Sample preparation in AFM for soft materials and life science

[1] Surface biology of DNA by atomic force microscopy.
Hansma, HG.
Annu. Rev. Phys. Chem. 52 (2001) 71-92.

[21] Probing biopolymers with the atomic force microscope: A review. Hansma, HG, Pietrasanta, LI, Auerbach, ID, Sorenson, C, Golan, R, Holden, PA.
 

The 72 page review article published in 1996 by Shao [17] gives a first insight into the requirements for sample preparation in AFM:

"It is clear that the major impediment to successful AFM imaging of biological materials is sample preparation. This is a particularly difficult issue as every sample requires a unique approach. The objective with any sample is to achieve a firm adhesion to a substrate of sufficiently minimal topography that the topography of the sample is easily discriminated. This fixing to the surface is to guarantee that the probe tip does not push the sample around during imaging."  •
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[17] Biological atomic force microscopy: what is achieved and what is needed.
Shao, Z, Mou, J, Czajkowsky, DM, Yang, J, Yuan, J-Y.
Advances in Physics 45 (1996) 1-86.
 

Substrates
Since AFM does not require the substrate to be conductive, the choice of substrates is almost unlimited. Both glass cover slips and cleaved mica sheets have been used to adsorb various macromolecules and organelles with varying degrees of success. However, the functionality of the adsorbed macromolecules on these surfaces has not been characterized by biochemical experiments. Within the resolution achieved by AFM so far, structural changes have not been observed when compared with that obtained by electron microscopy. Imaging of living cells in buffer solutions did not appear to affect the integrity of the cells either. This, of course, does not imply that structural changes at higher resolutions can be entirely ruled out, nor that the function of theses molecules is not in some way affected. Detailed comparison with X-ray structures or cryo-electron microscopy should be invaluable in addressing this question. [17]
 

[17] Biological atomic force microscopy: what is achieved and what is needed.
Shao, Z, Mou, J, Czajkowsky, DM, Yang, J, Yuan, J-Y.
Advances in Physics 45 (1996) 1-86.
 

Today, a number of specimen supports are used, among which the most important are epitaxially grown gold films, layered transition metal dichalcogenides, amorphous thin films, glass, silicon and mica. The support used depends on the specimens and on how they are to be immobilized: by physisorption, covalent bonding, self-assembly on van der Waals surfaces or the Langmuir-Blodgett technique. In the following, frequently used specimen supports and the most important immobilization techniques are described.[4]

Many sample preparation techniques used for AFM are derived from sample preparation in electron and light microscopy. For example samples are prepared on glass slides for light microscopy or on iron stubs used for electron microscopy. In 1968 Kleinschmidt [18] published a method for DNA preparation, long time before the AFM was invented and which today is still in use. The basics about substrates used for AFM sample preparation are described in [4]:

[4] Sample preparation techniques in scanning probe microscopy.
Amrein, M, Müller, DJ.
Nanobiology 4 (1999) 229-256.

Substrates: preferences
The biopolymers to be imaged in the AFM are normally dissolved in aqueous solution and then deposited onto the substrate. Compared with the size of the sample molecule the substrate has to be flat and easy to prepare.  •
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Mica
da Silva and Kindt described the properties of mica in [3, 20]:
The most commonly used substrate is mica. Mica is a non-conducting layered material. It is cheap and can easily be cleaved, usually with a pin or sometimes cellotape, to produce clean, atomically flat surfaces up to even millimetres in size. The commonest form of mica is Muscovite KAl2(OH)2AlSi3O10. The minimum step size which can be observed on the surface is the sickness of an individual layer (1 nm) and the hexagonal lattice constant within the layers, which can be used for calibration, is 0.52 nm." "The root-mean-square roughness is 0.06 ± 0.01 nm." [3] "Mica has been successfully used in numberless studies especially for AFM imaging of double stranded DNA and DNA-protein complexes, protein arrays, and densely packed proteins. Although the mechanism by which macromolecules absorb to this substrate still remains poorly understood, a large number of protein samples adhere tightly to this surface." [20]

[3] Biological probe microscopy in aqueous fluids.
Kindt, JH et al. In Jena, BP & Hörber, HJK. Atomic force microscopy in cell biology. Academic press, San Diego, London, 2002.

[20] Atomic force microscopy and proteins.
da Silva, LP.
Protein and Peptide Letters. 9 (2002)117-125.

Mica's property to be highly charged at the surface leads to the fact that it is always covered with a thin (0.5 nm) layer of water when exposed to ambient air. This water layer leads to a continuous adhesion between AFM tip and sample.

Despite the widespread use of these substrates, the mechanism of adsorption is not well understood. For both glass and mica in aqueous media, it is known that positive ions tend to dissociate from the surface to make them negatively charged. It is also known that most protein surfaces contain both positively and negatively charged residues at neutral pH and that the neutrality can be altered by changing the pH of the buffer solution. For example, ferritin has an isoelectric point of about 5 and is positively charged at pH < 5 and negatively charged at pH > 5. Therefore it seems plausible to assume that electrostatic interaction is primarily responsible for adsorption. However, we must also realize that most of these charged groups are shielded by counter-ions in solution. It is not clear whether it is the direct interaction between the oppositely charged groups o the salt bridges between like charged groups that is responsible for surface adsorption in each case. [17]  •
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[17] Biological atomic force microscopy: what is achieved and what is needed.
Shao, Z, Mou, J, Czajkowsky, DM, Yang, J, Yuan, J-Y.
Advances in Physics 45 (1996) 1-86.
 

Glass
Glass is flat enough for imaging cells or other large and relatively high samples. Apart from cells, also large molecules like tubulin molecules, chromosomes or cell organels. On the other hand glass is generally too rough for reliable visualization of DNA, especially under fluid.[3]

For preparation of AFM samples on glass coverslips, the round ones with a diameter of 25 mm and a thickness of 0.17 mm are widely used. Also the square cover slides are easily obtainable. Amrein describes a method for cleaning glass in [4]: "The slides can be used either unmodified or altered to change their physisorption or chemical properties. The surface can be almost featureless on the scale of macromolecular specimens. They are best suited for all experiments in which visible light is transmitted across the sample, as in scanning near field optical microscopy (SNOM) or in the combined light- microscopy and SPM. […] Before use, organic contaminants, dust or other particles are removed by washing on time with concentrated HCl/HNO3 (3:1 v/v) and 5 times for 1 min with Millipore water in an ultrasonic bath (50 kHz). This process makes the coverslips clean and smooth (rms-roughness ~ 0.5 nm). They show a mottled background with less than one particle/µm2 (particle size ~ 0.3 µm2).  •
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[3] Biological probe microscopy in aqueous fluids.
Kindt, JH et al. In Jena, BP & Hörber, HJK. Atomic force microscopy in cell biology. Academic press, San Diego, London, 2002.

[4] Sample preparation techniques in scanning probe microscopy.
Amrein, M, Müller, DJ.
Nanobiology 4 (1999) 229-256.

Glass cleaning
Using glass as a solid support in AFM experiments it requires cleaning. In [11] two glass cleaning methods are recommended:

Method 1

• rinse with ultrapure (Milli-Q) water several times
• rinse with ethanol
• rinse with Milli-Q- water
• air plasma treatment for 20 min
• dry in a nitrogen flow
• additional treatment in the plasma cleaner increases the charge density on the surface.

Method 2

Precleaning with detergent

• glass is pre-cleaned with commercial detergent
• rinse with Milli-Q water

Removal of organic contamination

• incubation for 5-10 min. in RCA1 solution at 70 deg C. RCA1 solution is a mixture of Milli-Q, ammonia (25%), hydrogenperoxide (30%) in 5:1:1 ratio.
• thoroughly rinse with Milli-Q water

Removal of inorganic contamination

• incubation for 5-10 min in RCA2 solution at 70 deg C.
RCA2 solution is a mixture of Milli-Q, hydrochloric acid (37%), hydrogenperoxide (30%) in 5:1:1 ratio.

• thoroughly rinse with Milli-Q-water
• dry in nitrogen flow

[11] Rasterkraftmikroskopische Untersuchungen von natürlichen und künstlichen Lipidmembranen und daran gebundener Proteine.
Eschrich, R. Thesis. Technische Universität München 1998.

Gold
The properties of gold surfaces used for AFM sample preparation are described in [4 ]:
Gold surfaces can be easily prepared by vapor deposition onto glass and mica. Gold is chemically inert against oxygen and stable against radicals. It binds organic thiols or bifunctional disulfides with high affinity, which can be used to covalently attach biological macromolecules.  •
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[4] Sample preparation techniques in scanning probe microscopy.
Amrein, M, Müller, DJ.
Nanobiology 4 (1999) 229-256.

Epitaxially grown gold surface Au(111)
Special preparation techniques deliver ultraflat gold surfaces as described in [17] and [5]: "For epitaxially grown gold surfaces, atomically flat areas of up to several micrometers can be found and the surface is hydrophilic if clean." "Ultraflat Au(111) surfaces have a mean roughness of 0.2-0.5 nm over areas larger than 25 µm2. "  •
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Silicon
Also parts of silicon wafers used in semiconductor industry can be used as substrate. They have a thin oxide layer on their surface which makes them hydrophilic because of the OH groups on the surface. [17]  •
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[5] Immobilization of native biomolecules onto Au(111) via N-hydroxysuccinimide ester functionalized self-assembly monolayers for scanning probe microscopy. Wagner, P, Hegner, M, Kernen, P, Zaugg, F, Semenza, G. Biophys. J. 70 (1996) 2052-2066.

[17] Biological atomic force microscopy: what is achieved and what is needed.
Shao, Z, Mou, J, Czajkowsky, DM, Yang, J, Yuan, J-Y.
Advances in Physics 45 (1996) 1-86.
 

Graphite - HOPG
HOPG is not widely spread among AFM sample techniques. Its chemical properties and its disadvantages are described in [4] and [5]:
The sheets were of HOPG were flat on an atomic scale over areas of microns. They were also well conductive, which was a prerequisite for STM. However, the adsorption of most biological specimens was very poor. Moreover, defects and the fine structure of step edges of pure graphite were very misleading, since they often resembled the expected structure of the specimen. Graphite is now used exclusively as a support for the investigation of self-assembly monolayers of organic molecules. " [4] "Furthermore, HOPG bind macromolecules by way of weak electrostatic or "adsorption" forces, and, moreover, it has fallen into, perhaps excessive disrepute when it was shown to yield artifactual images mimicking DNA [in STM investigations]." [5 ]  •
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[4] Sample preparation techniques in scanning probe microscopy.
Amrein, M, Müller, DJ.
Nanobiology 4 (1999) 229-256.

[5] Immobilization of native biomolecules onto Au(111) via N-hydroxysuccinimide ester functionalized self-assembly monolayers for scanning probe microscopy. Wagner, P, Hegner, M, Kernen, P, Zaugg, F, Semenza, G. Biophys. J. 70 (1996) 2052-2066.
 

Thermanox
A rough polymer like Thermanox is only suitable for AFM imaging of large structures like cells as described in [11]. Thermanox is hydrophobic and available from different distributors [11].  •
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[11] Rasterkraftmikroskopische Untersuchungen von natürlichen und künstlichen Lipidmembranen und daran gebundener Proteine.
Eschrich, R. Thesis. Technische Universität München 1998.

Functionalized surfaces
To strengthen further the adsorption, functionalized surfaces can be introduced. The simplest method is to treat the surface with certain molecules, such as poly-L-lysine or poly-L-arginine (van Holde: Chromatin, 1989), in order to change the charge characteristics of the surface. Therefore the adsorption can be enhanced or modified. One step further is to introduce cross-linking groups to the surface. So far, several schemes have been used for AFM imaging in solution. One method is based on silanizing a solid surface with 3-aminopropyltriethoxysilane (APTES) (Lyubchenko, 1992, 1993), which protonates at neutral pH. The silane group in APTES is highly reactive and silanizes the surface by forming covalent bonds with surface atoms. Karrasch and co-workers introduced another cross-linking group at the amino end of APTES on a glass surface, N-5-azido2-nitrobenzoyloxysuccinimide (ANB-NOS) [10]. The azide group, upon ultraviolet irradiation, can make non-specific covalent bonds to proteins on contact. Since the functionalized surface becomes hydrophobic, and soluble proteins do not come close enough to be cross-linked, a squeezing pressure of 10-500 atm must be used to force macromolecules to come within reach of the azide group. In another method, an ultraflat Au(111) surface is used as a substrate for N-hydroxysuccinimide terminated self-assembled monolayers of dithio-bis(succinidylundecanoate). This monolayer readily reacts with amino groups, covalently linking the protein to the substrate." [17]  •
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[10] Covalent binding of biological samples to solid supports for scanning probe microscopy in buffer solution.
Karrasch, S, Dolder, M, Schabert, F, Ramsden, J, Engel, A.
Biophys. J. 65 (1993) 2437-2446.

[17] Biological atomic force microscopy: what is achieved and what is needed.
Shao, Z, Mou, J, Czajkowsky, DM, Yang, J, Yuan, J-Y.
Advances in Physics 45 (1996) 1-86.
 

Silanized surfaces - the APTES method
AP-Mica has amino groups exposed to the surface. Aliphatic amino groups have a pK of approximately 10.6. Although the close packing of the aliphatic amino groups on the AP-mica surface decreases the pK, the surface will still be positively charged in solution at neutral pH. [8]  •
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[8] Adsorption of DNA to mica, silylated mica, and minerals: Characterization by atomic force microscopy.
Bezanilla, M, Manne, S, Laney, DE, Lyubchenko, YL, Hansma, HG.
Langmuir 11 (1995) 655-659.

AP glass
The method for APTES coated glass is described in [10]:

"Silanization and derivatization of coverslips were carried out in Petri dishes that had been washed in "piranha bath" (3.5 % H2O2 in 18 M H2SO4), followed by rinsing with water and acetone. The following steps were carried out at room temperature unless stated otherwise. Coverslips were washed once with concentrated HCl/HNO3 (3:1) and five times for 1 min with destilled water in an ultrasonic bath (50 kHz). They were etched with trifluoroacetic acid for 90 min and stored in vacuum over solid KOH for at least 10 h. Coverslips were then silanized with APTES (2 % in 95 % aqueous acetone) for 3 min followed by washing with acetone (12 times, 5 min each) […]. Curing of the silane linkages was carried out in an oven at 110 °C for 1 h. " [10]

[10] Covalent binding of biological samples to solid supports for scanning probe microscopy in buffer solution.
Karrasch, S, Dolder, M, Schabert, F, Ramsden, J, Engel, A.
Biophys. J. 65 (1993) 2437-2446.
 

3-aminopropyltriethoxysilane APTES

3'-glycidoxypropyltrimenthoxysilane GOPS

1-(3-Aminopropyl)silatrane APS

ANB-NOS
N-5-azido2-nitrobenzoyloxysuccinimide

Schematic reaction diagram of an APTES coated surface with crosslinker ANB-NOS. After [10].

AP-silicon
Möller et al. [6] described a modified protocol for the APTES method: "The [ silicon] chips were immersed for 15 min in 1 % APTES solution in 95 % acetone/water. Afterwards, the chips were washed five times (5 min each) with acetone and dried for 45 min at 110 °C. They were then incubated for 2 h with a solution 0.2 % 1,4-phenylenediisothiocyanate in 10 % pyridine/dimethyl formamide and washed with methanol and acetone. The activated chips may be stored in a vacuum desiccator containing anhydrous calcium chloride for a longer time without discernible loss of activity."

Möller's et al. modified procedure for the GOPS method is: "For substrate modification with 3'-glycidoxypropyl-trimethoxysilane (GOPS) the slides were suspended in dry toluene containing 1% silane at 80° C for 4-6 h, using a modified procedure from the literature [7]."  •
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[6] DNA probes on chip surfaces studied by scanning force microscopy using specific binding of colloidal gold.
Möller, R. Andrea Csáki, Köhler, M, Fritzsche, W. Nucleic Acids Research 28 (2000) 1-5.

[7] Direct detection of nucleic acid hybridization on the surface of a charge coupled device. Lamture, JB, Beattie, KL, Burke, BE, Eggers, MD, Ehrlich, DJ, Fowler, R, Hollies, MA, Kosicki, BB, Reich, RK, Smith, SR, Varma, RS, Hogan, ME. Nucleic Acids Res. 22 (1994) 2121-2125.

[8] Adsorption of DNA to mica, silylated mica, and minerals: Characterization by atomic force microscopy.
Bezanilla, M, Manne, S, Laney, DE, Lyubchenko, YL, Hansma, HG.
Langmuir 11 (1995) 655-659.

[22] Glutaraldehyde modified mica: a new surface for atomic force microscopy of chromatin. Wang, H., Bash, R., Yodh, J.G., Hager, G.L., Lohr, D., Lindsay, S.M. Biophys. J. 83 (2002) 3619-3625.
 

ANB-NOS modified surfaces
"All subsequent steps were performed in the darkroom using red safety light. The reaction of the NH2 groups with the succinimide ester group of ANB-NOS was carried out in 0.1 M Na2CO3, pH 9.0. The reaction mixture was prepared by adding 10 nmol ANB-NOS/cm2 glass surface dissolved in 1 ml dioxane to 20 ml Na2CO3 solution. This corresponds to a 10-fold molar excess of the photocross-linker with respect to the amino groups. The coverslips were incubated for 4 h. Excess reagent was then removed by washing the coverslips three times with distilled water, and two times with acetone. Coverslips were stored under vacuum and handled in the dark." [10]

[10] Covalent binding of biological samples to solid supports for scanning probe microscopy in buffer solution.
Karrasch, S, Dolder, M, Schabert, F, Ramsden, J, Engel, A.
Biophys. J. 65 (1993) 2437-2446.

A yet more detailed method was described by Liu [24] :
„As described by Karrash, APTES-coated coverslips were immersed in 0.1 M Na2CO3 (1 mL dioxane to 20 mL of Na2CO3 solution, pH 9.0) containing 3 µmol ANBNOS for at least 4 h. The concentration of ANBNOS was thus at least tenfold molar excess with respect to the amino groups of APTES on the coverslip (approx. 1 nmol NH2 groups/cm2). ANBNOS reacts with the exposed amino end of APTES and covalently binds to the coverslip surface […]. Excess ANBNOS was removed by washing the coverslip with gentle pipet squirting 3 times with 1 % n-butylamine in 0.1 M Na2CO3, 3 times with 0.1 M Na2CO3, 2 times with deionised water, and 2 times with acetone. Air-dried coverslips were stored in a vacuum-sealed desiccator and handled in the dark.” [24]  •
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[24] Sample preparation and imaging of erythrocyte cytoskeleton with the atomic force microscopy.
Liu, F, Burgess, J, Mizukami, H, Ostafin, A.
Cell Biochem.Biophys. 38 (2003) 251-270.
 

APS-mica
"Unlike the (3-aminopropyl)triethoxysilane (APTES) commonly used for silica surface modifications, 1-(3-aminopropyl)silatrane (APS) is not very reactive and is extremely resistant to hydrolysis and polymerisation at neutral pH. " [9]

APS synthesis:
1-(3-Aminopropyl)silatrane was prepared by vacuum evaporation of a mixture of 4.13 g of triethanolamine containing 1 mg of sodium as a catalyst and 6.12 g of (3-aminopropyl)triethoxysilane at 60 °C to constant weight (6.4 g). The compound obtained by this procedure can be used directly without purification for surface modification. A 50 mM solution of APS in deionised water was used as a stock solution. The stock was stored in refrigerator at 4 °C with no special precautions." [9]

"Preparation of APS mica for AFM: The APS stock solution diluted 1: 300 in deionised water was used for APS-mica preparation. Freshly cleaved mica strips of appropriate sizes were immersed into APS working solution for 30 min, rinsed thoroughly with deionised water, Ar dried and stored in Ar filled tubes." [9]  •
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[9] Silatrane-based surface chemistry for immobilization of DNA, protein-DNA complexes and other biological materials.
Shlyakhtenko, LS, Gall, AA, Filonov, A, Cerovac, Z, Lushnikov, A, Lyubchenko, YL.
Ultramicroscopy 97 (2003) 279-287.
 

Immobilization on poly-L-lysine-coated surface
Poly-L-lysine is a positively charged polymer with adsorbs very well to negatively charged glass or silicon dioxide leading to positively charged surfaces.

"Acid-washed coverslips were coated with a poly-L-lysine solution (10 mg/mL ; Mr 1000-4000), washed with water after 1 min and dried in air. The protein solution (3 mg/mL) was deposited on the dry surface and washed off after 15 min." [10]  •
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[10] Covalent binding of biological samples to solid supports for scanning probe microscopy in buffer solution.
Karrasch, S, Dolder, M, Schabert, F, Ramsden, J, Engel, A.
Biophys. J. 65 (1993) 2437-2446.
 

Hydrophobic /hydrophilic surfaces
"For AFM in solution, hydrophobic surfaces do not appear to be a very useful substrate, not only because many proteins could denature upon contact with a hydrophobic surface, but also because the Si3N4 tip interacts strongly with a hydrophobic surface, resulting in a noticeable adhesion force in solution. The exact nature of these interactions is not fully understood, and reliable results cannot be obtained. However, for AFM in air, hydrophobic surfaces can be adequate, and sometimes superior. For example, carbon-coated mica was shown to be a good substrate for imaging DNA with a very good lateral resolution and specimen stability. One problem with a hydrophilic surface in air is that a thin layer of water will be condensed onto the surface for medium to high humidity. This condensed water can interact with the tip giving rise to a quite large adhesion force (capillary force) and can reduce the stability of adsorbed macromolecules. " [17]  •
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[17] Biological atomic force microscopy: what is achieved and what is needed.
Shao, Z, Mou, J, Czajkowsky, DM, Yang, J, Yuan, J-Y.
Advances in Physics 45 (1996) 1-86.
 

[11] Rasterkraftmikroskopische Untersuchungen von natürlichen und künstlichen Lipidmembranen und daran gebundener Proteine.
Eschrich, R. Thesis. Technische Universität München 1998.

Method 2 - Silanization with n-octadecyltrimethoxysilane
"n-octadecyltrimethoxysilane (Gelest or United Chemical), methanol (Optima Grade, Fisher), and heptane (Optima Grade, Fisher) were used as received. Water was deionized, distilled over quartz, and filtered by a Milli-Q reagent water system (Millipore Co.), which resulted in a resistivity of 18 MΩ*cm.
Glass microscope slides (Fisher Scientific; rms surface roughness measured by AFM was 1.5 (0.2 Å) were soaked in concentrated NH4OH for 1 h and washed 5 times by ultrasonication in 18 MΩ*cm water. The surfaces were then dried for 1 h at 140 °C and placed directly in methanol to keep the surface free from water vapor and other unwanted adsorbates. Glass surfaces and SiO2-coated cantilevers were silanized by immersion in a 5 mM silane solution in heptane for 24 h. The silanized surfaces were washed 3 times with heptane to remove unreacted silane materials. The treated surfaces were then heated for 1 h at 70 °C. After being coated, they were rinsed with acetone to remove any unreacted silane and then stored in methanol until used." [23]

[23] Single-molecule bond-rupture force analysis of interactions between AFM tips and substrates modified with organosilanes.
Wenzler, L.A., Moyes, G. L., Olson, L. G., Harris, J. M., and Beebe, Jr, T. P.
Anal. Chem. 69 (1997) 2855-2861.

N-Hydroxysuccinimide ester functionalization
"The approach is based on the ex-situ synthesis of dithiobis(succinimidyl-undecanoate) DSU providing a long-chain N-hydroxysuccinimide-ester functionalized dialkyldisulfide, which is accessible for nucleophilic attack (e.g. amide bond formation with amino-group- containing molecules)." [12 ]

[12] Omega-functionalized self-assembled monolayers chemisorbed on ultraflat Au(111) surfaces for biological scanning probe microscopy in aqueous buffers. Wagner, P, Zaugg, F, Kernen, P, Hegner, M, Semenza, G.
J. Vac. Sci. Technol. B 14 (1996) 1466-1471.

Synthesis and self-assembly of DSU dithiobis(succinimidylundecanoate) [after 5]

Synthesis of 3
"Sodium thiosulfate (55.3 g, 350 mmol) was added to a suspension of 11-bromo-undecanoic acid 1 (92.8 g, 350 mmol) in 50 % aqueous 1,4-dioxane (1000 ml). The mixture was heated at reflux (90 °C) for 2 h until the reaction to the intermediate Bunte salt 2 was complete (clear solution). The oxidation to the corresponding disulfide was carried out in situ by addin iodine in portions until the reaction retitrated with 15 % sodium pyrosulfite in water. After removal of 1,4 dioxane by rotary evaporation the creamy acetate/tetrahydrofuran (THF) provided 3 as a white solid (73.4 g, 96.5 %): mp 94 °C." [5]

Synthesis of 4
"To a solution of 3 (1.0 g, 2.3 mmol) in THF (50 ml) was added N-hydroxysuccinimide (0.575 g, 5 mmol) followed by dicyclohexylcarbodiimide (DCC, 1.03 g, 5 mmol) at 0 °C. After the reaction mixture was allowed to warm to 23 °C and was stirred for 36 h at room temperature, the dicyclohexylurea (DCU) was filtered. Removal of the solvent under reduced pressure and recrystallization from acetone/hexane provided 4 as a white solid. Final purification was achieved by medium-pressure liquid chromatography (9 bar) using silica gel and a 2:1 mixture of ethyl acetate and hexane. The organic phase was concentrated and dried in vacuum to yield 4 (1.12 g, 78 %): mp 95 °C." [5].  •
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[5] Immobilization of native biomolecules onto Au(111) via N-hydroxysuccinimide ester functionalized self-assembly monolayers for scanning probe microscopy. Wagner, P, Hegner, M, Kernen, P, Zaugg, F, Semenza, G. Biophys. J. 70 (1996) 2052-2066.
 

Preparation of ultraflat gold
The preparation of ultraflat gold is described in [5]:
"[…] gold was evaporated onto mica and glued upside down to a silicon wafer or glass coverslip; the mica was then removed by soaking in tetrahydrofuran. These "template-stripped" gold surfaces (TSG) consisted, therefore, of the gold atom layer that had been deposited on the mica template first."

More detailed instructions for the preparation of ultraflat gold are described in [13],[14], [15] .  •
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Adhesive tape
Commercially available adhesive tape can be used for immobilization of powders or even biological samples. First a strip of tape is superglued with its uncovered side to a microscope glass slide.
When an inorganic powder has to be imaged it is trickled onto the glue side of the tape and the supernatant is removed with a stream of pure nitrogen.
Also red blood cells have been successfully immobilized from suspensions. In this case the suspension has been poured onto the tape. After a short incubation time of some minutes the cells adhered. In our lab, red blood cells and polyelectrolyte shells could be successfully adsorbed onto adhesive tape, even in aqueous solution.  •
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[13] Ultralarge atomically flat template-stripped Au surfaces for scanning probe microscopy. Hegner, M, Wagner, P, Semenza, G. Surf. Sci. 291 (1993) 39-46.

[14] Formation and in situ modification of monolayers chemisorbed on ultraflat template-stripped gold surfaces. Wagner, P, Hegner, M, Güntherodt, H-J, Semenza, G. Langmuir 11 (1995) 3867-3875.

[15] Procedures in scanning probe microscopy. Hegner, M, Wagner, P. Ultraflat Au surfaces. In: Colton, RJ, et al. (ed.) John Wiley 1998. ISBN 047195912X

Superglue
A drop of superglue is dabbed onto a glass slide and smeared like a blood smear. When an inorganic powder has to be imaged it is trickled onto the glue and the supernatant is removed with a stream of pure nitrogen after drying.
Note: Superglue cannot be used when AFM experiment is performed in aqueous environment.  •
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Binding of biological molecules to a solid substrate
Karrasch et al. [10] published a method to covalently bind biomolecules and supramolecular assemblies such as

- bacteriophage T4 polyheads
- eucariotic intermediate filaments
- HPI layer of deinococcus radiodurans

Their method is described as follows:
"For the covalent binding of samples to the modified glass surface, the [APTES-ANBNOS-covered] coverslips were squeezed between two glass disks (borosilicate safety sight glass; diameter = 12 cm, thickness = 2 cm) at a pressure of 100 to 5000 N/cm2 to bring the hydrophilic biological structures into close contact with the hydrophobic cross-linker. Covalent coupling of the samples was induced by activating the azide with ultraviolet (UV) irradiation at 366 nm (Sylvania F8T5) at a distance of 10 cm for 3 min. The extent of the reaction was determined from the change in the absorption band of ANB-NOS at 312 nm. Coverslips were rinsed thoroughly with water to remove excess protein and stored in water or buffer. " [10]

[10] Covalent binding of biological samples to solid supports for scanning probe microscopy in buffer solution.
Karrasch, S, Dolder, M, Schabert, F, Ramsden, J, Engel, A.
Biophys. J. 65 (1993) 2437-2446.
 

[24] Sample preparation and imaging of erythrocyte cytoskeleton with the atomic force microscopy.
Liu, F, Burgess, J, Mizukami, H, Ostafin, A.
Cell Biochem.Biophys. 38 (2003) 251-270.
 

The Kleinschmidt method
For sample preparation in electron microscopy Kleinschmidt in 1968 [ ] published a monolayer transfer method:
"The DNA solution is spread on the surface of a water subphase. The protein film, along with the adsorbed DNA molecules, is picked up directly by the substrate." [16]  •
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[16] Promises and problems of biological atomic force microscopy (review). Yang, J., Tamm, L. K., Somlyo, AP, Shao Z. J. Microscopy 171 (1993) 183-198.

DNA adsorption to APTES mica
The method to adsorb double stranded DNA to an APTES modified mica sheet is described in [19 ]:
"Modified mica strips were immersed into DNA in Tris/HCl buffer (pH 7) (10 mM Tris/HCl, 10-20 mM NaCl, 5 mM EDTA) and incubated at room temperature for between 1 and 2 h. Concentration of DNA was varied between 0.01 and 0.1 µg/mL. λ-DNA and HindIII fragments of λ-DNA were purchased from New England BioLabs and used without additional purification. After the adsorption stage had been completed, the samples were rinsed with deionized water, blotted at the edge and vacuum-dried."  •
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[19] Atomic force microscopy of long DNA: Imaging in air and under water. Lyubchenko, Y, Shlyakhtenko, L, Harrington, R, Oden, P, Lindsay, S. Proc. Natl. Acad. Sci. USA 90 (1993) 2137-2140.

Protein adsorption
AFM has the potential to make unique contributions to the study of (membrane) proteins, not only in generating nanometre resolution structures, but also in studying structural changes under various conditions.
For AFM, supported membranes have been shown to be most appropriate for achieving high resolution. Unsupported membranes, such as the plasma membrane in an intact cell, are too soft and easily deformed under the AFM tip, preventing any high-resolution imaging. So far, the best resolution achieved in solution on cell surfaces, either fixed or native, is only in the range of several 10 nm, which is insufficient to resolve membrane proteins and other membrane structures. With supported membranes on mica or glass, the best resolution is an order of magnitude higher, approaching subnanometre in some cases. [17]

[17] Biological atomic force microscopy: what is achieved and what is needed.
Shao, Z, Mou, J, Czajkowsky, DM, Yang, J, Yuan, J-Y.
Advances in Physics 45 (1996) 1-86.

[20] Atomic force microscopy and proteins. da Silva, LP. Protein and Peptide Letters. 9 (2002)117-125.

[21] Probing biopolymers with the atomic force microscope: A review. Hansma, HG, Pietrasanta, LI, Auerbach, ID, Sorenson, C, Golan, R, Holden, PA.
 

Substrate supported lipid membranes
For decades, the use of the Langmuir trough to transfer mono- and bilayers of phospholipids, fatty acids or other amphiphilic compounds onto glass, mica or silicon is well established. It depends on the properties of the substrate surface, if it is hydrophilic or hydrophobic, to influence the orientation of the molecular layer(s). By this method, a surface coverage of nearly 100 % can be achieved. [16]  •
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[16] Promises and problems of biological atomic force microscopy (review). Yang, J., Tamm, L. K., Somlyo, AP, Shao Z. J. Microscopy 171 (1993) 183-198.
 

Sources of supply
Glass coverslips: Plano, W. Planet GmbH, Marburg, Germany.
Plasma cleaner: Harrick Plasma cleaner
UV-lamp: Pen-ray lamp
Mica: Mica New York Corp. 75 Varick Street, NY 10013.
PLL: Poly-L-lysine hydrobromide, Sigma P0879, mol wt 1,000 to 4,000.
Dichlorodimethylsilane (CH3)2SiCl2: Aldrich 44,027-2.
3-aminopropyltriethoxysilane: Aldrich 440140
1,4-phenylenediisothiocyanate C6H4(NCS)2: Aldrich: 25,855-5
3'-glycidoxypropyltrimethoxysilane: Power Chemical Corporation (www.powerchemical.net): PC3100
N-5-azido2-nitrobenzoyloxysuccinimide ANB-NOS: Pierce Biotechnology, USA (www.piercenet.com) 21451, Apollo Scientific, UK (www.apolloscientific.co.uk) BIPA110
Thermanox: Miles Scientific, Nunc GmbH, Wiesbaden, Germany.
Dithiobis(succinimidyl-undecanoate) DSU: Dojindo, Japan (www.dojindo.com): code D539.
11-Amino-1-undecanethiol, hydrochloride: Dojindo, Japan (www.dojindo.com): code A 423. •
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