by Robert A. Meyers.Copyright 2007 Wiley-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 978-3-527-31608-3

104 3 Electron Microscopy of Biomolecules

Negative Staining 126Immunolabelling of Spread Preparations 126Immunoelectron microscopy (cell biology and viruses) 127Image Reconstruction 127Electron Spectroscopic Imaging (ESI) 128

Keywords

Cryoelectron MicroscopyImaging of unfixed, unstained biomolecules in a hydrated state after rapid freezing tolow temperature (−160 ◦C).

Heavy-metal ShadowingEvaporation of a film of heavy metal (e.g. platinum–palladium or tungsten–tantalum)onto a dehydrated preparation. The deposit of metal particles around the biomoleculesimproves contrast and gives a shadowed, three-dimensional appearance.

High-resolution Autoradiography (EM ARG)Detection of radiolabeled molecules by coating a stained or shadowed preparation witha film of photographic emulsion. Radioactive emissions hit silver halide crystals in theemulsion, which can be developed into silver grains.

Immunoelectron MicroscopyThe application of antibodies to map specific sites on biomolecules. The antibodymolecules may be visible after shadowing or negative staining, although detection canbe improved by binding of a secondary antibody linked to an electron-dense tag (e.g.ferritin or colloidal gold particles).

Miller SpreadDeposition of dispersed chromatin onto a carbon-coated grid by centrifugation througha denser phase containing sucrose and formaldehyde.

Negative StainingInstead of staining the biomolecules themselves (positive staining), a solution ofheavy-metal salt (e.g. uranyl acetate or sodium phosphotungstic acid) is deposited inthe hydrated spaces around and within the molecules.

Replica CastingAdsorption of biomolecules to a mica surface followed by heavy-metal shadowing andcoating of the preparation with a film of carbon. The carbon–metal replica (minus thebiomolecules) is then removed from the mica and is mounted on an electronmicroscope grid for viewing.

Support FilmA thin film of plastic or carbon that is attached to an EM grid and provides a substrateonto which biomolecules can be adsorbed.

3 Electron Microscopy of Biomolecules 105

� Electron microscopy (EM) is a method appropriate for examining details of thesizes and shapes of biological macromolecules and is particularly useful in studyingisolated or reconstituted macromolecular assemblies. Small amounts of material(often <1 µg) can be used and prepared in a state suitable for viewing in as short atime as several minutes. The basic procedure involves adsorption of the biomoleculesonto a support film, followed by staining or shadowing of the preparation and viewingof the dehydrated molecules in vacuo in an electron beam. A resolution of 1 to 2 nmis routine with conventional microscopes. The basic procedure can be adapted togive information about sites of specific epitopes, location of newly synthesizedcomponents, internal structures, and atomic composition.

Structures most suitable for EM analysis are nucleoprotein complexes, includingribosomes, spliceosomes, nucleosomes, and virus particles. DNA and RNAmolecules are also suitable, and their lengths can be directly related to the number ofbase pairs or nucleotide residues determined biochemically. In general, it is difficultto visualize small proteins (<50 000 Da), but good detail can be obtained if they areisolated as multimeric complexes or if they can be induced to form filaments orcrystalline arrays.

A wide range of applications are available using EM techniques, includingvirus identification, mapping of hybridized regions in heteroduplexes, detailingof macromolecular interaction, and analysis of the organization of molecularcomponents in replication, transcription, splicing, and translation complexes.

1Principles

The transmission electron microscope(TEM) consists of a metal column fromwhich air is evacuated and through which alinear beam of electrons is accelerated andfocused by electromagnetic lenses. Thebiomolecules are adsorbed onto a supportfilm, stained and dehydrated (or frozenin an aqueous film on the grid), and in-troduced into the electron beam throughan air lock. Whereas some of the elec-trons collide with atoms in the specimen,lose energy, and are scattered, the remain-ing electrons pass through the preparationand are focused to form an image on aphosphorescent screen (for direct view-ing) or on a photographic plate (for laterexamination). Under ideal conditions, aresolution of 0.1 to 0.2 nm can be obtained;

however, limitations are imposed by thenaturally low masses of atoms (primarilyhydrogen, carbon, and oxygen) containedin biomolecules, by distortions and arti-facts created during sample preparation,and by radiation damage. Techniques aredesigned with the following objectives:

1. To minimize distortion by immobiliza-tion of the molecules, in their nativestate, onto an appropriate support.

2. To stabilize molecular complexes bysuitable chemical fixation.

3. To improve contrast by staining thepreparation with heavy-metal salt or byshadowing with heavy metal.

Irrespective of the method adopted, dataderived from the electron microscopicexamination of biomolecules should be


entirely consistent with the known bio-chemical and biophysical properties ofthe particles. Whenever possible, appar-ent sizes should be checked by indepen-dent measurement of sedimentation rate,electrophoretic mobility, or gel filtrationelution. Also, features revealed by EM ofisolated molecules should be comparedwith observations made on them in situ,by EM of cell or tissue sections.

2Techniques

2.1General

Similar principles of sample preparation(Fig. 1) apply for nucleic acids, proteins,and nucleoprotein complexes, since mostof these are less than 20 nm thick and canbe adsorbed directly from solution ontothe support matrix. The concentration ofmolecules must be high enough to permitseveral examples to be viewed together inone field. The efficiency of uptake ontothe support is not always predictable,and a range of initial concentrationsshould be tried. It is important that

the biomolecules be held in a solutionknown to maintain the proper structuralfeatures directly prior to applying to thesupport.

The grid consists of a fine mesh-work, usually of copper, which mustbe coated with a thin support film ofplastic (e.g. collodion, parlodion) or car-bon. Carbon films are preferable be-cause they are thin (down to 2 nm)and contribute little to the image. How-ever, they are frequently hydrophobic andshould be subjected to ionizing gases(by ‘‘glow discharging’’) or should betreated chemically (e.g. with Alcian blue8GX) to render them hydrophilic be-fore use.

2.2Nucleic Acids

Nucleic acids are used at a concentra-tion of 1 to 5 µg/µL, and as little as0.1 µg is required; detailed procedures forworking with these molecules may befound in the literature. The moleculescan be native DNA or RNA, cloned DNA,single-stranded or double-stranded forms,partially denatured duplexes, or denaturedand hybridized structures (Figs. 2–8). A

Preparecoated grids

Prepareaqueoussample

Make hydrophilic(carbon grids)

(Fix)Adsorb

(spread, spray diffuse or centrifuge)

Drain

(Fix)

Stain

Dehydrate

(Metal shadow)

View

Contrast

Fig. 1 Flow diagram of the basic procedure. Steps in parentheses are notalways used.


key step in their preparation for EM is thespreading out of the molecules so that theycan be adsorbed in an extended, nonaggre-gated form onto the support film. The mostsuccessful method of spreading is that de-vised originally by Kleinschmidt and Zahn(Table 1). The negatively charged nucleicacid is mixed with a basic protein (usuallycytochrome c) in a solution appropriatefor maintaining the required conforma-tion. This spreading mix, or hyperphase,is spread, via a glass ramp, onto the surfaceof a second solution, the hypophase, anda molecular monolayer of nucleic acid andprotein is formed at the liquid–air inter-face. Alternatively, the molecular mono-layer can be formed by diffusion to thesurface of the spreading mix contained ina small vessel or even in a droplet sittingon a hydrophobic surface (Table 2). The

nucleic acid–protein complex is adsorbedthrough brief contact with the supportfilm, stained with ethanolic uranyl ac-etate, rinsed in ethanol, and air-dried.Protein-free spreading is applied in specialcircumstances, for instance, to visualizevery large DNA molecules that have beenstabilized by polyamines (Table 3, Fig. 5).To improve contrast, spread preparationsare rotary-shadowing, usually with plat-inum–palladium, at an angle of 6 to 9◦from the plane of the metal vapor. Itshould be noted that although metal shad-owing greatly exaggerates the thickness ofthe molecules, double-stranded and single-stranded regions can still be differentiated(Figs. 6 and 7).

Cytochrome c should not be usedin spreading solutions when experimen-tally bound proteins are being studied

Fig. 2 (a) Simian virus 40 (SV40) DNAspread in the presence of cytochrome cdown a glass slide and across a waterhypophase. (b) Xenopus laevis ribosomalDNA–derived plasmid DNA spread bythe cytochrome c microdiffusion droplettechnique. The DNA–protein films werepicked up on parlodion-coated (a) andcarbon-coated (b) copper grids. The useof carbon-coated grids yields asignificantly higher proportion ofsupercoiled molecules and results in asmoother background than is the casefor parlodion-coated grids. However, theattachment of molecules to the grid issignificantly higher with parlodion. Forcontrast enhancement, the specimenswere rotary-shadowed withplatinum/palladium (80 : 20) at an angleof 8◦. Note that an additional, very brief,unidirectional shadowing in (a) leads tofurther contrast enhancement. The barsrepresent 1 µm.

(a)

(b)


Fig. 3 Herpes simplex virus (HSV)DNA as revealed by the cytochrome cmicrodiffusion droplet technique,picked up on a carbon-coated grid andshadowed as described in Fig. 2a.Because shearing and stretching forcesare minimal when the droplet techniqueis used, it is possible to spread thisextremely large viral genome(150–160 kbp) to its full length.Bacteriophage PM2 circular DNA servesas size markers. The ends of the HSVgenome are denoted by arrowheads. Barrepresents 1 µm.

Fig. 4 The ‘‘unraveling’’ of the HSVgenome out of the viral core as shownby cytochrome c spreading. Completeintact virus particles (see Fig. 17d) werebriefly lysed in water and immediatelyspread onto a water hypophase. Thewhite dotlike structure anchoring thespread DNA represents the unraveledpart of the genome, surrounded by fuzzymaterial, most probably remnants of theviral core. Bar represents 1 µm.

because it obscures fine details of nu-cleic acid structure. Thus, to permitdetection of proteins bound to specificregions of nucleic acid molecules, otherprotein-free spreading methods have been

devised (Fig. 8e). These methods em-ploy low–molecular weight inorganic sub-stances (e.g. benzyldimethylalkylammo-nium chloride or ethidium bromide) inplace of cytochrome c. In working with


Tab. 1 Sequence of steps for spreading nucleic acids.

1. Mix nucleic acid,a cytochrome c, and buffer.b

2. Pour hypophase solutionc into a spreading trough.3. Insert a glass slide in the trough with one end resting on the rim.4. Apply spreading solution on the glass slide.5. Wait until solution has run down the ramp and spread out.6. Touch the film side of a gridd to the surface of the hypophase.7. Stain the grid.e

8. Dehydrate the specimen.9. Rotary shadow for contrast enhancement.f

aThis can be dsDNA (Figs. 2a and 4), ssDNA (Fig. 6a), RNA (Fig. 6c), or heteroduplex molecules(Fig. 7).bSpreading of ssDNA, RNA, or heteroduplex molecules needs denaturing agents, such as formamideand/or urea (Figs. 6 and 7).cUsually, water or ammonium acetate is used; for highly denaturing conditions, formamide canbe included.dNormally, carbon- or parlodion-coated grids are used (Fig. 2).eEthanolic uranyl acetate is widely used.f Usually, platinum/palladium (80 : 20) is used for rotary shadowing at an angle of 6 to 9◦; specimenscan also be viewed without rotary shadowing (Fig. 8c).Note: These preparation steps are also used with slight modifications for protein-free spreadingprocedures (e.g. in the presence of protamines, Fig. 5, or benzyldimethylalkylammonium chloride,Fig. 8e).

Tab. 2 Sequence of steps for spreading nucleic acids: microdiffusion droplet technique.

1. Mix nucleic acid, cytochrome c, and buffer.a

2. Put a small drop of the solution on a hydrophobic surface.3. Leave for 10 to 30 min for diffusion.4. Pick up nucleic acid–protein film by allowing the gridb to touch the surface of the drop.5. Stain, dehydrate, and shadow.

aThe procedure yields best results with double-stranded nucleic acids (Figs. 2b, 3, and 8a–d). Forspreading single-stranded nucleic acids, denaturing agents, such as formamide and/or urea, have tobe added to the solution.bCarbon- or parlodion-coated grids may be used (Fig. 2).

protein molecules bound to nucleic acids,care must be taken to stabilize the com-plexes before spreading by cross-linkingthe molecules, in solution, with glu-taraldehyde (Fig. 8) or formaldehyde (forchromatin, see Section 2.3). Good detailof the association can be obtained bymaking a carbon–metal replica of themolecules bound to the surface of freshlycleaved mica. The use of platinum–carbon

or tungsten for shadowing reveals finerdetail.

2.3Chromatin

Chromatin spreading techniques aremostly adapted from the procedure de-vised by Miller and coworkers (Table 4).Large chromatin units are obtained by


Tab. 3 Sequence of steps for protein-free spreading of long DNA molecules.a

1. Dilute DNA (0.25–1.6 Mbp) to 4 to 10 ng/µL in microinjection buffer.b

2. Apply a 10 µL samplec to a freshly glow-discharged carbon-coated grid.3. Allow DNA to adsorb for 1 min and remove excess liquid by touching the drop with the edge of a

filter paper.4. Immediately add a 20 µL drop of 2% (w/v) aqueous uranyl acetate solution and leave to stain for

20 s.5. Remove staining solution by touching with the edge of the filter paper and air-dry grid.6. Rotary shadowd and view.

aAppropriate for chromosomal lengths of yeast DNA and artificial chromosomes to check molecularintegrity prior to microinjection (see Fig. 6).b10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA (pH 8), 100 mM NaCl, 30 mM spermine, 70 mMspermidine.cTransfer of DNA can be achieved without shearing by using a plastic pipette tip with the endcut off.dFor instance, platinum : palladium (80 : 20) at an angle of 5◦.

Tab. 4 Sequence of steps for spreading large chromatin units: Miller spreading.

1. Incubate cells, isolated nuclei, chromosomes, or chromatin under low ionic strengthconditions for chromatin dispersal.

2. Keep on ice for 15 to 30 min.3. Glow-discharge carbon-coated grids.4. Fill the well of the centrifugation chamber with a sucrose solution containing

formaldehyde.5. Insert the carbon-coated grid, which is now hydrophilic.6. Layer a small volume of chromatin solution on top of the sucrose cushion.7. Centrifuge the chamber (speed and time depend on the equipment used).8. Remove grid and dry in Photoflo detergent.9. Stain with phosphotungstic acid and dehydrate.

10. Rotary shadow.

Note: For details see Figs. 9, 10, and 11.

Fig. 5 Full-length chromosomal DNAmolecules of approximately 1 Mbisolated by pulse-field gelelectrophoresis from Saccharomycescerevisiae strain AB1380 and spreadfrom a buffer containing polyaminesand sodium chloride (see Table 3). Thelong DNA fibres may be stabilized by acoating of polyamines in the presence ofsodium chloride and appear to gatherinto compact bundles. The two ends ofa single molecule are indicated byarrows. Bar represents 0.5 µm.


(a) (b) (c)

Fig. 6 (a) Double-stranded and single-strandedDNA of bacteriophage fd spread in 40%formamide, 1 mM EDTA, 10 mM Tris-HCl (pH8.5) onto a water hypophase. The cytochromec–DNA film was picked up on a parlodion-coatedgrid and was shadowed as described in Fig. 2b.The double-stranded DNA molecules are widerwith smoother contours than the single-strandedmolecules. (b) Replicative intermediate ofplasmid pA2Y1, derived from a parvovirus, AAV(adeno-associated virus), spread by themicrodiffusion technique in the presence of 10%

formamide (parlodion-coated grids, rotaryshadowing). The replication eye is denoted by anarrowhead. For contrast enhancement, (a) and(b) are printed in reverse contrast. (c) Tobaccomosaic virus (TMV) RNA was diluted in aspreading solution made from 4 M ureadissolved in ‘‘pure’’ formamide, 1 mM EDTA,and 10 mM Tris-HCl, (pH 8.5), heated to 80 ◦C(5 min), and spread on a 60 ◦C water hypophase,picked up on parlodion-coated grids, andshadowed. The bars represent 1 µm in (a) and0.5 µm in (b) and (c).

Tab. 5 Sequence of steps for spreading small chromatin units.

Part A1. Dilute chromatin for dispersal under conditions of low ionic strength.2. Place a drop of chromatin solution on a hydrophobic surface.3. Place a glow-discharged carbon grid on top of the drop.4. After 15 min, remove the grid and place it on top of a drop of double-distilled water.5. After 10 min, transfer it to a second drop of water.6. After 10 min, remove the grid; negative-stain and air-dry the preparation.7. Rotary shadow for contrast enhancement (see Figs. 12a, b, and d)

Part B1. Hold a glow-discharged, carbon-coated grid with forceps.2. Apply a drop of chromatin solution.3. Allow the chromatin to adsorb onto the film for 1 to 2 min.4. Stain and air-dry (alternatively shadow) (see Fig. 12c).

lysing cells or nuclei in a low saltconcentration, high pH buffer (often re-ferred to as ‘‘pH 9 water’’). The chromatinis left to disperse and is then centrifugedat low speed, through a denser solu-tion containing fixative (formaldehyde),onto the surface of a carbon-coated grid.

The grid is dipped into a solution ofwetting agent (Kodak Photoflo 200) and air-dried. The preparations can be positivelystained (with ethanolic phosphotungsticacid), negatively stained (with aqueousuranyl acetate), or metal-shadowed. Chro-matin spread from avian erythrocytes


shows the typical nucleosomal configu-ration of dispersed chromatin (Fig. 9).The features of actively transcribing chro-matin can be seen in nucleolar (pre-rRNA)units (Fig. 10). In contrast, nonnucleo-lar transcription units normally containmore widely spaced nascent transcripts(Fig. 11b).

For small chromatin units, such aschromatin fragments and viral chromatin

(minichromosomes), high-speed centrifu-gation is required to deposit the chromatinonto the coated grid. Alternatively, smallchromatin units can be adsorbed directlyby floating the grid, carbon film down,on the surface of a droplet containingthe sample (Table 5). The nucleosomalconfiguration of simian virus 40 chro-matin (the SV40 minichromosome) isshown in Figs. 12a to c. Supranucleosomal

Cap site

3′ end of last exon

(a)

a′

3′

5′

12

7

8

6

543

B

A

I

H

ED

F

C

G

(b)

b′


particles – for instance, those obtained bybrief nuclease digestion of avian ery-throcyte chromatin (Fig. 12d) – are alsoprepared by direct adsorption.

2.4Proteins

The most commonly used method forcontrast enhancement of proteins is neg-ative staining (Table 6). This methodis extremely rapid and involves dryingof an aqueous solution of heavy-metalsalt (e.g. uranyl acetate) with the pro-tein molecules on the coated grid. Themetal salt occupies hydrated regions inand around the biomolecules and, upon

drying, forms a dense cast. Since thebiomolecules themselves are not stained,but the background is stained, a neg-ative image is created. This techniquehas been used extensively for examin-ing protein filaments. Negatively stainedintermediate filaments, in this example,glial fibrillary acidic protein (GFAP), areshown in Fig. 13a. Details of the arrange-ment of individual collagen moleculeswithin the collagen fibril can be ob-tained from preparations like that seenin Fig. 14.

An alternative to the adsorption of pro-teins from droplets is to spray them, inaerosols, onto the surface of carbon-coatedgrids. The spraying is most easily achieved

Tab. 6 Sequence of steps for negative staining.

1. Hold a coated grida with forceps.2. Apply a drop containing biological material to the grid.3. After 10 to 60 s, drain off excess liquid.4. Apply a drop of negative stainb for 10 to 60 s.c

5. Remove liquid and air-dry the preparation.

aCarbon, parlodion, or sandwich coats (made from Formvar and carbon) may be used. Glowdischarge increases the amount of biological material adsorbed onto the film; plastic films, however,are destroyed by glow discharge.bUranyl acetate and phosphotungstic acid are widely used for negative staining.cAlternatively, the grid can be transferred through several drops of stain.Note: For details on the various procedures, see Figs. 13–18.

Fig. 7 (a) A DNA : DNA heteroduplex formed between cloned mouse tyrosine aminotransferase(TAT) and rat TAT. Heteroduplexes were formed after denaturation in 0.1 M NaOH, 20 mM EDTA at20 ◦C for 10 min and renaturation in the presence of 50% formamide, 20 mM Tris-HCl (pH 7.2) at20 ◦C for 90 min. Heteroduplexes were then prepared from a spreading solution containing 4 M ureadissolved in ‘‘pure’’ formamide, 1 mM EDTA, 10 mM Tris-HCl, (pH 8.5) on water hypophase,followed by rotary shadowing. (a′) The interpretive drawing of (a): the positions of the cap and of the3′ end of the last exon are indicated. (b) A heteroduplex formed between poly(A+) RNA from bovinemuscle epidermis and a genomic clone that contains the gene coding for epidermal bovine keratin(KBla). Heteroduplexes were formed in 70% formamide, 0.3 M NaCl, 1 mM EDTA, 20 mM Tris-HCl(pH 8.0). The sample was kept for 17 h at 62 ◦C, followed by incubation at 64 ◦C for 2 to 4 h. Thehybrids were prepared for electron microscopy as described in (a). (b′) The interpretative drawing of(b): DNA is represented by a continuous line, RNA by an interrupted line; the 5′ end of mRNA isidentified by a change in molecular diameter and the 3′ end by a projecting [i.e. nonhybridized,poly(A) tail]. Exons are denoted by capital letters, introns by arabic numerals. For contrastenhancement, (a) and (b) have been printed as negatives. Bars represent 100 nm.


(a)

(c)

(e)

(b)

(d)

Fig. 8 (a, b) Binding of monoclonal Z-DNA antibodies to DNA frombovine papillomavirus (BPV-1). Antibodies against left-handed Z-DNAwere incubated and cross-linked with 0.1% glutaraldehyde (2 h) to thepML2d-BPV-1 plasmid DNA (a). To determine the position of thebinding site, the DNA was digested with the single-cutting restrictionenzyme Xba I (b). Arrows indicate the position of Z-DNA antibodieson the plasmids. (c, d) Localization of large-tumor antigen (T antigen)on replicative intermediates of SV40 DNA. The molecules wereimmunostained with either ferritin-labeled protein A (c) orferritin-labeled goat antimouse antibodies (d) after cross-linking with0.1% glutaraldehyde (20 min, 37 ◦C). To localize T antigen on thereplicated section, the complex was cleaved with the single-cuttingenzyme Bgl I. Arrows indicate the ferritin-labeled T antigen on DNA.These complexes (a–d) were prepared for electron microscopyaccording to the cytochrome c droplet diffusion technique. Thespecimens were rotary-shadowed, except the one shown in (c), whichis only stained by ethanolic uranyl acetate. (e) DNA-relaxing enzymemolecules linked to single-stranded SV40 DNA (arrow). After enzymereaction, the complexes were spread from a solution containing0.25 M NaOH, indicating that the protein dot is stable in alkali and istherefore linked via a covalent bond to the end of the single-strandedDNA. Enzyme–DNA complexes were prepared with a protein-freespreading procedure using benzyldimethylalkylammonium chloride(BAC) and were shadowed. Bars represent 100 nm.


Fig. 9 Appearance of the nucleosomalchromatin configuration of chickenerythrocytes lysed and swollen in0.5 mM sodium borate buffer (pH 8.8)for 5 min, spread by the Millertechnique, positively stained, andmetal-shadowed. The dispersedchromatin shows the characteristicnucleosomal ‘‘beads-on-a-string’’organization. Bar represents 1 µm.

Fig. 10 Organization of nucleolarchromatin from oocytes of the newtPleurodeles waltlii, after spreading usingthe Miller technique, positive staining,and rotary shadowing. The transcribedchromatin segments are densely packedwith RNA polymerases and nascentribonucleoprotein (RNP) fibrils. Thetandemly arranged active pre-rRNAgenes are interspersed by the spacerregions. The axis of the spacer isrelatively thin and does not shownucleosome-sized particles. Barrepresents 1 µm.

by touching the tip of a glass capillarytube, containing both sample and stain,into a stream of nitrogen. Elongate, orrod-shaped, proteins can be mixed withglycerol (and no stain) and sprayed onto

the surface of a piece of freshly cleavedmica. The deposit is then shadowed finelyand carbon-coated to produce a replica castof the protein molecules. (Details are pro-vided in the literature.) Unidirectionally


(a)

(b)

Fig. 11 High-resolutionautoradiography of chromatin spreadfrom mouse P815 cells after labeling for5 min with [3H]uridine. (a) Radioactivity(recorded as dense silver grains)associated with densely transcribingregions showing the gradient-likefeatures of pre-rRNA transcription units(cf. Fig. 10). The background consists ofmany strands of nucleosomalchromatin. (b) Radioactivity associatedwith individual RNP fibrils characteristicof those found in nonnucleolartranscription units. In sparselytranscribing units, nucleosomes can beseen on the chromatin strand betweenthe nascent transcripts. Bothpreparations were stained withphosphotungstic acid, rotary-shadowedwith platinum, and exposed with IlfordL4 emulsion. Bars represent 0.5 µm.(Courtesy of S. Fakan, with permissionfrom Springer-Verlag.)

Fig. 12 Small chromatin units such as SV40 chromatin complexes (a–c) or supranucleosomalparticles from chicken erythrocyte chromatin (d) are prepared by direct adsorption to freshlyglow-discharged, carbon-coated grids. The chromatin is allowed to adsorb onto the film for 1 to2 min, stained with 2% uranyl acetate (c), and rotary-shadowed (a, b, d). Under spreading conditionsinvolving buffers of low ionic strength (2 mM EDTA, 1 mM Tris-HCl, pH 8.4), the SV40 nucleoproteincomplexes (‘‘minichromosomes’’) show the typical ‘‘beads-on-a-string’’ morphology of the circularnucleosomal chain (a, b). The foreshortening of DNA in the SV40 ‘‘minichromosomes’’ due tonucleosomal organization is demonstrated by comparison with the viral DNA, which is included inthe preparation (b). This packaging of the SV40 genome, at first-order level into nucleosomes, resultsin a foreshortening ratio of about 5.5 : 1. (d) At the second level of packaging, chromatin is organizedinto supranucleosomal structures. Fractions of supranucleosomal granular subunits, obtained bybrief digestion of chromatin with micrococcal nuclease at physiological salt concentration, can bespread after fixation for 15 min at 4 ◦C with glutaraldehyde (final concentration 0.2%). (e)Nonnucleosomal forms of chromatin beside nucleosomal chains are observed in Miller spreadpreparations of African green monkey kidney cells (RC37) after infection with herpes simplex virus(HSV). The ‘‘bubblelike’’ configurations, thickly coated with a single-strand DNA-binding protein,alternate with unbranched thin intercepts (staining and shadowing as in Figs. 9 and 10). Barsrepresent 500 nm in (a), (d), and (e), 200 nm in (b), and 50 nm in (c).


shadowed molecules, prepared by thespray/replica technique and representingearly stages in the assembly of GFAP fila-ments, are shown in Fig. 13b.

2.5Macromolecular Assemblies

Multimeric enzyme complexes, ribo-somes, viruses, and other particles can be

treated in ways similar to those describedfor protein filaments or for small chro-matin units. Particles in the size range of10 to 200 nm diameter are excellent tar-gets for negative staining (Figs. 15–18),which often reveals considerable detail ofsurface structure (Fig. 17). Nevertheless,the size of the particles of dried stain(or of the metal particles after shadow-ing) limits the resolution of structural

(a)

(b)

(d)

(e)

(c)


(a)

(b)

Fig. 13 Intermediate filaments as visualized bynegative staining and partly assembled monomersas visualized by metal shadowing. (a) GFAPintermediate-sized filaments spread onto carbonfilm and negatively stained with 1% w/v uranylacetate. GFAP was prepared from spinal cord andassembled in vitro by dialysis against the followingbuffer: 10 mM Tris-HCl (pH 7.0), 1 mM MgCl2,50 mM NaCl, and 25 mM 2-mercaptoethanol. Notethat the intermediate filaments are 10 nm in widthand are long with smooth edges. (b) The assemblyof intermediate filaments can be arrested at variouspoints along the assembly pathway by choosing theappropriate buffer conditions. In this preparation,GFAP molecules containing chains of four proteinswere formed in 10 mM Tris-HCl (pH 8.5). A sampleof this was made 50% v/v in glycerol, sprayed ontofreshly cleaved mica, and unidirectionally shadowedat 10◦ with platinum. The replica was then floated onwater and picked up onto grids. The molecules ofGFAP appear as short rodlets, between 45 and65 nm long and 2 to 3 nm wide (arrows). Latexbeads demonstrate the direction of shadow andlocate the position of the droplets on the replica(arrowheads). Both micrographs are shown at thesame magnification; bar represents 100 nm.(Courtesy of R. Quinlan and A. M. Hutcheson.)

Fig. 14 A collagen fibril, negativelystained with 4% phosphotungstic acid,on a glow-discharged, carbon-coatedgrid. The staggered arrangement of thecollagen molecules in the fibril results inthe striated appearance after negativestaining. The bars represent 100 nm.

detail. A superior technique cryoelectronmicroscopy involves the rapid freezingof a thin (100 nm) aqueous film con-taining the specimen particles. Below−143 ◦C, water can be held in a vit-rified state, which resembles the liquidstate and avoids formation of ice crys-tals. In this condition, macromoleculesand macromolecular assemblies can beviewed free of artifacts from fixation,dehydration, and staining. Under-focusphase contrast is used to produce a high-resolution image.


3Applications

3.1Use of Antibodies

Antibody molecules (IgG and IgM)are large enough to be resolved inmetal-shadowed or negatively stainedpreparations. Monoclonal antibodies canbe used to map sites on nucleic acid orprotein molecules and to identify individ-ual components within macromolecularassemblies. Antibodies can also be used tobuild up denser structures around small

Fig. 15 Examples of filamentousbacteriophages and rodlike viruses. Thebacteriophage fd was negatively stainedwith either 4% phosphotungstic acid(a) or 2% uranyl acetate (b). Thetobacco mosaic viruses in (c) arestained with 4% phosphotungstic acid.All preparations were made oncarbon-coated, glow-discharged grids.The bars represent 0.5 µm.

(a)

(b)

(c)

Tab. 7 Sequence of steps for immunogold labeling: identification of individual components inmacromolecular assemblies.

1. Hold a coated grida with forceps.2. Apply a drop containing biological material to the grid.3. After 1 min, wash the grid with 20 drops of double-distilled water from a Pasteur pipette and

drain off excess liquid.b

4. Place the grid (film side down) on a dropc of the primary antibody diluted in phosphate-bufferedsaline (PBS), 1% bovine serum albumin (BSA).d

5. After 30 min, remove the grid and place it on top of a drop of PBS, 1% BSA.6. After 5 min, transfer the grid to a fresh drop of PBS, 1% BSA; repeat this washing step a

third time.7. After 5 min, transfer the grid to a drop of the gold-labeled secondary antibodye diluted in PBS,

1% BSA.8. After 30 min, place the grid on top of a drop of PBS, 1% BSA and continue as described in step 6.9. Wash the grid as described in step 3.

10. Stain the specimen with 2% aqueous uranyl acetate for 1 min and air-dry.

aThe procedure yields best results with glow-discharged carbon-coated grids.bNever drain the grids completely.cDroplets are placed on a piece of Parafilm.dPBS, 1% BSA; filtered through a 0.22 µm Millipore filter.eAlternatively, a protein A–coated colloidal gold probe may be used.


(a)

(b)

(c)

Fig. 16 Examples of tailed (a, b) andglobular (c) bacteriophages.Bacteriophage λ (a) and the globularbacteriophage fr (c) were stained with4% uranyl acetate. The bacteriophage T5(b) was stained with 2% uranyl acetate.Note the empty phage heads in (b). Allpreparations were made on freshlyglow-discharged, carbon-coated grids.The bars represent 200 nm.

proteins, which are themselves difficult toresolve. Secondary antibodies tagged withelectron-dense markers such as ferritin

(Figs. 8c and d) or colloidal gold particlesare routinely used to improve detection(Table 7). For instance, gold-conjugatedsecondary antibodies can be used to de-tect a range of epitopes in recombinantviral coat particles (Fig. 18). A quite dif-ferent application is the use of anti-biotinto detect biotinylated nucleic acid probesthat have been hybridized to complemen-tary sequences in chromatin preparationsor in situ in isolated chromosomes. In theexample shown in Fig. 19, two differentantibody-binding sites are enhanced forEM detection by addition of a commonsecondary antibody tagged with colloidalgold particles. In this way, specific genescan be detected within complex chro-matin masses.

Tab. 8 Sequence of steps for high-resolution autoradiography of biomolecules.

1. Radioactively labela living cells or subcellular fractions in vitro.2. Isolate molecules or molecular complexes and prepare by direct adsorption on the grid or by

spreading techniques.3. Stain,b dehydrate, and rotary shadow.4. Evaporate a thin layer of carbon (4–8 nm) on the specimen.c

5. Coat the specimen with a layer of photographic emulsion.6. Follow the gold latensification–Elon–ascorbic acid (GEA) procedure.d

7. Transfer to Elon–ascorbic acid developer.8. Transfer into the fixing bath.9. Wash and air-dry.

aGenerally, radioactive tritium is used.bNegative or positive staining; shadowing with platinum/palladium.cThe fine coat prevents chemical interactions between specimen and emulsion.dThe GEA development procedure increases the sensitivity and gives rise to silver grains of rathersmall size.


(a) (b)

(c) (d)

Fig. 17 Details of virus structure revealed in negatively stainedpreparations on carbon-coated, glow-discharged grids. (a) Hepatitis Bviruses (HBV) were stained with 2% uranyl acetate. (b) Poliovirusesand (c) bovine papilloma viruses (BPV) were stained with 4%phosphotungstic acid. (d) Herpes simplex virus (HSV) was stainedwith 2% uranyl formate. HBV was obtained from a patient’s serum andreveals, in addition to the mature ‘‘Dane’’ particles, globular andfilamentous forms of the viral S protein (a). Polioviruses show afine-textured surface (b), while BPVs reveal a moruloid structure, whichis due to the capsomere architecture (c). This organization is alsovisible in the HSV capsid, even when surrounded by the envelope (d).The micrographs are magnified to the same scale to give animpression of the size difference between the virus families. Barrepresents 100 nm.

3.2Detection of Radiolabeled Molecules

Although high-resolution autoradiography(EM ARG) is applied mostly to the

detection of newly synthesized (radiola-beled) components in situ at the cellularlevel, application is possible with spreadmolecules and molecular complexes. How-ever, EM ARG is limited to the use


1

2

3

Fig. 18 Detection of different epitopesin recombinant viral core particles. Thevector cassette pB–CHG–HBc (C,cytomegalovirus (CMV) promoter; H,histidine tag; G, green fluorescentprotein; HBc, core protein of hepatitis Bvirus) was transfected into HeLa cells.The recombinant core particles wereisolated by sucrose gradientcentrifugation. Epitopes in the fusionprotein were detected byimmunoelectron microscopy afteradsorption of the particles ontocarbon-coated grids. All incubation andwashing steps were carried out on thegrids. The antibodies used were(1) rabbit anti-HBc/goat antirabbit−5 nm gold; (2) mouseanti-histidine/goat antimouse −5 nmgold; and (3) rabbit anti-GFP/goatantirabbit −5 nm gold. Afterimmunoreactions, the grids werenegatively stained with uranyl acetate.Bar represents 100 nm.

of radioisotopes emitting soft β-particles,usually 3H. Molecules that have been ra-diolabeled either in vivo or in vitro areprepared for EM by the method most ap-propriate for that type of sample (Table 8).

After staining, the preparation is covered,first with a thin protective coat of car-bon and then with a layer of photographicemulsion. Ideally, the emulsion shouldconsist of a homogeneous monolayer of


Fig. 19 Whole-chromosome mount fromXenopus culture cells after in situ hybridizationwith a mixture of biotinylated DNA encodingtRNA and oocyte-specific 5S RNA. Thechromosomes are partially unfolded, and thesites of hybridization are detected usinganti-biotin and secondary antibodies tagged with

colloidal gold. The gold particles are seen todecorate DNA loops containing tRNA genes(arrows) and 5S RNA genes (arrowheads). Thebar represents 2 µm. (Courtesy of S.Narayanswami and B. A. Hamkalo, withpermission from Oxford University Press.)

the silver halide crystals. After being keptin the dark, to allow a sufficient amountof radioactive decay, the emulsion is devel-oped, leaving silver grains above the sitesof radiolabeling in the molecules. EM ARGis subject to two severe limitations:

1. The accuracy with which the devel-oped silver grain can be located tothe source of radiation in the speci-men molecule. This depends on thethickness of the preparation and thediameter of the silver halide crystal inthe emulsion and results in scatteringof grains with a half distance of about100 nm.

2. The efficiency in detecting radioactivedisintegrations. Even molecules labeledto high specific activities require expo-sure times of generally more than afew weeks.

In spite of these limitations, EM ARGhas been used successfully in study-ing sites of replication in isolated DNAmolecules and the location of active

transcription complexes in spread chro-matin (Fig. 11).

3.3Electron Spectroscopic Imaging (ESI)

By adapting the electron microscope to in-clude an imaging electron energy filter,positional information can be recoveredfrom electrons that have lost a specificand characteristic amount of energy incolliding with atoms in the specimenmolecules. This approach has been usedsuccessfully in the location of phosphorusatoms, particularly those in the phos-phodiester bonds of nucleic acids. Theresulting electron spectroscopic imaging(ESI) has been used to delineate the path ofthe sugar–phosphate backbone in nucleo-somes and the configuration of SRP–RNA(7SL RNA) within the signal recognition(ribonucleoprotein) particle. Along withrelated techniques in element mappingwithin biomolecules and their complexes,ESI is becoming increasingly sophisticatedand promises many diverse applications.


3.4Image Reconstruction

Individual molecules, under optimumconditions of spreading and staining,give weak and ill-defined images. Toimprove on the amount of structuraldetail, information from many moleculescan be combined to smooth out randomvariations between images. To do this,it is necessary to use molecules (e.g.protein filaments), which contain regular,repeating arrays of subunits. Alternatively,the biomolecules can be induced to formcrystalline arrays of regular, tightly packed,oriented units. Electron micrographs ofarrays of these types can then be usedfor image processing to produce enhancedstructural detail.

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