Atomic Force Microscopy in Cell Biology Episode 1 Part 9 doc

In our experiments the cantilever scans in constant height exerting a horizontal force to the mechanosensory structures of outer hair cells.. The applied force results in a horizontal di

of parallel oriented actin filaments, called the cuticular plate, forming a core and a root embedded in a platform made of actin on top of the cell (Tilney and Tilney, 1986) At the base, the number of actin filaments is reduced from a few hundred to about a dozen form-ing a point of flexform-ing The actin filaments are crosslinked by smaller filaments, probably fimbrin, preventing bending of stereocilia along their length This construction allows stereocilia to flex around their pivot point located at the bottom where actin filaments penetrate the cuticular plate A fibrous layer, called the tectorial membrane, overlies the organ of Corti The tips of the tallest stereocilia of the OHC but not of the IHC touch the bottom side of the tectorial membrane The origin of stereocilia displacement is a rela-tive displacement between the basilar and the tectorial membrane forming together with the hair cells a layered or sheet-like structure Bending of the stereocilia results in stretch-ing the tip links and openstretch-ing of the transduction channel allowstretch-ing an influx of cations into the hair cell (Gillespie, 1995; Markin and Hudspeth, 1995) The ultrastructure of sensory hair bundles of the mammalian inner ear has been extensively studied by scanning and

transmission electron microscopy (SEM and TEM) (Pickles et al., 1984; Lenoir et al.,

1987; Russell and Richardson, 1987) These methods are restricted to fixed and dehy-drated specimens, however Structural details of living hair cells have been visualized

by light microscopy with a limited spatial resolution due to the limitation of the optical system Investigation of hair cells with atomic force microscopy (AFM) seems attractive since this method may combine three advantages: the high spatial resolution of scanning

probe microscopy (Binnig et al., 1986), the possibility to work under physiological con-ditions (H¨orber et al., 1992), and the opportunity to record mechanical properties of the

structures being imaged (Hoh and Schoenenberger, 1994) AFM has already been used for simultaneous imaging and elasticity measurements on living cells under

physiologi-cal conditions (H¨orber et al., 1992; Hoh and Schoenenberger, 1994; Shroff et al., 1995; Radmacher et al., 1996) Therefore, it seems to be an appropriate technique for studying

the mechanical characteristics of the hair bundle under physiological conditions

B Preparation

Organs of Corti were isolated from 4-day-old rats (Wistar, Interfauna, Germany)

according to the methods used by Sobkowicz et al (1975) and by Russell and Richardson

(1987) for mice They were cut into three segments (basal, medial, apical) and placed with the basilar membrane onto Cell-TAK-coated glass coverslips with a diameter of

10 mm The sample was transferred into Ø 35-mm Falcon dishes filled with 3 ml culture

medium MEM D-VAL with 10% heat-inactivated FCS and 10 mM Hepes buffer, (pH 7.2)

supplemented at 37◦C and 5% CO2 Using this preparation, hair bundles are oriented perpendicular to the surface of the supporting glass coverslip For experiments, specimens were transferred from the incubator to a specimen chamber Cochlear cultures were bathed at room temperature (22◦C) in a solution containing (millimolar concentrations)

144 NaCl, 0.7 NaH2PO4, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 5.6D-glucose, 10 Hepes– NaOH, pH 7.4, osmolarity 304 mOsmol The solution was exchanged before and after investigation using a conventional gravity-driven perfusion system to keep cells in good condition At 4 to 6 days after birth the tectorial membrane of rats is already partially developed In some preparations, the tectorial membrane directly touched the hair bundles

of OHC To guarantee free access of the AFM tip to the hair bundles, the tectorial membrane was removed with a cleaning pipette under the light microscope Cultures were investigated for a maximum of 2 h

III AFM Technology

A Short Introduction to AFM

The theories of AFM, the technological aspects, imaging techniques, and applications, have already been the topic of numerous publications (e.g., Meyer, 1992) Therefore, only the aspects necessary for understanding the contents of this chapter, not aspects just briefly described in other publications, will be discussed The AFM experiments described here concentrate on force spectroscopy at the extracellular surface of hair cells The capability of imaging is only used for correlation of force and position of the

sensor tip on the specimen As described by Binnig et al (1986), AFM locally measures

the force between a small tip and the specimen surface using a free-moving cantilever

with the force constant kL Scanning the cantilever line for line across the sample while the tip interacts with the surface, we can obtain an image of the sample topography The vertical deflection of the cantilever can be assigned to each point of the object The AFM tip is scanned with a piezo-electric tube The outer electrode is segmented into eight commensurate electrodes electrically connected in a way that the walls at the end

of the piezo-electric tube remain perpendicular to the direction of motion (Siegel et al.,

1995) The AFM tip consequently moves on a plane surface in contrast to piezo-electric tube scanners with only four outer electrodes scanning on a curved surface Typically the scan range was between 1 and 8μm The force F exerted to the sample depends on

the vertical cantilever deflection Las follows:

Typical force constants of commercially available cantilevers vary from 10−3to 102N/m Different methods for detection of the cantilever deflection such as heterodyne

interfero-meters (Martin et al., 1987), capacitive detection (Neubauer et al., 1990; G¨oddenhenrich

et al., 1990; Miller et al., 1990), and tunneling current detection (Binnig et al., 1986)

have been reported In our instrument, the most common detection method was used:

the optical lever method (Meyer and Amer, 1988, Alexander et al., 1989) The light of

a laser diode is focused on the gold-coated surface of the AFM cantilever The reflected light is detected on a position-sensitive four-segmented photo-diode Deflection of the cantilever beam results in displacement of the laser spot on the photo-diode The dif-ference in intensity between the pairs of upper and lower segments encodes the vertical cantilever deflection Torsional forces are detected subtracting the intensity at the left pair of segments from the intensity detected at the right pair of segments AFM pro-vides a wide range of microscopic techniques, which allow the measurement of different surface and material properties These techniques can briefly be classified into two dif-ferent operation modes: the contact and noncontact mode Both principles, discussing

the measurement of a force versus distance curve, can easily be explained (F versus d).

Fig 3 Principle of force versus distance measurements The deflection versus elongation of the piezo-electric scanner vertically moving the sample The force corresponds to the product of cantilever deflection and force constant Before examination the sample is fully retracted, not interacting with the AFM cantilever (inset: free-moving cantilever) As the scanner extends (arrows), the tip

and sample surface attract each other (position z1 ) and the cantilever starts to bend downward This attraction decreases with increasing scanner position The force goes to zero (equilibrium between attractive and repulsive forces, piezo position 0) As the scanner continues to extend the total force becomes positive (inset: repulsive forces) At the maximum of the curve the scanner stops and retracts With increasing contraction of the piezo-electric scanner (dashed lines with arrowhead) the cantilever follows this motion beyond zero position and

loses contact at position z2 At this point the tension of the cantilever beam exceeds the attractive forces (see inset), thereby losing contact with the sample surface.

A F versus d is a plot of the deflection of the cantilever versus the extension of the

piezo-electric scanner (Fig 3) On the left side of the curve, the scanner is fully retracted and the cantilever is in its resting position since the tip does not touch the sample As the scanner moves, the cantilever remains undeflected until it comes close enough to the sample surface As the atoms of tip and surface are gradually brought together, they first weakly attract each other This attraction increases until the atoms are so close together

that their electron clouds begin to repel each other electrostatically (position z1) This

electrostatic repulsion progressively weakens the attractive force as the interatomic sep-aration continues to decrease The force approaches zero when the distance between the atoms reaches a couple of angstroms, about the length of a chemical bond (position 0)

As the scanner continues to extend, the total van der Waals force becomes positive (repulsive), and the atoms are in contact This situation corresponds to the contact mode For the noncontact mode AFM measurements have to be performed in the attractive

regime (position z1)

B Principle of Hair Cell Measurements

Normally, force versus distance curves are recorded perpendicular to the sample sup-port In our experiments the cantilever scans in constant height exerting a horizontal force to the mechanosensory structures of outer hair cells The applied force results in a horizontal displacement of the stereocilia along the axis of symmetry of V-shaped hair

bundles This kind of measurement (Fig 4A) corresponds to a force versus distance curve performed in the horizontal direction The feedback electronics normally keeping the measured force at a constant level was switched off during investigation Every data point in an AFM image represents a spatial convolution (in the general sense, not in the sense of Fourier analysis) of the shape of the tip and the shape of the feature imaged For most imaging applications this convolution leads to unwanted artifacts In our mea-surements, we took advantage of the convolution of AFM tip and rod-like stereocilia geometry permitting us to calculate the stiffness of stereocilia The van der Waals force curve (Fig 3) represents just one contribution to the cantilever deflection Local

varia-tions in the form of the F versus d curve indicate variavaria-tions in the local elastic properties These local variations in the slope of F versus d were used as a measure for the elasticity

of the investigated sample Langer et al (2000) showed for stereocilia that a steep slope

of the force curve indicates a high stiffness of the sample, while a shallow slope indicates

a soft sample For examination, the AFM tip was adapted to the axis of symmetry of the stereociliary bundles of OHC rotating the specimen chamber A certain hair bundle was visually selected in the light microscope and directly positioned below the AFM tip adjusting the specimen stage For the vertical fine approach, the specimen chamber was elevated with the help of a piezo-electric stack while scanning the cantilever tip (scan range: 4.5μm) The approach is stopped as soon as stereocilia of the OHC cause

signif-icant deflection of the AFM cantilever (about 20 to 50 nm) For stiffness measurements, the AFM tip successively scans each stereocilium within a hair bundle (Fig 4A) Only the tallest stereocilia of the hair bundle are touched with the frontal lateral face of the

pyramidal tip and deflected in excitatory and inhibitory directions (Langer et al., 2000).

Fig 4 Principle of stereocilia examination by AFM (A) The AFM tip scans with the feedback electronics switched off across the hair bundle, thereby deflecting a stereocilium of the tallest row of stereocilia (see dashes stereocilium) Relative displacement between this taller and the adjacent shorter stereocilium (smaller dashes stereocilium) results in a strain of the connecting tip link possibly pulling at a transduction channel Interaction with the stereocilium results in vertical deflection of the AFM cantilever beam indicated as a broken line for the excitatory scan direction (arrow) (B) Representative example of an AFM curve The vertical deflection of the AFM cantilever was recorded versus time while scanning an individual stereocilium The scan frequency was set to 2 Hz The peak represents a force interaction between AFM tip and stereocilium (between 1700 and

2250 nm).

Figure 4B shows a typical example of an AFM trace The ordinate displays the vertical deflection of the AFM cantilever while scanning an individual stereocilium An increase

in stiffness as a result, for example, of chemical fixation (Fig 6 in Langer et al., 2000)

leads to an increase in the positive slope of the force curve This increase in slope reflects

that for the same horizontal force FLexerted by an AFM tip, a stiff stereocilium does not horizontally move as much as a soft stereocilium Assuming that friction is very low,

FLcan be calculated from the measured vertical deflection “a” (Fig 4B) using

FL = FN · tan α = kCant · a · tan α, [2] whereα is the angle between the lateral face of the pyramidal-shaped AFM tip and the scan direction, “a” is the vertical deflection of the cantilever beam, and kCantis the force constant of the AFM cantilever

It was shown (Langer et al., 2001) that the lateral force constant kLof a stereocilium

is given by

where c is the distance the AFM tip moves from the first point of contact to the point where the AFM cantilever reaches deflection “a” The denominator in Eq [3]

corre-sponds to the horizontal displacement of the tip of the stereocilium Using the horizontal deflection method described earlier, it is essential to know the angleα between the lateral

face of the AFM tip and the scan plane Therefore, pyramidal-shaped Si3N4tips with a well-defined tip angle of 70◦were used For calculation of the force constant the scan line with maximum peak amplitude was chosen At this particular scan line the AFM tip is in contact with the center of the stereocilium Contaminants and lubricants may affect our measurements by inducing adhesion and friction forces that distort the stiff-ness measurements Such adhesive and friction forces were detected using a horizontal modulation technique

The investigated hair bundles were scanned adding a sinusoidal modulation signal (peak-to-peak amplitude: 100 nm; frequency: 98 Hz) to the fast scan signal of the piezo-electric tube scanner of the AFM This results in an additional horizontal forward and backward movement of the AFM tip at higher frequency When being in contact with

a stereocilium, this technique (G¨oddenhenrich et al., 1994) allows the detection of

fric-tional and attractive forces For negligible friction, the AFM tip slides up and down

on identical paths while scanning the tip forward and backward at this fast modulation frequency If frictional forces acting between AFM tip and sample surface increase, the cantilever moves on small loops indicating a speed- and direction-dependent interaction between tip and sample

C Basic Requirements for an AFM/Patch-Clamp Setup

Section II reported the unique features of hair cells Not even their functional prop-erties, as the possibility to transform a mechanical into an electrical signal, mani-fest not only their extraordinary characteristics but also their high degree of structural organization within the organ of Corti The orientation of hair bundles of isolated hair

cells may vary in a wide range when being attached to a supporting glass coverslip, thereby complicating the detection of the hair bundle orientation in the light microscope and access with the AFM tip In contrast, cultures of the organ of Corti offer a nice way to hold hair cells in upright position Accordingly, the instrument used to study the mechanical properties of hair bundles must support the identification of individual hair bundles of about 4 to 5μm in width within the whole organ of Corti (diameter:

about 300μm) The identification exclusively by AFM at big scan ranges is insufficient.

Contact between the AFM tip and the microvilli, situated between the hair cells (see Fig 2A), promptly results in adsorption of contaminants at the tip surface Accordingly, both proper selection of individual hair bundles under light microscopic control and precise adjustment of the AFM tip above the selected bundle are inevitable The exami-nation of stereocilia stiffness and gating of the transduction channel require acquisition

of all physical values such as force and current as a function of time In contrast to most commercial AFM instruments, our setup is able to record the correlation in time

of all recorded signals and not only the spatial position of the AFM tip Therefore, the AFM/patch-clamp hardware was combined with data acquisition hardware allowing the simultaneous generation of up to four stimulation patterns such as the scan signal for AFM and time-correlated acquisition of data such as the transduction current For maximum flexibility, the data acquisition software provides the possibility of generating user-defined stimulation patterns

D AFM Setup

Attachment of the organ of Corti with the basilar membrane to the supporting glass coverslip results in orientation of hair cell bodies and stereocilia perpendicular to the specimen support The identification of stereociliary bundles requires high optical res-olution in both vertical and horizontal directions Stereocilia are about 1.5 to 3.0μm

in length (postnatal rat; age: 3 days) located on top of a 40- to 50-μm-thick cell layer

To allow stable patch-clamp measurements simultaneously to AFM scans, as soon as the microelectrode has sealed to the lipid membrane of a hair cell, we must prevent relative movement between the cell body and the pipette Therefore, the AFM tip, rather than the sample, is scanned Figure 5 shows the three major units of the instru-ment: optical microscope, AFM, and patch-clamp device The whole setup is mounted

on a regulated air-damped table isolating mechanical vibrations at low frequencies

(15–100 Hz) An upright optical microscope is mounted on a custom-made xy-translation

stage allowing two-dimensional horizontal adjustment This translation stage provides

an independent positioning of the objective with respect to the separately mounted AFM cantilever and the patch-clamp pipette Optical imaging is done using a water immersion objective (40×/0.75, Achroplan, Zeiss) with a working distance of 1.92 mm The outside

is coated with a nonconductive material allowing electrical recordings at low noise Mechanical noise of the experimental setup was reduced by mounting AFM and the patch-clamp head stage on a separate platform For additional higher mechanical stability eyepiece and CCD-camera were separated from the optical microscope Cells were investigated in a liquid chamber filled with physiological solution A micrometer

Fig 5 Mechanical components of the experimental setup It consists of three major units: the optical

microscope, the AFM, and the patch-clamp head stage 1, xyz-translation device allowing precise positioning

of the AFM sensor; 2, electrically shielded piezoelectric tube scanner of the AFM equipped with a cantilever

holder made of titanium; 3, patch-clamp headstage mounted on a xyz-translation device; 4, microelectrode

holder; 5, halogen light source illuminating the sample from below The liquid chamber includes a piezo-electric actuator allowing fine approach of the sample to the AFM tip.

screw-driven xy translation device allows horizontal displacement of the specimen

chamber in two dimensions The chamber is additionally adjustable in a vertical direction using a piezo-electric stack providing a resolution of better than 1 nm and a maxi-mum vertical displacement of 20μm The patch-clamp setup, the object chamber, and

the AFM head are fixed on a separate support, at a height of 190 mm, made of alu-minum with a 20-mm-thick U-shaped steel plate on top to prevent mechanical vibrations The AFM cantilever is mounted on a piezo-electric tube scanner introduced by Binnig and Smith (1986) The maximum horizontal scan size at maximum driving voltage of

±160 V is 6.5 μm The maximum elongation is 3.7 μm in the z direction The AFM

cantilever is attached to the end of a rigid titanium beam, which is small enough to po-sition the cantilever between objective and specimen In the coarse approach, the whole

scanning unit is adjustable in the horizontal plane and vertical direction by a xy- and z-translation stage The light microscope (Axioskop FS I, Zeiss) uses infrared differential

interference contrast (DIC) and a water immersion objective providing information from

a thin optical plane of the organ of Corti The combination of an AFM with such optics

is essential for the precise vertical approach of the AFM tip to the top of hair bundles

of postnatal rats Due to the limited working distance between objective and speci-men, bending of the AFM cantilever had to be detected through the objective (Langer

et al., 1997) Detection from below through the condenser of the optical microscope was

critical because the laser beam would have to penetrate the optically quite inhomoge-neous cell tissue Moreover, it was necessary to use a collimated instead of a focused laser beam for detection of the AFM cantilever deflection A collimated laser beam ex-clusively allows detection of changes in angle rather than in linear movements of the cantilever Movement of the cantilever along the optical axis of the collimated laser beam does not affect the electrical signal of the photodiode The reflected beam propagates the same way back to the AFM detector Only a deflection of the AFM cantilever results in

an off-axis angle of the reflected beam shifting the laser spot on the photodiode Never-theless, AFM cantilevers had to be controlled in the light microscope for contaminants potentially causing changes in the detector signal

E Patch-Clamp Setup

The patch-clamp technique is an electrophysiological method that allows the recording

of whole-cell or single-channel currents flowing across biological membranes through ion channels Using patch clamp we have the possibility to control and manipulate the voltage (voltage clamp) of membrane patches or whole cells such as hair cells The basic approach to measuring small ionic currents in the picoampere range as the transduction currents in hair cells requires a low-noise-recording technique combined with a precise mechanical positioning of the patch-clamp microelectrode This paragraph concentrates

on the AFM-specific requirements for a patch-clamp setup For a detailed and general description of the patch-clamp technology, see, e.g., Sakmann and Neher (1995) The electrical connection between microelectrode and preamplifier headstage is kept as small

as possible keeping the noise at a low level Patch-clamp experiments require a precise and drift-free control of the movement of the microelectrode Therefore, the head stage

is attached to the top of a three-dimensional micrometer screw-driven translation stage with a spatial resolution of about 1μm in each direction For vertical fine

position-ing of the patch pipette, the same piezo-electric device as that used for the specimen chamber was implemented into this translation stage Thus, the vertical movement of the patch pipette and the investigated specimen are synchronized A Faraday cage made of a metal grate shielding the microelectrode from electrical noise surrounds the whole setup The piezo-electric tube scanner of the AFM is separately shielded preventing electro-magnetic cross-talk between scanner and patch-clamp microelectrode In voltage-clamp experiments the voltage across the membrane is measured with respect to a bath elec-trode Unfortunately, the AFM cantilever holder consists of metal (titanium) which in principle could lead to interfering potentials between the bath electrode and the AFM holder Therefore, the AFM holder and the bath electrode were electrically connected with a small cable Supporting cells surrounding the outer hair cells had to be removed using a big cleaning pipette (diameter: 10–15μm) allowing free access to the cell bodies

with the patch-clamp microelectrode Cleaning pipettes were fabricated of Ø 2 mm borosilicate glass and mounted on a hydraulic-driven three-axis manipulator providing

an adjustment range of 10× 10 × 10 mm Patch-clamp pipettes were fabricated of Ø 1-mm quartz glass using a laser-based puller Stimulating voltage steps and intracellular

DC potentials were generated using custom-made software

IV Applications

Although hair bundles and their cross-links have been extensively examined by SEM and TEM many questions about their function remain unanswered Filamentous links were identified in SEM and TEM, but their elastic and functional properties have not yet been directly measured Local AFM measurements at individual stereocilia are presented providing information on the mechanical properties of side links and their possible func-tion for hearing For the further understanding of the mechanoelectrical transducfunc-tion of stereociliary bundles in the inner ear, two experiments were performed In the initial ex-periment, we examined the force transmitted by side-to-side links connecting stereocilia

of the same row The strength of side links determines the magnitude of displacement of adjacent stereocilia not directly interacting with the AFM tip Results should allow the number of stereocilia displaced when an individual stereocilium is stimulated by AFM

to be determined In a second set of experiments the mechanical properties of stereocilia were correlated to the gating properties of the transduction channel

A Effect of Lateral Links on Hair Bundle Mechanics

Force transmission between adjacent stereocilia was examined deflecting one or two stereocilia of a hair bundle with a fine modulating glass fiber tip (diameter: 229±

21 nm (mean± SD)) while scanning the entire stereociliary bundle with the AFM tip The force transmitted via the side links was measured at different stereocilia detecting the magnitude of the AFM signal at the modulation frequency with a lock-in amplifier Organs of Corti were rotated around their vertical axis until the direction of motion of AFM tip and stimulator were aligned parallel to the axis of symmetry of OHC bundles Stimulation fibers were attached to a piezo-electric tube actuator mounted on a three-axis translation stage tilted by 27◦ For parallel alignment of fiber and specimen support, the fiber was appropriately angled close to the tip melting the glass capillary with a heated wire The fiber was coarsely placed under light microscopic control to the axis of symmetry of the OHC bundle using the three-axis translation stage The voltage at the piezoelectric stack of the specimen chamber was adjusted for vertical fine positioning of the hair bundle For the horizontal approach of the stimulating fiber, the piezo-electric tube was axially elongated until the fiber tip touched the top of the stereocilium The relative arrangement of fiber, AFM cantilever, and OHC bundle during the approach is shown in Fig 6A Vertical and horizontal fine positioning of fiber and stereocilium was

Fig 6Approaching a glass fiber tip to the top of an individual stereocilium (A) A fine glass fiber is coarsely approached under the optical microscope toward the major axis of a hair bundle of an OHC For controlling the approach with nanometer precision, the AFM tip was successively scanned across the hair bundle and the upper edge of the glass fiber (dashed line corresponds to recorded force curve) While scanning, the glass fiber was horizontally moved with a piezo-electric positioning device to the top of an individual stereocilium (B) AFM trace displaying the interaction with a stereocilium ( ∗) and the upper edge of the fiber tip (∗∗) during fine approach The cantilever deflection is plotted versus scan size The distance between stereocilium ( ∗) and pipette ( ∗∗) is about 200 nm (C) AFM trace recorded after completing the approach The pipette (∗∗) already touches the stereocilium ( ∗).