Sample preparation procedures for biological atomic force microscopy

Since the late 1980s, atomic force microscopy (AFM) has been increasingly used in biological sciences and it is now established as a versatile tool to address the structure, properties and functions of biological specimens. AFM is unique in that it provides three-dimensional images of biological structures, including biomolecules, lipid films, 2D protein crystals and cells, under physiological conditions and with unprecedented resolution. A crucial prerequisite for successful, reliable biological AFM is that the samples need to be well attached to a solid substrate using appropriate, nondestructive methods. In this review, we discuss common techniques for immobilizing biological specimens for AFM studies.     

A simple method for producing flattened atomic force microscopy tips

We describe a simple and reliable procedure for obtaining a flat plateau on top of standard silicon nitride atomic force microscopy tips by scanning them over the focus of a high-numerical-aperture objective illuminated by near-infrared ultrashort laser pulses. Flattened tips produced this way exhibit a plateau that is parallel to the substrate when the cantilever is mounted. They represent a valid and cost-effective alternative to commercially available plateau tips.     

Scanning electron microscopy studies of protein-functionalized atomic force microscopy cantilever tips

Protein-functionalized atomic force microscopy (AFM) tips have been used to investigate the interaction of individual ligand-receptor complexes. Herein we present results from scanning electron microscopy (SEM) studies of protein-functionalized AFM cantilever tips. The goals of this study were (1) to examine the surface morphology of protein-coated AFM tips and (2) to determine the stability of the coated tips. Based on SEM images, we found that bovine serum albumin (BSA) in solution spontaneously adsorbed onto the surface of silicon nitride cantilevers, forming a uniform protein layer over the surface. Additional protein layers deposited over the initial BSA-coated surface did not significantly alter the surface morphology. However, we found that avidin-functionalized tips were contaminated with debris after a series of force measurements with biotinylated agarose beads. The bound debris presumably originated from the transfer of material from the agarose bead. This observation is consistent with the observed deterioration of functional activity as measured in ligand-receptor binding force experiments.     

Real-time atomic force microscopy in lubrication condition

We have studied frictional force and wear problem in real-time atomic force microscopy in contact-mode using a resonator type mechanical scanner allegedly reported. The fast scanning may cause wear in the sample surface or the tip, and may deteriorate the image quality. Mineral oil was used to make a lubricious surface on a polycarbonate sample, and it was found that the interfacial frictional force was decreased. A Si tip which was coated with a hydrophobic film by means of chemical modification was confirmed to diminish the frictional force in the fast scanning process. The resultant image quality was improved due to reduced friction and wear.     

Promises and problems of biological atomic force microscopy

Recent developments in the biological applications of atomic force microscopy are reviewed, and the great potential of this novel, high-resolution imaging technique, including its limitations and possible future directions for biological research, are discussed.     

Scanning (atomic) force microscopy imaging of earthworm haemoglobin calibrated with spherical colloidal gold particles

Scanning (atomic) force microscopy (SFM) permits high-resolution imaging of a biological specimen in physiological solutions. Untreated extracellular haemoglobin molecules of the common North American earthworm, Lumbricus terrestris, were imaged in NH₄Ac solution using calibrated SFM. Individual molecules and their top and side views were clearly identified and were comparable with the images of the same molecule obtained by scanning transmission electron microscopy (STEM). A central depression, the presumed mouth of the hole, was detected. We analysed 75 individual molecules for their lateral dimensions. Compression varied for different molecules, presumably because of the variation of the interaction between the SFM tip and the protein molecule. Two effective heights which correspond to the heights of the points of the haemoglobin molecules first and last touched by the tip, h₁ and h₂, respectively, were measured for each protein and ranged between 1.58 and 16.2 nm for h₁ and 1.23 and 13.6 nm for h₂. The apparent diameter was measured and ranged from 44.9 to 86.6 nm (63.2±10.5 nm, n =75), which is about twice the diameter of the molecule reported by STEM for the top view orientation. The higher the measured effective heights, the worse was the tip convolution effect. In order to determine the tip parameters (semivertical angle, curvature of radius and the cut-off height) and to calibrate images of earthworm haemoglobin molecules, spherical gold particles were scanned as standards. The tip sectional radii at distances of h₁ and h₂ above the tip apex were subtracted from the apparent diameter of the protein. The calibrated lateral dimension was 29.1 ±3.85 nm, which is close to the reported scanning transmission electron microscopy data 30.0 ±0.8 nm. The results presented here demonstrate that the calibration approach of imaging gold particles is practical and relatively accurate. Calibrated SFM imaging can be applied to the study of other biomacromolecules.     

Combined atomic force and scanning reflection interference contrast microscopy

A sphere attached to a cantilever is used simultaneously as an atomic force microscope (AFM) tip and as a curved reflective surface for producing scanning reflection interference contrast microscope (RICM) images of fluorescent beads dried onto a glass slide. The AFM and RICM images are acquired in direct registration which enables the identification of individually excited beads in the AFM images. The addition of a sharp, electron beam-deposited tip to the sphere gives nanometer resolution AFM images without loss of optical contrast.     

Phase imaging with intermodulation atomic force microscopy

Intermodulation atomic force microscopy (IMAFM) is a dynamic mode of atomic force microscopy (AFM) with two-tone excitation. The oscillating AFM cantilever in close proximity to a surface experiences the nonlinear tip-sample force which mixes the drive tones and generates new frequency components in the cantilever response known as intermodulation products (IMPs). We present a procedure for extracting the phase at each IMP and demonstrate phase images made by recording this phase while scanning. Amplitude and phase images at intermodulation frequencies exhibit enhanced topographic and material contrast.     

Research on double-probe, double- and triple-tip effects during atomic force microscopy scanning

Information obtained by atomic force microscopy (AFM) depends strongly on the kind of probe or tip used; therefore, probe and tip effects have to be taken into account when verifying or interpreting the data acquired. In many papers, double-tip effects have been mentioned while other research was done; however, there are only a few special reports on double- or triple-tip effects, especially double-probe effects. In our paper, metaphase chromosomes of Chinese hamster ovary (CHO) cells, aggregates of pectin molecules, membrane surface of mouse embryonic stem cells, and R-phycoerythrin-conjugated immunoglobulin G complexes were imaged by AFM with high-quality probes, double-probe cantilever, and double-tip and triple-tip probes, respectively, in order to determine double-probe, double-tip, and triple-tip effects during AFM scanning. We found that the double-probe, double-tip, and triple-tip effects share the same principle, and that these effects correlate with distance and height differences between probes of double-probe cantilever or tips of double-tip or multiple-tip probes. Since many other factors influence double-probe or double-tip effects, more in-depth studies must be undertaken. However, this initial research will make all users of AFM techniques aware of double-probe and double-tip or triple-tip effects during AFM scanning and aid in verifying or interpreting the data acquired.     

Fabrication of ball-shaped atomic force microscope tips by ion-beam-induced deposition of platinum on multiwall carbon nanotubes

Ball-shaped atomic force microscope (AFM) tips (ball tips) are useful in AFM metrology, particularly in critical dimension AFM metrology and in micro-tribology. However, a systematic fabrication method for nano-scale ball tips has not been reported. We report that nano-scale ball tips can be fabricated by ion-beam-induced deposition (IBID) of Pt at the free end of multiwall carbon nanotubes that are attached to AFM tips. Scanning electron microscopy and transmission electron microscopy analyses were done on the Pt ball tips produced by IBID in this manner, using ranges of Ga ion beam conditions. The Pt ball tips produced consisted of aggregated Pt nano-particles and were found to be strong enough for AFM imaging.     

Method of imaging low density lipoproteins by atomic force microscopy

This short paper reports a simple method to image low density lipoproteins (LDL) using atomic force microscopy (AFM). This instrument allows imaging of biological samples in liquid and presents the advantage of needing no sample preparation such as staining or fixation that may affect their general structure. Dimensions (diameter and height) of individual LDL particles were successfully measured. AFM imaging revealed that LDL have a quasi-spherical structure on the x and y axis with an oblate spheroid structure in the z axis (i.e., height). LDLs were found to have an average diameter of 23 +/- 3 nm. The obtained mean height was 10 +/- 2 nm.     

Magnetic refinement of tips for magnetic force microscopy

Silicon cantilever probes with monolithically integrated tips are commercially available and are routinely used for atomic force microscopy (AFM). For such probes, amagnetic refinement of the silicon tip has been developed and results in a deposition of ferromagnetic material such as nickel or CrCoTa in the top area of the tip. The method consists of essentially three steps: (1) A broad-area sputter deposition of a ferromagnetic material; (2) a selective electron beam-induced carbon deposition at the top of the tip; (3) a broad-area ion-beam sputter etching, which removes the magnetic layer everywhere except underneath the carbon cap. The method allows to control the total amount and extension of the magnetic material left at the tip. It is applicable to all kinds of ferromagnetic materials which can be deposited as a thin layer by sputter deposition or evaporation. Experiments indicate that the method is reliable and improves the resolution of magnetic force microscopy (MFM). With such magnetically refined tips on silicon cantilevers, MFM measurements have been performed in contact mode as well as in dynamic and static noncontact modes. In this paper, the method for magnetic tip refinement is described and MFM measurements with these tips are presented.     

High-aspect ratio metal tips attached to atomic force microscopy cantilevers with controlled angle, length, and radius for electrostatic force microscopy

We demonstrate a method to fabricate a high-aspect ratio metal tip attached to microfabricated cantilevers with controlled angle, length, and radius, for use in electrostatic force microscopy. A metal wire, after gluing it into a guiding slot that is cut into the cantilever, is shaped into a long, thin tip using a focused ion beam. The high-aspect ratio results in considerable reduction of the capacitive force between tip body and sample when compared to a metal coated pyramidal tip.     

Magnetic domain imaging of nano‐magnetic films using magnetic force microscopy with polar and longitudinally magnetized tips

Perpendicular or parallel magnetic fields are used to magnetize the tips used in magnetic force microscopy (MFM). In this process, perpendicular or parallel magnetic dipole moments are produced on the tip plane, thus leading to the formation of polar magnetized tips (PM‐tips) or longitudinally magnetized tips (LM‐tips), respectively. The resolution of an MFM image of a magneto‐optic disk is used for calibration of these tips, and the saturated magnetic fields of the PM‐ and LM‐tips are found to be 2720 Oe and 680 Oe, respectively. Because both tips can simultaneously magnetize the sample during the scanning process when measuring a Co thin film, clear MFM images are captured, which enable the identification of magnetizable regions and the distribution of the magnetic domains on the sample surface. These results will be useful for improving the manufacturing processes required for soft nano‐magnetic film production.     

Identification and ultrastructural imaging of photodynamic therapy-induced microfilaments by atomic force microscopy

Atomic force microscopy (AFM) is an emerging technique for imaging biological samples at subnanometer resolution; however, the method is not widely used for cell imaging because it is limited to analysis of surface topology. In this study, we demonstrate identification and ultrastructural imaging of microfilaments using new approaches based on AFM. Photodynamic therapy (PDT) with a new chlorin-based photosensitizer DH-II-24 induced cell shrinkage, membrane blebbing, and reorganization of cytoskeletons in bladder cancer J82 cells. We investigated cytoskeletal changes using confocal microscopy and atomic force microscopy. Extracellular filaments formed by PDT were analyzed with a tandem imaging approach based on confocal microscopy and atomic force microscopy. Ultrathin filaments that were not visible by confocal microscopy were identified as microfilaments by on-stage labeling/imaging using atomic force microscopy. Furthermore, ultrastructural imaging revealed that these microfilaments had a stranded helical structure. Thus, these new approaches were useful for ultrastructural imaging of microfilaments at the molecular level, and, moreover, they may help to overcome the current limitations of fluorescence-based microscopy and atomic force microscopy in cell imaging.     

A force-matching method for quantitative hardness measurements by atomic force microscopy with diamond-tipped sapphire cantilevers

We present a new method to improve the accuracy of force application and hardness measurements in hard surfaces by using low-force (<50 μN) nanoindentation technique with a cube-corner diamond tip mounted on an atomic force microscopy (AFM) sapphire cantilever. A force calibration procedure based on the force-matching method, which explicitly includes the tip geometry and the tip-substrate deformation during calibration, is proposed. A computer algorithm to automate this calibration procedure is also made available. The proposed methodology is verified experimentally by conducting AFM nanoindentations on fused quartz, Si(1 0 0) and a 100-nm-thick film of gold deposited on Si(1 0 0). Comparison of experimental results with finite element simulations and literature data yields excellent agreement. In particular, hardness measurements using AFM nanoindentation in fused quartz show a systematic error less than 2% when applying the force-matching method, as opposed to 37% with the standard protocol. Furthermore, the residual impressions left in the different substrates are examined in detail using non-contact AFM imaging with the same diamond probe. The uncertainty of method to measure the projected area of contact at maximum force due to elastic recovery effects is also discussed.     

Development and testing of hyperbaric atomic force microscopy (AFM) and fluorescence microscopy for biological applications

A commercially available atomic force microscopy and fluorescence microscope were installed and tested inside a custom-designed hyperbaric chamber to provide the capability to study the effects of hyperbaric gases on biological preparations, including cellular mechanism of oxidative stress. In this report, we list details of installing and testing atomic force microscopy and fluorescence microscopy inside a hyperbaric chamber. The pressure vessel was designed to accommodate a variety of imaging equipment and ensures full functionality at ambient and hyperbaric conditions (≤85 psi). Electrical, gas and fluid lines were installed to enable remote operation of instrumentation under hyperbaric conditions, and to maintain viable biological samples with gas-equilibrated superfusate and/or drugs. Systems were installed for vibration isolation and temperature regulation to maintain atomic force microscopy performance during compression and decompression. Results of atomic force microscopy testing demonstrate sub-nanometre resolution at hyperbaric pressure in dry scans and fluid scans, in both contact mode and tapping mode. Noise levels were less when measurements were taken under hyperbaric pressure with air, helium (He) and nitrogen (N(2) ). Atomic force microscopy and fluorescence microscopy measurements were made on a variety of living cell cultures exposed to hyperbaric gases (He, N(2) , O(2) , air). In summary, atomic force microscopy and fluorescence microscopy were installed and tested for use at hyperbaric pressures and enables the study of cellular and molecular effects of hyperbaric gases and pressure per se in biological preparations.     

Levels of plant cell wall structural organization revealed by atomic force microscopy

We used an atomic force microscope to image cell wall isolated from needles of Serbian spruce tree and that synthesized from cell wall components. We also observed the structure of lignin model polymer (DHP), as a best substitute for the natural lignin. A tendency of aggregate formation was observed in all samples. Cell wall was revealed as a laminated fibrous structure. General organization is similar in both isolated and synthesized cell wall samples, with dominating globular motifs arranged regularly as rods and forming cavities. The synthesized cell wall has a more regular structural organization than isolated cell wall. The dimensions of individual globular aggregates and pores differed between the two samples. DHP showed a similar, regular organization, with globular aggregates and holes. Globules and pores are smaller in size than the corresponding structures in both the isolated and synthesized cell walls. Such modular organization of cell walls may have a physiological role in response to the external mechanical stress caused to plant cells.