and this thiol group can bind to agold coated silicon nitride tip. Anamine group at the other end of thePEG molecule attaches proteins(antibodies for example) via acovalent bond41.

• To coat the tip with biotin, it ispossible to first coat the tip withbovine serum albumin (BSA), whichallows the attachment of biotin.

• Oligonucleotides can be covalentlyattached to the tip and to probesequence specific interactions.

• Cells can be grown onto tiplesscantilevers or onto cantilevers withsmall beads at the end. They can also be chemically attached viapolyethyleneimine (PEI) interactions for example42.

Important probe Parameters

Two fundamental parameters of anAFM probe are tip shape andmechanical parameters (the cantileverspring constant, resonance frequencyand quality factor “Q”).

Plasma treatmentPlasma processing can change the hydration properties of the tip. Glow discharge in a hexafluoro–propene–atmosphere, resulting in aTeflon-like coating of the tip, renderssilicon nitride cantilevers hydrophobicdue to the hydration properties ofTeflon27. One minute glow discharge in air renders silicon nitride probeshydrophilic23,28 by “cleaning” theorganically contaminated surface.

SilanizationSilanization (Figure 2) is anotherpossibility for changing the wettingproperties and the surface charge of atip. Organochloro- or organoalkoxy-silanes are chemically bound to theprobe surface. Non-reactive groupssuch as alkyls provide hydrophobicity.For example, trimethoxysilylpropyl-diethylene- triamine (DETA) renders asilicon oxide surface (oxide-sharpenedcantilevers) positively charged andhydrophobic29. Lubricant coating – N-3(3- triethoxysilylpropyl)perfluoro(polyisopropoxy 2-methylacethyl)amide for example – can also be used to render silicon nitride tipshydrophobic and, thus, reduceadhesion forces that might damage the sample30.

Note that Silanization is also useful formodifying the surface chemistry of thesubstrates used for sample support inbiology, for example to promote the

binding of molecules to the substrate.A typical example is the binding ofDNA molecules onto a mica surfacetreated with APTES(Aminopropytriethoxysilane), whichrenders the surface positively charged31.

Tip Functionalization

Functionalization of tips by coatingthem with molecules has opened anew research area for studyingspecific interactions on a molecularlevel, e.g. ligand-receptor pairs or cell-cell-interactions (Figure 3).

• Chemical coating of probes ismainly done by silanization or byfunctionalized thiols and is often a first step before biologicalfunctionalization.

• Biological coating has been mostlyused for mapping the distribution ofbinding partners on samples32-34, aswell as for force measurements13,35,36.It becomes therefore possible toinvestigate molecular forces, like:– forces between a receptor and

a ligand13,37, including antigen-antibody-pairs38.

– forces between molecules and cells.

– forces between cells36,39.Intramolecular forces have also beenmeasured with great success40.

Many protocols can be performed toattach proteins to a tip. Sometimesnon-specific binding is enough, butmore complicated protocols may berequired. Most of them use a spacerto covalently bind the proteins. Theadvantage is that it is possible to orientthe protein in order to expose specificsite(s) of the proteins (enzymatic site forexample). Here are a few examples:

• Polyethyleneglycol (PEG) is acommon spacer. A terminal thiolgroup can be first attached to PEG

Figure 3. Functionalized cantilever for forcemeasurements on ligand-receptor pairs14.

Figure 2. Schematic representation of thesilanization process.

2

For force measurements it is crucial toknow the value of the spring constant.The accuracy of the force measurementis determined by the error of the forceconstant and by any errors in thedetection system.

Different methods have been proposedto determine the spring constant:

• Acquiring the thermal vibrationspectrum of a cantilever anddeducing the spring constant of the cantilever from theequipartition theorem56,57.

• Measuring the change of theresonance frequency while loading the cantilever with small weights58.

• Measuring the resonance frequencyand calculating the spring constantfrom the geometrical dimensions of the cantilever59 or from its quality factor,Q60.

• Measuring the deflection of thecantilever when loading with smallweights61 or while exerting a force with another cantilever of known force constant62.

synthetic calibration standard likecolloidal gold particles48, latexbeads49 and calibration arrays, or byscanning well known biomoleculeslike DNA50. Note that the TipEvaluation Option available on theDigital Instruments NanoScope®

software contains a calibrationstandard to estimate tip shape.

• The sample topography can also becalculated by estimating the tip shapewith mathematical morphologyoperations without using a knownstandard topography (“blind”mathematical restoration)51-55. Thesenon-linear mathematical operations(dilation and erosion) consist in anover- and under-estimation of the tipbroadening effect (Figure 5).

Due to variations in etching duringproduction, double tips can sometimesbe generated, yielding images in whichthe topography appears repeated(Figure 6). This is a very commonartifact, and any user should be able to recognize it.

Spring constantThe spring constant k is defined as theratio between the applied force F andthe cantilever deflection ∆d:

Tip shapeThe radius of curvature of the tip, or tip sharpness, determines the lateralresolution. An AFM image is always a‘combination’ between the tip shapeand the sample topography. Thereforethe duller the tip is, the wider thetopography appears (“tip broadening”effect). Most of the time, optimalresolution on biomolecules requires the minimum possible tip radius43.Nevertheless, it is sometimespreferable to use dull tips rather thansharp tips, when imaging cells forexample, because the pressure exertedon the sample is less44. A sharp tip canpoke through the membrane anddamage the cell45,46 (Figure.4).

To help quantify the tip broadening it may be useful to do a qualityassessment of the probe. Knowing thetip shape is then essential to restore thesample surface. The following methodshelp for studying the tip shape andrestoring the true sample surface:

• Imaging and measuring the tips withelectron microscopy47, or with fieldion emission microscopy.

• It is also possible to estimate the tipshape by scanning a sample ofknown topography such as a

Figure 4. Dissecting a cell membrane locally by applying too high forces.

a.

Figure 5. Morphological filter applied to an AFM image (a) to reconstruct the DNA shape (b).

b.

(Hooke’s Law)

3

Resonance frequency and quality factorThe resonance frequency is the first natural mode of vibration of a cantilever and is determined by the material, geometry and theenvironment (air or liquid).TappingMode is performed with acantilever oscillating preferentially close to its first natural mode ofvibration. In air, typical values ofresonance frequencies are 900Hz to88kHz for silicon nitride cantileversand 60kHz to 400kHz for siliconcantilevers, depending upon thecantilever geometry. In liquid, withsilicon nitrides probes (100µm long,narrow legs, spring constant =0.06N/m), we recommend choosinga frequency between 8 and 10kHz.Note that it is important to tune thefrequency very close to the surfacebecause there can be a frequency shiftas the tip approaches the surface.

The quality factor Q of the cantilever isanother parameter of interest affectingthe scan speed and the sensitivity. Q isdefined as

where:m = cantilever mass

= resonance frequency∆F = width of the resonance peakb = damping factor

Q is a measure of the dissipationmechanisms that damp the oscillationof the cantilever. A high Q is desirablefor TappingMode to optimize thesensitivity63. Typical values of Q forcantilevers in air are 100 to 300, and around 1 in water due tohydrodynamic damping64,65.

Problems of Tip Contamination

Tips can easily become contaminatedduring the scanning process. This isespecially the case for biologicalsamples that can easily detach from

the surface or from the sample itself,like proteins. This can result in doubletip images (Figure 6) and/or reductionof the lateral resolution. Imaging tips with electron microscopy afterimaging with an AFM can show thecontamination of the tip47. A change in the image during a scan shouldalways alert the user about analteration of tip quality.

Cleaning tipsCleaning a tip is a good way todecrease contamination from thefabrication process or storage. Thisprecaution can increase the imageresolution. The most popular cleaningmethod is UV-light treatment thatproduces ozone and removes organicdebris66. Another possibility is toincubate the cantilevers in a piranhasolution (mixture of sulfuric acid andhydrogen peroxide) for 30 minutes.This procedure is used to clean siliconand silicon nitride surfaces in theelectronics industry and also removesthe silicone oil contamination oftenintroduced from the cantilever packing material67.

Plasma ashing/etching, also derivedfrom the electronics industry, is knownto remove organic contaminants. Here,hydrogen, oxygen and argon plasmareact with carbon compounds or

oxides on the surface68. For example,exposing cantilevers to argon plasma(80W) for 30 seconds seems tosufficiently clean probes for tipfunctionalization28. CO2 snow alsoremoves organic debris from thesurface because of a transientformation of liquid CO2

69.

Note that a “cleaned” probe isgenerally hydrophilic because of theremoval of the organic contaminantsthat are generally present in ambient air.

Choosing Probes

Table 1 is a guide to choosing probesfor various and typical biologicalsamples as deduced from a wealth of user experience. As previouslymentioned, the main choice of probesis between two families — silicon and silicon nitride probes, differingprimarily by their spring constant. The table indicates the probe as afunction of sample type, as well as the preferred imaging mode(TappingMode vs. Contact Mode).Unless otherwise noted, whendiscussing TappingMode in liquid, weare referring to the indirect drive fluidcell. Magnetic actuated TappingModeis discussed separately. For moredetail about these and other probes,please visit www.veecoprobes.com.

Figure 6. “Double tip” image of two DNA molecules.

4

LIQ

UID

AIR

TAPP

ING

CON

TACT

SAMPLE TYPE

OTESPARTESPTESP

(D)NP-S

NP-STTOTR4

(D)NP

TESP(D)NP(D)NP-S

FESP

OTESPATESP

(D)NP-S

(D)NP

MSCT

PROBEFAMILY/MODEL

Biomolecules (NucleicAcids, Proteins, Lipids,Carbohydrates, etc.)

Biomolecules (NucleicAcids, Proteins, Lipids,Carbohydrates, etc.)

Cells

Tissues

Tissues

Biomaterials

Biomaterials

Force Measurements

Force Measurements

Table 1. Recommended probe types for specific biological samples and imaging modes.

Silicon

SiliconNitride

SiliconNitrideSilicon

SiliconNitride

Silicon

SiliconNitride

SiliconNitride

xxx———

—

x——xxx—

—

—

———xxxx

—xx———x

x

x

xxxxxx

x

xxxxxxx

—

—

———x

xx

x

———

—

——

—

x

—

EXPERIMENT AFM MODE

Key to Probe Model NamesSilicon Probes:OTESPA: TappingMode Etched SiliconProbe. General purpose, air,TappingMode probe with backsidecoating for increased laser reflectivity.RTESP: TappingMode Etched SiliconProbe. General purpose, air,TappingMode probe with the tiprotated to optimize sidewall anglesymmetry in the fast scan direction.While advantageous on some sampleswith tall, sharp step features, thisfeature is not generally important onmost biological samples.

TESP: TappingMode Etched SiliconProbe. General purpose, air,TappingMode probe, interchangeable with RTESP in most applications.

FESP: Force Modulation Etched SiliconProbe. Originally intended for forcemodulation experiments, this probe also works well for general purposeTappingMode in air in case where its lower spring constant is advantageous (i.e. soft samples).

Silicon Nitride Probes:

(D)NP-S: Oxide-Sharpened SiliconNitride Probe. General purposeprobe for both contact mode andTappingMode in fluid. Four differentcantilevers on each substrate allow anappropriate choice of spring constantfor different experiments. Optional “D” prefix indicates low stresscantilevers recommended for DigitalInstruments Dimension-series scanners.

(D)NP: Silicon Nitride Probe. Same as (D)NP-S probes, except that the tipis not oxide-sharpened, resulting in aless sharp tip. This is advantageous on some samples, especially cells,where a sharp tip may damage the sample. Optional “D” prefixindicates low stress cantileversrecommended for Digital InstrumentsDimension-series scanners.

NP-STT: Oxide-Sharpened SiliconNitride Probe Twin Tip. These probesare very similar to the NP-S probesexcept their tips are formed by aprocess that produces a considerably

sharper tip. However, a second,shorter tip is also formed near themain tip as result of the process. The second tip is not intended forimaging and can result in “doubletip” artifacts if it contacts the sample.For this reason the NP-STT probes are restricted to samples with smallvertical features (typically less than 100nm).

OTR4: Oxide-Sharpened SiliconNitride Probes. General purposeprobe for both contact mode andTappingMode in fluid. Two differentcantilever spring constants. Generallyinterchangeable with NP-S probes.

MSCT: Sharpened ContactMicroLevers with Backside GoldCoating, oxide sharpened tip.General purpose probe for bothcontact mode and TappingMode in fluid. Six different cantilevers offera wide range of spring constants.These are particularly popular forpiconewton-scale for measurements as some of the spring constants arevery low. Also unique in that one of the levers is rectangular, rather than triangular like most silicon nitride levers.

Conclusion

Choosing the correct probe is acrucial part of working on biologicalsamples. The chemical and physicalparameters of the probe greatly affect AFM measurements and canreduce resolution. A large number of commercially available probesalready enable many differentapplications and skilled users areincreasingly modifying these probesto achieve their specific goals.

5

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