Sample preparation and imaging of erythrocyte cytoskeleton with the atomic force microscopy

INTRODUCTION

For atomic force microscopic studies of bio-logic membranes in air and liquid environ-ment, a novel method to covalently bondbiological cells onto a glass coverslip is intro-duced. The effectiveness of the method isdemonstrated using the spectrin network ofhuman erythrocytes as a model with quantita-tive evaluation of images.

The potential to resolve biological samplesat the molecular level by using atomic forcemicroscopy (AFM) has been slowed becauseof the difficulty of mounting them on a solidflat substrate with reliable stability (2–6).Much effort has already been aimed at estab-lishing secure mounting of biological samplesby using poly-L-lysine (7–9), 3-aminopropyl-trimethoxysilane (APS) (10,11), 3-aminopropyl-triethoxysilane (APTES) (12,13), N-5-azido-2-nitrobenzoyloxysuccinimide (ANBNOS) (1) orby growing cells directly on microscopic cover-slips (6,14–16). Although these methods havebeen used successfully for analysis of macro-

Sample Preparation and Imaging of ErythrocyteCytoskeleton with the Atomic Force Microscopy

Fei Liu,1 Joel Burgess,1 Hiroshi Mizukami,2 and Agnes Ostafin1,*

1Department of Chemical Engineering, University of Notre Dame, Notre Dame, IN; 2Department of Biological Science, Wayne State University, Detroit, MI

Abstract

A novel method for the covalent attachment of erythrocytes to glass microscope coverslips thatcan be used to image intact cells and the cytoplasmic side of the cell membrane with either solidor liquid mode atomic force microscopy (AFM) is described. The strong binding of cells to the glasssurface is achieved by the interaction of cell membrane carbohydrates to lectin, which is bound toN-5-azido-2-nitrobenzoyloxysuccinimide (ANBNOS)–coated coverslips (1). The effectiveness ofthis method is compared with the other commonly used methods of immobilizing intact erythro-cytes on glass coverslips for AFM observations. Experimental conditions of AFM imaging of bio-logic tissue are discussed, and typical topographies of the extracellular and the cytoplasmicsurfaces of the plasma membrane in the dry state and in the liquid state are presented. Comparisonof the spectrin network of cell age–separated erythrocytes has demonstrated significant loss in thenetwork order in older erythrocytes. The changes are quantitatively described using the pixelheight histogram and window size grain analysis.

Index Entries: Erythrocyte; cytoskeleton; spectrin; atomic force microscopy; biological membranes.

*Author to whom all correspondence and reprintrequests should be addressed. E-mail: [email protected]

ORIGINAL ARTICLE

© Copyright 2003 by Humana Press Inc.All rights of any nature whatsoever reserved.1085-9195/03/38/251–270/$20.00

Cell Biochemistry and Biophysics 251 Volume 38, 2003

molecules with AFM, they have been less suc-cessful for reproducible high-resolution analy-sis of cell membranes and cells. Here, wedescribe a modified method for attachment ofcell membranes onto glass coverslips via spe-cific affinity interactions between lectin andglycophorin, a major component of the junc-tional complexes of the cytoskeleton.

Glycophorin in human erythrocytes is atrans-membrane protein, possessing threedomains: an extracellular N-terminal domainwith 16 carbohydrate chains; a hydrophobicdomain, spanning the lipid bilayer; and acytoplasmic C-terminal domain with chargedand polar residues. Each human erythrocytehas approx 500,000 copies of glycophorinembedded in its plasma membrane (17).Erythroagglutinating phytohemagglutinin (E-PHA), one of the polypeptide subunits of lectinisolated from Phaseolus vulgaris (red kidneybean), binds with high affinity to the bisectedbiantennary oligosaccharide of glycophorinmolecules on human erythrocytes (18,19). If E-PHA is covalently bonded to ANBNOS, whichis already covalently bonded on the surface ofthe coverslips, erythrocytes and other cellswith glycophorins will be firmly bound on thecoverslips, and may be subjected to furtheranalysis with AFM in the air and liquid.Replacing E-PHA with another type of lectin,only those cells with the specific oligosaccha-ride will be captured on the coverslips.Therefore the lectin attaching method could beextended to the study of other cell types.

Erythrocytes bound firmly to the coverslipsmay be subsequently lysed to expose thecytoskeleton network on the cytoplasmic sideof the membrane. The erythrocyte membraneis one of the most extensively studied cellmembranes. It is supported by the erythrocytecytoskeleton, a protein meshwork that under-lies the plasma membrane and helps to retainoptimal biochemical functions of metabolicproteins, while allowing sufficient flexibilityfor cells to move through small capillaries inthe body. The currently accepted model for thestructure and composition of the erythrocytecytoskeleton has been derived from the results

of a large number of biochemical and analyticalstudies (20–22). From these studies, we knowthat the major component of the erythrocytecytoskeleton is a fibrous polypeptide calledspectrin (23). Two isoforms of spectrin, α and β,form a loosely wound helix (24,25), and twospectrin dimers are linked head-to-head toform a single tetramer (26). The tetramer hasbinding sites for short actin filaments, band 4.1,glycophorin, and several other minor proteincomponents (27–29). The arrangement of junc-tional complexes in the cytoskeleton is sug-gested by the results of high-resolutiontransmission electron microscopy (TEM) studieson expanded cytoskeleton networks supportedon carbon-coated copper grids. Each complexis interlinked to adjacent complexes by multi-ple spectrin tetramers, with five to eight spec-trin tetramers gathered around each actin core(21,22,30). However, these expanded-spectrin-based results have been questioned. Studies onunexpanded cytoskeleton reveal different net-works of 3 or 6 filaments connected to eachactin core (31,32). To further investigate thosediscrepancies, a number of researchers haveused AFM in the analysis of the erythrocytecytoskeletal structure and properties in physio-logic conditions. In most of the early work,membranes from intact erythrocytes or ghostswere absorbed onto the surface of glass cover-slips or freshly cleaved mica through electro-static interactions. In addition to cleanacid-washed glass coverslip surfaces (33,34),coverslip surfaces coated with poly-L-lysine(7,8) or APS (10,11) have been used. AFMimages of the erythrocyte cytoskeleton wereobtained mostly in air (10,33,34). Only a fewliquid-mode imaging studies of the erythrocytecytoskeleton were reported (8,11). Besides ery-throcytes, AFM analysis on other cellcytoskeleton, such as those of glial cells (2), aci-nar cells (5), fibroblasts (35,36), cardiocytes(37), and macrophages (38), have beenreported. Because cell membranes are fluidphases under physiologic conditions, the reso-lution of AFM images obtained in liquid isrestricted. Several factors influence the qualityof imaging in liquid, such as the force applied

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during scanning (2), the cell type, the complex-ity of the topography, and the composition ofthe surface (39,40). Of key importance is thecell’s adherence to the substrate (41).

In this study, E-PHA was attached to thecoverslips coated previously with ANBNOS,forming a hydrophilic surface with affinityattractions to the outer surface of erythrocyte.The effectiveness of this method was comparedwith some of the other commonly used meth-ods of immobilizing intact erythrocytes onglass coverslips for AFM observation. Theattached erythrocytes were osmotically lysedto break the cell membrane and expose thecytoplasmic surface. The exposed membranes,including those of cell age-separated erythro-cytes, were then imaged and analyzed withAFM both in air and in liquid.

METHODS

Materials

Poly-L-lysine solution (0.1%, v/v), 3-amino-propyltriethoxysilane (APTES), ANBNOS,erythroagglutinating phytohemagglutinin (E-PHA, lectin from P. vulgaris, red kidney bean),and Percoll were obtained from Sigma (St.Louis, MO). Reagent grade lactose; sodiumchloride; dibasic and monobasic potassiumphosphate; sodium carbonate; n-butylamine;1,4-dioxane; hydrogen peroxide; sulfuric acid;and acetone were obtained from FisherScientific (Fair Lawn). All chemicals were usedwithout further purification. Deionized waterat 18 MΩ (Millipore E-Pure D4641 System,Barnstead, Dubuque, IA) was used for allmanipulations.

Modification of Glass Coverslip Surface

Silica glass coverslips with diameter of 15mm (Fisher Scientific, Pittsburgh, PA) wereused as the substrate to immobilize cells (root-mean-square deviations of the surface [RMS],approx 0.45 nm) (1) rather than mica (RMSapprox 0.2–0.3 nm) (42) because of its betteroptical transparency. Figure 1 shows the chem-

Atomic Force Microscopy 253


Fig. 1. (A) Acid-washed glass coverslip sur-face is positively charged. (B) The negativelycharged peptide oxygen of poly-L-lysine elec-trostatically interacts with the glass surface,while the positively charged amine may bind tocell membrane. (C) The silane groups of APTESform covalent bonds with the glass surfaceleading to exposed amino groups. Thus, thesurface is hydrophobic around slightly alkalineto higher pH, but becomes positively charged inacidic pH. (D) ANBNOS reacts with the amineof APTES and thus the reactive azide onnitrobenzoyloxysuccinimide becomes indi-rectly, but covalently bound to the coverslipsurface. (E) Upon UV photolysis (at approx 302nm) aryl azides on ANBNOS generate reactivenitrenes, thereby producing insertions via theC-H groups of proteins, or forming bonds withnucleophilic groups in proteins. The E-PHAprotein molecule is not drawn to scale. (Figurecontinues.)


Fig. 1. (continued)

ical structures of reagents used in this studyand their reactions to glass coverslip surface.

POLY-L-LYSINE COATING

Glass coverslips were cleaned by immersingin 30% (v/v) hydrogen peroxide in concen-trated sulfuric acid for 30 min, rinsed withdeionized water, and air-dried for 1 h (43).Clean coverslips were immersed for 3 min in0.1% poly-L-lysine solution, washed withdeionized water, and air-dried. The coverslipswere stored in a vacuum-sealed desiccator(Fig. 1A,B).

3-AMINOPROPYLTRIETHOXYSILANE COATING

Following the method of Aebersold et al.(44), acid-washed glass coverslips prepared aspreviously described were immersed in APTESsolution (2% in 95% aqueous acetone, v/v) for3 min, followed by gentle pipet squirting fivetimes with acetone to remove excess unboundAPTES. Coverslips were dried at 110°C, thenstored in a vacuum-sealed desiccator (Fig. 1C).

N-5-AZIDO-2-NITROBENZOYLOXY-SUCCINIMIDE COATING

As described by Karrash et al. (1), APTES-coated coverslips were immersed in 0.1 MNa2CO3 (1 mL dioxane to 20 mL of Na2CO3solution, pH 9.0) containing 3 µmol ANBNOSfor at least 4 h. The concentration of ANBNOSwas thus at least tenfold molar excess withrespect to the amino groups of APTES on thecoverslip (approx 1 nmol NH2 groups/cm2).ANBNOS reacts with the exposed amino endof APTES and covalently binds to the coverslipsurface as shown in Fig. 1D. Excess ANBNOSwas removed by washing the coverslip withgentle pipet squirting 3 times with 1% n-buty-lamine in 0.1 M Na2CO3, 3 times with 0.1 MNa2CO3, 2 times with deionized water, and 2times with acetone. Air-dried coverslips werestored in a vacuum-sealed desiccator and han-dled in the dark.

LECTIN COATING

Ten microliters of E-PHA lectin (2 mg/mL inphosphate-buffered saline [PBS] buffer: 145mM NaCl and 5 mM NaH2PO4/Na2HPO4, pH

at 7.4) were compressed between twoANBNOS-coated glass coverslips under irradi-ation at 302 nm (8 Watt, UVP ultraviolet transil-luminator TMW-20 Upland, CA) at 10 cm fromthe light source for 3 min to bring thehydrophilic lectin in close contact with thehydrophobic ANBNOS. Completion of light-activated crosslinking was confirmed spec-trophotometrically (1). The coverslips wererinsed with PBS five times and stored in PBSand used for erythrocyte attachment within 1 h.The crosslinking between ANBNOS and lectinis via either an azide insertion reaction withCH2 groups or an azide addition reaction withNH2 groups in the protein (Fig. 1E).

Hydrophobicity of Modified GlassCoverslip Surface

The relative hydrophobicity of the glass sur-faces following the described treatments wascompared by measuring the diameter of 10-µLwater droplets deposited on the coverslipsaccording to the method of Engel (45) using aplastic ruler with gradations of 1 mm.

Preparation of Erythrocytes

Blood from healthy human volunteers wasdrawn using a venipuncture into Vacutainers(Becton Dickinson, Franklin Lakes, NJ) withethylenediamine tetraacetic acid (EDTA), andstored at 4°C. Ten milliliters of whole bloodwas centrifuged at 3564g for 15 min at 4°Cusing a SORVALL SuperT21 Superspeed cen-trifuge and SORVALL ST-H750 rotor. The yel-lowish supernatant containing plasma and thewhite fluffy coat on the pellet were discarded.The erythrocytes were resuspended andwashed 3 times in PBS with centrifugation (33).The packed cells were stored at 4°C until usedwithin 1 wk.

Separation of Light and DenseErythrocytes

Erythrocytes were separated on the basis oftheir buoyant-density using Percoll discontinu-ous-density-gradient-separation (46,47). Percollin PBS solution corresponding to densities



from 1.091 to 1.121 g/cm3 were filled into a 50-mL polyethylene centrifuge tube to form 5 dif-ferent layers with 10 mL each. Three millilitersof packed RBC were placed on the top and thetube was centrifuged 15 min at 3564g at roomtemperature. The light cells (approx 1.09g/cm3) on the top and the dense cells (approx1.11 g/cm3) on the bottom were removed fromthe column using a pipet, and the Percoll ineach portion removed by centrifuging withPBS as previously described. The packed cellswere resuspended with PBS for mounting ontothe glass coverslips.

Comparison of the Erythrocyte Adhesion Strength Among Different Coverslip Coatings

Twenty microliters of the packed erythro-cytes were resuspended in 40 mL of PBS at4°C. The acid-washed uncoated coverslips,and other coverslips coated with poly-L-lysine, APTES, ANBNOS, and lectin wereimmersed into their respective aliquots of ery-throcyte suspension and incubated for 4 h. Tocompare the strength of adhesion amongthese coatings, each erythrocyte-bound cover-slip was dip-washed in fresh PBS buffer 10times in 30 s. The number of remaining cellson the coverslip was recorded by a photo-graph taken with a CCD camera (XC-999,CCD video camera module, Sony) mountedon a light microscope (Axiovert 135 TV, Zeiss)and compared with identically prepared cov-erslips that had not undergone the washingprocess. The average number of erythrocytesin 10 regions spanning 135 µm × 102 µm inarea was used to determine the initial andretained number of erythrocytes on the cover-slips.

Lectin Binding Specificity

The adhesion strength procedure wasrepeated exactly with a lectin-coated cover-slip as described, except that before cellattachment, the coverslip was incubated in20-mM lactose solution (in PBS) for 20 min.

Lysis of the Erythrocytes and Exposure of their Cytoplasmic Sides

The erythrocytes mounted on lectin-coatedcoverslips were lysed by immersing them in ahypotonic buffer solution 5P8–10 (5 mM phos-phates at pH 8.0 with 10 mM NaCl) for 30 minat 4°C, and then washed with fresh 5P8–10 toremove the cell debris.

Atomic Force Microscopy Imaging and Analysis

The AFM topographic images of both theextracellular and the cytoplasmic side of ery-throcyte plasma membranes were recorded inboth the contact-mode and the tapping-modewith the Nanoscope III SPMTM system(Digital Instruments, CA). A scanning tube forscan widths of 15 µm was used. StandardSilicon Nitride ProbesTM NP-20 (DigitalInstruments, CA) with a tip radius of 20–60 nmand a spring constant k = 0.12 N/m were usedfor contact-mode imaging. NanoSensorsTappingModeTM Etched Silicon Probes TESP(Digital Instruments, CA) with a tip radius of5–10 nm, a spring constant k = 20–100 N/mand a resonance frequency f = 200–400 kHzwere used for tapping-mode imaging.

For imaging in the air (solid-mode), the ery-throcyte-coated coverslips were attached to 1-mm-thick steel plates with a diameter of 15mm using double-sided adhesive tape, andmounted on the magnetic AFM scanningstage. The AFM measurements were per-formed at an ambient pressure in air and atroom temperature. In the contact mode, a typ-ical contact force of approx 6 nN was applied.The scan rate was approx 1–2 Hz for scansizes from 15 to 1 µm. In the tapping-mode,the resonant frequency was near 300 kHz.Images of the extracellular surface of the ery-throcyte membrane were obtained by scan-ning similarly prepared coverslips with intactcells attached on the surface. For both cases,the erythrocyte-bound coverslips were air-dried before imaging.

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For imaging in liquid, the bottom side ofeach coverslip was dried with a piece of filterpaper and attached onto a steel plate with dou-ble-sided adhesive tape and mounted on themagnetic AFM scanning stage. The Digital™Fluid Cell (Digital Instruments, CA) was used.Standard Silicon Nitride Probes™ NP-20 wereused in both contact-mode and tapping-modein liquid. The measurements were performedat room temperature and ambient pressure. Aseries of images of the same area on the cyto-plasmic surface of the cell membrane wereobtained using different contact forces (0.9 toapprox 6.0 nN) for a fixed scan rate of 0.6 Hzand scan size of 5 µm in the contact-mode indeionized water to determine the contact forcethat yielded the optimal image lateral resolu-tion. For tapping-mode in liquid, a typical res-onant frequency of approx 8.9 kHz was chosenfor all the samples.

The AFM images were analyzed using theNanoscope Image III 4.42r4 software (DigitalInstruments, CA). The “Section” process wasused to estimate the sample dimensions. Theapparent width of the spectrin molecule at halfmaximum height was measured as a represen-tation of the image lateral resolution to evaluatethe image quality. The “Bearing” process wasused to generate pixel height histogram and the“Grain size” process was used to measure thenumber of “windows” framed by spectrin mol-ecules within a specific area to evaluate thecytoskeleton meshwork dimensions.

RESULTS

Hydrophobicity of Coverslip Surfaces

The relative hydrophobicities of coverslipstreated with acid, poly-L-lysine, APTES,ANBNOS, and lectin are summarized in Table1. Wetting implies that water completely cov-ered the whole surface of the coverslip, indi-cating the surfaces of acid-washed andlectin-coated coverslips are hydrophilic, whilethose treated with poly-L-lysine, APTES, andANBNOS are hydrophobic (1).

THE MEAN SURFACE ROUGHNESS

OF THE COVERSLIPS

The RMS roughness of the lectin-coated cov-erslip surfaces within an area of 1 × 1 µm2

observed in the liquid mode was about 1.60 nm(Fig. 2). In comparison, the acid-washed cover-slip surfaces had an RMS roughness of 0.45nm, the APTES-coated surfaces 1.02 nm, andANBNOS-coated surfaces 1.54 nm (1). Thepoly-L-lysine coated surface had the highestRMS roughness of at least 2.2 nm (48).

EFFECTIVENESS OF ERYTHROCYTE

ATTACHMENT

The numbers of erythrocytes attached to theuncoated acid-washed clean glass coverslip andthose coated with APTES, ANBNOS, poly-L-lysine, and lectin before and after washing withPBS are shown in Fig. 3. These numbers were



Table 1Hydrophobicity of Modified Coverslip Surfaces

Surface modification Diameter (mm) Hydrophobicity

Untreated 4.3 ± 0.1 HydrophobicAcid-washed Wetting HydrophilicAPTES 4.0 ± 0.1 HydrophobicANBNOS 4.5 ± 0.1 HydrophobicLectin Wetting HydrophilicPoly-L-lysine 6.1 ± 0.1 Hydrophobic

APTES, 3-aminopropyltriethoxysilane; ANBNOS, N-5-azido-2-nitrobenzoy-loxysuccinimide.

taken as an average over 10 (135 × 102 µm)regions. The order of initial binding of the ery-throcytes is lectin > APTES > acid-washed >poly-L-lysine > ANBNOS, while the order ofretention percentage is lectin > ANBNOS >poly-L-lysine > APTES > acid-washed. Theresults demonstrate that the lectin-coated cover-slips offer the highest retention of erythro-cytes after washing. Although hydrophobicANBNOS-coated coverslips have one of thehighest retention percentages after washing,their original adhesion number is the smallest ofall coatings tested; therefore, the smallest num-

ber of cells were found to be bound after wash-ing. A modest to significantly large number ofcells was adhered originally to the acid-washedcoverslips, but they had the least retention withwashing. The coverslips coated with poly-L-lysine and APTES have modest numbers of cellsretained after washing.

Specificity of Lectin Attachment

The number of erythrocytes remainingattached to the lectin-coated coverslip pretreatedwith lactose solution and washed with PBS is

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Fig. 2. AFM image of the glass coverslip surface coated with lectin as described in Materials andMethods. The average surface roughness (RMS) of the whole image (1 × 1 µm) is approx 1.6 nm.Images were obtained in water and the RMS was calculated with the software provided by DigitalInstruments.

shown in Fig. 3. The number of cells retained onthe lactose-treated coverslip was reduced signif-icantly compared to the untreated coverslip.

Atomic Force Microscopy Analyses ofInside-Out Erythrocyte Membrane

After mounting and lysing erythrocytes on alectin-coated coverslip, the cytoplasmic side of

erythrocytes was exposed for AFM analysis(Fig. 4A). The opened cells were closely packedside by side, with the flat regions being sur-rounded with bright edges corresponding tocurled or overlapped membranes. The averagearea of the flat regions was 41.6 ± 5.0 µm2,which is close to the half of the surface area of a7.5-µm diameter discoid erythrocyte, approx


Fig. 3. Binding efficiency of different surface modifications. Histogram values were obtained bycounting the average number of cells on the coverslips in 10 (135 × 102 µm) regions after washingthe samples as described in the text.

Fig. 4. The AFM topographic images of cytoplasmic side of the erythrocyte membrane obtainedusing tapping-mode AFM. (A) The concentration of erythrocytes is significantly higher than that in(B). The cells are packed side-by-side and overlapping membranes create the bright lines. (B)Reduced concentration of erythrocytes produces the AFM images of isolated cell membranes. Themembrane thickness ranges between 10 and 18 nm.


44.2 µm2. The presence of closely packed flatareas suggests strong attachment of the cells tothe substrate. By using a reduced concentrationof erythrocyte suspension, separated cell mem-branes were obtained (Fig. 4B). The averagemembrane thickness, as measured from thecross-section lines, is 10–18 nm. An image withhigher magnification of Fig. 4B is shown in Fig.5A. The textured structure observed resemblesthe spectrin-actin network observed by otherswith TEM (30,49). The dimensions of the net-work were measured by drawing section linesat different regions of the digitized image usingthe Digital Instrument software. The averageheight of the spectrin-actin network (bright

area) relative to the phospholipid bilayer (darkarea) was 7.91 ± 2.55 nm and was close to theheight of cytoskeleton network in fixed ery-throcyte membrane samples of 5–9 nm reportedin other AFM studies (10,34). The averagelength of the elongated molecules in the net-work was 162 ± 13 nm, and the average appar-ent width was 64.5 ± 5.0 nm. Length valueswere obtained by measuring the distancebetween junctional complexes (the intersectionsof 2 or more spectrin molecules). As a compari-son, an image of the outer surface of an ery-throcyte is shown in Fig. 5B. The extracellularmembrane topography is relatively smooth andno distinguishable structure can be identified.

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Fig. 5. (A) An AFM image with higher-magnification of a small area in Fig. 4B with a scan rangeof 1 µm. The average height of the spectrin-actin network (bright area) relative to the phospholipidbilayer (dark area) is 7.91 ± 2.55 nm. (B) An AFM image of extracellular surface of intact erythro-cyte membrane. The slight unevenness may be attributed to the carbohydrate chains of glycopro-teins in the membrane.

Comparison of Tapping and Contact ModeImaging of Samples in Air and Liquid

Typical solid-mode AFM images of the ery-throcyte cytoskeleton network obtained withtapping-mode and contact-mode (contact forceapprox 6 nN) are shown in Fig. 6. The averagewidth of the spectrin molecule at half maxi-mum height was similar in tapping mode andin contact-mode using a relatively high contactforce of approx 6 nN. However, as expectedtapping-mode reveals more details within themeshwork area and the spectrin networkappears to be more complex than could beobserved in contact-mode.

A tapping-mode AFM image of the erythro-cyte cytoskeleton network obtained in liquid isshown in Fig. 7A and a contact-mode AFMimage obtained with a contact force of approx2 nN is shown in Fig. 7B. Similar to solid-modeanalyses, the tapping-mode image providedmore details of the spectrin network than didcontact-mode.

Solid-Mode AFM Images of Light andDense Cells

Shown in Fig. 8 are the solid-mode AFMimages of the cytoplasmic side of membranesurface of light (A) and dense (B) erythrocyteswith a scan size of 2.5 µm (A1 and B1) obtainedusing a contact force of 6 nN. The lower images(A2 and B2) are a magnification of a portion ofthe previous images. The cytoskeleton networkstructure is significantly different between thelight and dense erythrocytes. In the light cells,the cytoskeleton appears to be plainer, andevenly distanced from the underlying phos-pholipid bilayer, while in the dense cells, theheight of the cytoskeleton is irregular. Thisirregularity may be the result of spectrin col-lapsing directly onto the surface of the phos-pholipid bilayer.

To compare structural differences betweentwo cells, their images are converted to his-tograms of pixel percentage vs relative heightin nanometers. The relative height of the sam-ple, from the surface of the coverslip, is diffi-cult to obtain because assignment of the

coverslip surface as the reference point of zeromay vary. For convenience, two histogramscan be arbitrarily adjusted so that the phos-pholipid bilayers relative heights coincide.

Figure 9 shows typical pixel height his-tograms of the cytoskeleton AFM images ofcells obtained at low and high density regionsof the Percoll density step gradient. Two distinc-tive peaks appear in the lighter cells, the peak atthe higher location corresponds to the spectrinnetwork and that at the lower location is fromthe “windows” framed by spectrin molecules,which expose the surface of the phospholipidbilayer. For the dense cell, only one distinctivepeak could be isolated. This may be the repre-sentation of the cytoskeleton collapsing ontothe phospholipid bilayer increasing the num-ber of pixels of lower height. Additionalcurves obtained from different cells are shownin Fig. 10.

In addition to height distortion, the numberof “windows” created by the spectrin networkappears to be different between the two typesof cells. The number of such windows in seven1 × 1 µm imaged areas for each type of cellswere counted. The number of windows for thelighter density cell was 27 ± 3 per square µmand that for the denser cells was 21 ± 4 persquare µm.

DISCUSSION

As demonstrated in Table 1, coating the acid-washed glass coverslips with a layer of poly-L-lysine, APTES, ANBNOS, or lectin changes thecoverslip surface charge and hydrophobicity,affecting the cell membrane binding proper-ties. Acid washing generates a high density ofhighly hydrophilic hydroxyl groups on thecoverslip surface capable of forming hydrogenbonds with water (Fig. 1A), thus 10 µL of waterwets the entire surface of the coverslip (50). Onthe other hand, APTES coating (Fig. 1C) gener-ates an amine layer with a density of approx 1-nmol NH2 groups/cm2 glass surface (6 NH2groups/nm2) (1). When air-dried, the protona-tion state of the amine is NH2 and makes the



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Fig. 6. The solid mode AFM images obtained with different scan ranges in (A) the tapping-modeand (B) the contact-mode with a contact force of approx 6 nN. The images are similar, but the tap-ping-mode image appears to reveal slightly more detailed molecular structures than the contact-mode image, as expected.


surface hydrophobic (50), forming a waterdroplet of 4.0 mm in diameter with 10 µL ofwater. The poly-L-lysine–coated coverslip alsohas amine groups on the surface when air-dried (Fig. 1B), but with less density and thusforms a slightly larger diameter water droplet(6.1 mm). For the ANBNOS-coated slides, thedensity of azide groups on the surface has notbeen determined, but the surface is stillhydrophobic because the diameter of the waterdroplet with similar volume is only 4.5 mm. Asfor the lectin-coated coverslips, the averageprotein density on ANBNOS-coated glass sur-face is approx 0.01 nmol molecules/cm2 (1)and results in a highly hydrophilic surface.

Although hydrophobicity may play animportant role in determining the adhesion ofmolecules on the surface of coverslips, by com-paring the hydrophobicity in Table 1 and thenumber of cells adhered in Fig. 3, it is evidentthat additional factors must also determine thebinding stability of cells. For nucleated cells,their attachment, spread and cytoskeletal reor-ganization are significantly greater onhydrophilic surfaces than on hydrophobic sur-faces. This observation is used to suggest thatthe communication between the extracellularmatrix and cytoplasm depends on the nature ofsurface on which the cells grow (43). Althoughno similar activities are necessarily anticipatedin enucleated erythrocytes, their initial attach-ment to the hydrophilic surface is also strong.On the other hand, retention percentage of thecells after washing in buffer is significantlylarge only in lectin-coated and ANBOS-coatedsurfaces, again suggesting that once adhered,erythrocytes are preferentially retained byforces other than hydrophilicity.

All other attaching processes that have beenused are driven by a balance between van derWaals interactions, the electrostatic double-layer forces, as well as hydrophobic effects.Unlike van der Waals interaction, the electro-static force depends strongly on the concentra-tion and valency of charged solutes, as well asthe surface charge density of both the substratesurface and specimen (51). Therefore, for AFMstudy in physiologic conditions, electrostaticforces should not be the dominant mechanismof attachment. ANBNOS forms covalent bondswith macromolecules (Fig. 1D,E) when they arepressed against the ANBNOS surface andlinked by photoactivation (1). However,because ANBNOS creates a highly hydropho-bic surface, it inhibits adsorption of some sam-ples, as well as interferes with AFM imaging(1,52). Moreover, covalent linkage of the softand fragile cells and tissue to the ANBNOSsurface is difficult because the samples must becompressed against it.

Lectin coating via covalent linkage throughan ANBNOS-coated coverslip surface seems toovercome all of the above attachment prob-



Fig. 7. Tapping mode (A) and contact mode(using approx 2 nN contact force) (B) AFMimages in liquid.

lems. The red kidney bean, P. vulgaris, contains5 tetrameric lectins designated L4, L3E1, L2E2,L1E3, and E4 based on the number of L and Epolypeptide subunits/molecule (53–55). Epolypeptide subunits/molecule accounts forthe erythroagglutinating activity of the lectins.E-PHA refers to the lectin species containingone or more E subunits (55,56). Previous stud-ies indicate that E-PHA binds to glycophorinmolecules on human erythrocytes and that the

binding results from the high affinity interac-tion of E-PHA with the bisected biantennaryoligosaccharide of glycophorin (18,19). Thisaffinity interaction makes the binding of ery-throcytes on the E-PHA coated surface strongerthan those formed by electrostatic interactions.As shown in Fig. 1E, the azide groups on theANBNOS-coated surface on UV photolysisgenerate reactive nitrenes, which can forminsertion bonds with C-H groups and other

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Fig. 8. Solid-mode AFM images of the cytoplasmic side of membrane surface of light (A) anddense (B) erythrocytes with a scan size of 2.5 µm (A1 and B1) and a scan size of 1.0 µm (A2 and B2)obtained using a contact force of 6 nN. The cytoskeleton network structure is significantly differentbetween the light and dense erythrocytes. In the light cell, the cytoskeleton appears to be plainer,and evenly distanced from the underlying phospholipid bilayer, while in the dense cell, the heightof the cytoskeleton is irregular.

nucleophilic groups in the E-PHA proteins.After lectin attachment, the surface property ischanged from hydrophobic to hydrophilic.

The affinity interaction between PHA-E andthe cell membrane was sufficiently strong tohold a large number of cells on the treated cov-erslips, while the cells were washed, and whenthey were subsequently osmotically lysed. Theattached cell membrane exposed its cytoplas-mic side for AFM imaging. As indicated in Fig.2, modification of the coverslip surface intro-duces roughness of 1.6 nm RMS, but theincrease is insignificant when compared with1.54-nm RMS for ANBNOS treatment alone (1).To demonstrate that the cell attachment ishighly E-PHA specific, the lectin-coated cover-slip surface was incubated with lactose (GalGlcNAc). This oligosaccharide is known to beone of the oligosaccharides with E-PHAinhibitory activity and is a major component ofthe bisected biantennary oligosaccharide ofglycophorin (18). When the coverslip was incu-bated with lactose solution, the binding sites ofE-PHA were occupied and their binding to gly-

cophorin of the erythrocyte membrane inhib-ited as indicated in Fig. 3.

The use of lectin as a binding linker betweenthe erythrocyte membrane and the glass cover-slips is a general procedure that has broaderimplications. Lectins are a highly diversegroup of proteins with carbohydrate recogni-tion. A large number of plant and animallectins have been found and characterized, andeach of them shows a specific lectin-carbohy-drate interaction to a certain type of cell surfaceoligosaccharides (58–61). There are 9 commoncell surface monosaccharides assembled inprototypical structures. These units can belinked to each other at numerous positionsaround the sugar ring and in 2 distinct geome-tries at the glycosidic linkage to form differentglycosylation on the cell surface, and varieswith cell type (57). By choosing different typesof lectin molecules, the attachment methoddescribed in this study might be extended toother types of cells.

Tapping-mode AFM has been verified toimprove the resolution of both solid and liq-



Fig. 9. Pixel height histograms of cytoskeleton adjusted to the height of membrane surface. Theheight shown in the X-axis is from the surface of the coverslip, but the absolute value may not bevalid because assignment of the coverslip surface as the reference point of zero is difficult. Instead,the histograms of the 2 samples are adjusted so that the phospholipid bilayers will coincide.

uid sample AFM (62,63). In tapping-modeAFM, the cantilever is vibrated as it passesover the sample surface and the probe tip con-tacts the sample intermittently, eliminatingshear forces that can damage soft samples andreduce image resolution (62,64). In addition,the tip radii of tapping-mode probes are usu-ally smaller (approx 5–10 nm) than that ofcontact mode probes (approx 20–60 nm).Figures 6 and 7 show similar resolution forcontact-mode AFM and tapping-mode AFMin air and liquid, but with a slightly moredetail in the latter. This demonstrates that

lectin attachment works for different opera-tion conditions.

There exists a positive correlation betweencell age and density, due to the loss of surfacesialic acid (part of the surface anionic sites),associated water (65,66), and cell dehydration(67). The solid-mode AFM analysis of light anddense cells revealed distortion of cytoskeletonin dense cells. Visual observation of Fig. 8 indi-cates more unevenness in the height of thecytoskeleton, and larger average size of win-dows framed by the cytoskeleton network inthe dense cells than in light cells. The analysis

266 Liu et al.


Fig. 10. Pixel height histograms of light and dense cells from different samples. Among the 2 dis-tinct peaks appearing in light cells, the peak at the higher location corresponds to the spectrin net-work and that at the lower location is from the “windows” framed by spectrin molecules, which isthe surface of the phospholipid bilayer. While in the dense cell, no distinctive peaks can be isolated.The relative distance (in nm) between the two peaks is indicated for light cells. Dense cells representcells in the last 20 d of their lifespan and exhibit a range of surface topographies.

based on the pixel height histogram (Figs. 9,10)and the numbers of windows created by thespectrin network are intended to assess thesedifferences quantitatively. The pixel height his-togram is a representation of vertical heightchange of the cytoskeleton molecules and thenumber of windows their horizontal extentchange. Together, they represent the extent ofmolecular shift and indicate cytoskeleton dam-age. The cytoskeleton of dense erythrocyteshas been exposed to many days of mechanicalstress and oxidative attack in the body. Theweak bonds may eventually be broken andcause damage to the integrity of the cytoskele-ton. The breakage may take place at the bond-ing site of a spectrin molecule to the junction,where actin, tropomyosin, tropomodulin, band4.9, adducin, and other molecules are found.The resulting image may have a different num-ber of windows framed by the spectrin mole-cules per unit area as the broken off spectrinmoves away from its original position. Thesechanges should be reflected in Figs. 9 and 10.The dense samples include cells that are in thelast 20 d of their lifespan. Thus a large variationamong them is expected leading to the his-tograms with various abnormalities.

CONCLUSIONS

Lectin-bonded glass coverslips stronglyattach erythrocyte membranes and are usefulfor both solid- and liquid-mode AFM analyses.The adhesion is the strongest when comparedwith several conventional methods and, evenafter lysis of the cell, the firmly attached mem-brane exposes the interior of the cell for theAFM analyses. Using the spectrin network as amodel, it has been demonstrated that the opti-mal contact force in liquid-mode resolution isbetween 0.9 and 2.0 nN, whereas a suitablecontact force in solid mode for air-dried sam-ples is 6 nN. The AFM images of the young andold erythrocytes in solid-mode have revealedsignificant deteriorations in the spectrincytoskeleton of the older cells than the youngand the results are quantitatively defined using

the pixel height histogram and window sizegrain analyses.

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Sample preparation and imaging of erythrocyte cytoskeleton with the atomic force microscopy