Combining AFM and FRET for high resolution fluorescence microscopy

Here we demonstrate a new microscopic method that combines atomic force microscopy (AFM) with fluorescence resonance energy transfer (FRET). This method takes advantage of the strong distance dependence in Förster energy transfer between dyes with the appropriate donor/acceptor properties to couple an optical dimension with conventional AFM. This is achieved by attaching an acceptor dye to the end of an AFM tip and exciting a sample bound donor dye through far-field illumination. Energy transfer from the excited donor to the tip immobilized acceptor dye leads to emission in the red whenever there is sufficient overlap between the two dyes. Because of the highly exponential distance dependence in this process, only those dyes located at the apex of the AFM tip, nearest the sample, interact strongly. This limited and highly specific interaction provides a mechanism for obtaining fluorescence contrast with high spatial resolution. Initial results in which 400 nm resolution is obtained through this AFM/FRET imaging technique are reported. Future modifications in the probe design are discussed to further improve both the fluorescence resolution and imaging capabilities of this new technique.     

Feedback based simultaneous correction of imaging artifacts due to geometrical and mechanical cross-talk and tip-sample stick in atomic force microscopy

This paper presents a feedback scheme that simultaneously corrects, in real time, for the imaging artifacts caused by cantilever and photosensor misalignments as well as misinterpretations in relative lateral position of the tip with respect to the sample due to the tip-sample stick in atomic force microscopy (AFM). The optical beam bounce method, typically used in AFM for imaging, is sensitive to inaccuracies of cantilever geometry and the relative misalignment of the laser source, cantilever, and the laser sensitive diode from the intended design. These inaccuracies, which contribute to the geometrical cross-talk between the normal and the lateral signals, become prominent at the atomic and subnanometer scales, and thereby impede high resolution imaging studies. The feedback scheme accounts for these artifacts and makes imaging insensitive to, in fact, practically independent of these inaccuracies. This scheme counteracts the lateral twisting dynamics of the cantilever, and as a result, it avoids the misinterpretation problem of the relative lateral position of the cantilever tip from the sample and thereby avoids the corresponding imaging artifacts that are typically prominent in contact mode friction force microscopy (FFM). The feedback scheme consists of simultaneously regulating the normal as well as the lateral cantilever deflection signal at their respective set points. This not only removes the imaging artifacts due to geometrical misalignments, mechanical cross-talk, and irregular sliding but also the corresponding compensatory control signal gives a more accurate real time measure of the lateral interaction force between the sample and the cantilever as compared to the lateral deflection signal used in FFM. Experimental results show significant improvement, and in some cases, practical elimination of the artifacts. The design and implementation of a split piezoassembly needed for the lateral actuation for the feedback scheme are also presented.     

Atomic and magnetic force microscopy imaging of thin-film recording heads

Investigations on the use of the scanning probe microscope (SPM) in the atomic force microscopy (AFM) mode for topography imaging and the magnetic force microscopy (MFM) mode for magnetic imaging are presented for a thin-film recording head. Results showed that the SPM is suitable for imaging the surface profile of the recording head, determining the width of the pole gap region, and mapping the magnetic field patterns of the recording head excited under current bias conditions of different polarity. For the cobalt sputter-coated tips used in MFM imaging, it was found that the magnetic field patterns obtained under different polarities of the current bias to the recording head were similar. This can be explained by the nature of the thin-film MFM tip, in which the direction of the tip magnetic moment can follow the stray magnetic field of the sample as the current bias to the recording head reverses in direction.     

Dual resonance excitation system for the contact mode of atomic force microscopy

We propose an improved system that enables simultaneous excitation and measurements of at least two resonance frequency spectra of a vibrating atomic force microscopy (AFM) cantilever. With the dual resonance excitation system it is not only possible to excite the cantilever vibrations in different frequency ranges but also to control the excitation amplitude for the individual modes. This system can be used to excite the resonance frequencies of a cantilever that is either free of the tip-sample interactions or engaged in contact with the sample surface. The atomic force acoustic microscopy and principally similar methods utilize resonance frequencies of the AFM cantilever vibrating while in contact with the sample surface to determine its local elastic modulus. As such calculation demands values of at least two resonance frequencies, two or three subsequent measurements of the contact resonance spectra are necessary. Our approach shortens the measurement time by a factor of two and limits the influence of the AFM tip wear on the values of the tip-sample contact stiffness. In addition, it allows for in situ observation of processes transpiring within the AFM tip or the sample during non-elastic interaction, such as tip fracture.     

Second harmonic atomic force microscopy imaging of live and fixed mammalian cells

Higher harmonic contributions in the movement of an oscillating atomic force microscopy (AFM) cantilever are generated by nonlinear tip–sample interactions, yielding additional information on structure and physical properties such as sample stiffness. Higher harmonic amplitudes are strongly enhanced in liquid compared to the operation in air, and were previously reported to result in better structural resolution in highly organized lattices of proteins in bacterial S-layers and viral capsids [J. Preiner, J. Tang, V. Pastushenko, P. Hinterdorfer, Phys. Rev. Lett. 99 (2007) 046102]. We compared first and second harmonics AFM imaging of live and fixed human lung epithelial cells, and microvascular endothelial cells from mouse myocardium (MyEnd). Phase–distance cycles revealed that the second harmonic phase is 8 times more sensitive than the first harmonic phase with respect to variations in the distance between cantilever and sample surface. Frequency spectra were acquired at different positions on living and fixed cells with second harmonic amplitude values correlating with the sample stiffness. We conclude that variations in sample stiffness and corresponding changes in the cantilever–sample distance, latter effect caused by the finite feedback response, result in second harmonic images with improved contrast and information that is not attainable in the fundamental frequency of an oscillating cantilever.     

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.     

Study of the tip mass and interaction force effects on the frequency response and mode shapes of the AFM cantilever

The vibrational characteristics of an atomic force microscope (AFM) cantilever beam play a key role in dynamic mode of the atomic force microscope. As the oscillating AFM cantilever tip approaches the sample, the tip–sample interaction force influences the cantilever dynamics. In this paper, we present a detailed theoretical analysis of the frequency response and mode shape behavior of a cantilever beam in the dynamic mode subject to changes in the tip mass and the interaction regime between the AFM cantilever system and the sample. We consider a distributed parameter model for AFM and use Euler–Bernoulli method to derive an expression for AFM characteristics equation contains tip mass and interaction force terms. We study the frequency response of AFM cantilever under variations of interaction force between AFM tip and sample. Also, we investigate the effect of tip mass on the frequency response and also the quality factor and spring constant of each eigenmodes of AFM micro-cantilever. In addition, the mode shape analysis of AFM cantilever under variations of tip mass and interaction force is investigated. This will incorporate the presentation of explicit analytical expressions and numerical analysis. The results show that by considering the tip mass, the resonance frequencies of the cantilever are decreased. Also, the tip mass has a significant effect on the mode shape of the higher eigenmodes of the AFM cantilever. Moreover, tip mass affects the quality factor and spring constant of each modes.     

MAC mode atomic force microscopy studies of living samples, ranging from cells to fresh tissue

Magnetic AC mode (MAC mode) atomic force microscopy (AFM), a novel type of tapping mode AFM in which the cantilever is driven directly by a magnetic field, is a powerful tool for imaging with high spatial resolution and better signal-to-noise in liquid environment. It may largely extend the application of AFM to living samples, especially those are sensitive to cantilever forces, even to multilayer tissue samples. However, there are few reports on the imaging of living cells by MAC mode AFM previously. In our present study, we explore the optimal imaging conditions of MAC mode AFM on living astrocytes and fresh arterial intima surface. We also used nude tips for PicoTREC panel (i.e., Aux in BNC, a new data collecting channel) to image living samples and discussed its difference with phase imaging. We show that living biological samples can be imaged by MAC mode AFM at details of comparable resolution as those by high resolution scanning electron microscopy. Furthermore, the combination of height, amplitude, phase and TREC panel signals provide abundant informations for the characteristics of living samples, such as topography, profile, stiffness and adhesion.     

Control of tip-to-sample distance in atomic force microscopy: a dual-actuator tip-motion control scheme

The control of tip-to-sample distance in atomic force microscopy (AFM) is achieved through controlling the vertical tip position of the AFM cantilever. In the vertical tip-position control, the required z motion is commanded by laser reading of the vertical tip position in real time and might contain high frequency components depending on the lateral scanning rate and topographical variations of the sample. This paper presents a dual-actuator tip-motion control scheme that enables the AFM tip to track abrupt topographical variations. In the dual-actuator scheme, an additional magnetic mode actuator is employed to achieve high bandwidth tip-motion control while the regular z scanner provides the necessary motion range. This added actuator serves to make the entire cantilever bandwidth available for tip positioning, and thus controls the tip-to-sample distance. A fast programmable electronics board was employed to realize the proposed dual-actuator control scheme, in which model cancellation algorithms were implemented to enlarge the bandwidth of the magnetic actuation and to compensate the lightly damped dynamics of the cantilever. Experiments were conducted to illustrate the capabilities of the proposed dual-actuator tip-motion control in terms of response speed and travel range. It was shown that while the bandwidth of the regular z scanner was merely a small fraction of the cantilever's bandwidth, the dual-actuator control scheme led to a tip-motion control system, the bandwidth of which was comparable to that of the cantilever, where the dynamics overdamped, and the motion range comparable to that of the z scanner.     

Nanometer-scale heat-conductivity measurements on biological samples

With semiconductor structures reaching the nanometer scale, heat conductivity measurements on the mesoscopic range of some tens of nanometers become an increasingly important aspect for the further improvement in digital processing and storage. Also the attempt to use atomic-force microscopy (AFM) technology for high-density data storage by writing information bits as nanometer-sized indentations into a polymer substrate with a heated cantilever tip asks for a careful investigation of the nano-scale heat-conductivity properties of polymers. Furthermore, in many AFM imaging applications, heat conductivity can provide additional information about the material the imaged structures consist of. In this respect, heat conductivity can also become very interesting in studies of usually quite heterogeneous biological samples, if the resolution can attain the nanometer range. In standard scanning thermal microscopy application, the tip forms a thermocouple, which precludes high-resolution imaging, as thermocouples cannot be made sufficiently small. In this paper, which focuses on biological applications, we demonstrate that by using an ultra sharp AFM cantilever with a Joule heating element above the tip structure different molecular components can be distinguished thanks to their different heat-conductivity properties. In this case, the resolution is determined by the actual tip size, and it can reach 10nm.     

Topography imaging with a heated atomic force microscope cantilever in tapping mode

This article describes tapping mode atomic force microscopy (AFM) using a heated AFM cantilever. The electrical and thermal responses of the cantilever were investigated while the cantilever oscillated in free space or was in intermittent contact with a surface. The cantilever oscillates at its mechanical resonant frequency, 70.36 kHz, which is much faster than its thermal time constant of 300 micros, and so the cantilever operates in thermal steady state. The thermal impedance between the cantilever heater and the sample was measured through the cantilever temperature signal. Topographical imaging was performed on silicon calibration gratings of height 20 and 100 nm. The obtained topography sensitivity is as high as 200 microVnm and the resolution is as good as 0.5 nmHz(1/2), depending on the cantilever power. The cantilever heating power ranges 0-7 mW, which corresponds to a temperature range of 25-700 degrees C. The imaging was performed entirely using the cantilever thermal signal and no laser or other optics was required. As in conventional AFM, the tapping mode operation demonstrated here can suppress imaging artifacts and enable imaging of soft samples.     

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.     

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.     

Constant tip-surface distance with atomic force microscopy via quality factor feedback

The atomic force microscope (AFM) is a powerful and widely used instrument to image topography and measure forces at the micrometer and nanometer length scale. Because of the high degree of operating accuracy required of the instrument, small thermal and mechanical drifts of the cantilever and piezoactuator systems hamper measurements as the AFM tip drifts spatially relative to the sample surface. To compensate for the drift, we control the tip-surface distance by monitoring the cantilever quality factor (Q) in a closed loop. Brownian thermal fluctuations provide sufficient actuation to accurately determine cantilever Q by fitting the thermal noise spectrum to a Lorentzian function. We show that the cantilever damping is sufficiently affected by the tip-surface distance so that the tip position of soft cantilevers can be maintained within 40 nm of a setpoint in air and within 3 nm in water with 95% reliability. Utilizing this method to hover the tip above a sample surface, we have the capability to study sensitive interactions at the nanometer length scale over long periods of time.     

Atomic force microscopy imaging of living cells: a preliminary study of the disruptive effect of the cantilever tip on cell morphology

Recent studies have demonstrated that atomic force microscopy (AFM) is a potential tool for studying important dynamic cellular processes in real time. However, the interactions between the cantilever tip and the cell surface are not well understood, and the disruptive effect of the cantilever tip on cell morphology has not been well characterized. In this study, the disruptive effect of the scanning cantilever tip on cell morphology, in the AFM contact mode, has been investigated. The aims of this study are to identify what kinds of cell morphological changes generally occurred under normal AFM imaging conditions and to find out how long cells remain viable during scanning. Two cell lines, SK-N-SH (human neuroblastoma cells) and AV12 (Syrian hamster cells) were studied in the experiment because these are widely used in biomedical research as an expression system for studying cellular functions of neuronal receptors. The experimental results suggest that the sensitivity of cells to the cantilever disruptive effect is dependent on cell type and that there are patterns observed in the changes of cell morphology induced by the cantilever force in these two cell lines.     

Improving tapping mode atomic force microscopy with piezoelectric cantilevers

This article summarizes improvements to the speed, simplicity and versatility of tapping mode atomic force microscopy (AFM). Improvements are enabled by a piezoelectric microcantilever with a sharp silicon tip and a thin, low-stress zinc oxide (ZnO) film to both actuate and sense deflection. First, we demonstrate self-sensing tapping mode without laser detection. Similar previous work has been limited by unoptimized probe tips, cantilever thicknesses, and stress in the piezoelectric films. Tests indicate self-sensing amplitude resolution is as good or better than optical detection, with double the sensitivity, using the same type of cantilever. Second, we demonstrate self-oscillating tapping mode AFM. The cantilever's integrated piezoelectric film serves as the frequency-determining component of an oscillator circuit. The circuit oscillates the cantilever near its resonant frequency by applying positive feedback to the film. We present images and force-distance curves using both self-sensing and self-oscillating techniques. Finally, high-speed tapping mode imaging in liquid, where electric components of the cantilever require insulation, is demonstrated. Three cantilever coating schemes are tested. The insulated microactuator is used to simultaneously vibrate and actuate the cantilever over topographical features. Preliminary images in water and saline are presented, including one taken at 75.5 μm/s—a threefold improvement in bandwidth versus conventional piezotube actuators.     

Dual frequency atomic force microscopy on charged surfaces

The cantilever is mechanically driven at two resonant frequencies in a bimodal atomic force microscope (AFM). To generate the feedback signal for topography measurement the deflection signal is demodulated at one frequency and for compositional surface mapping at the other. In particular, the second mode amplitude and phase signals are used to map surface forces such as the van der Waals interaction. On electrically charged surfaces both, van der Waals forces and electrostatic forces contribute to the second eigenmode signal. The higher eigenmode signal in bimodal AFM reflects the local distribution of electrical charges. Mechanically driven bimodal AFM thus also provides a valuable tool for compositional mapping based on surface charges.     

Bias-induced forces in conducting atomic force microscopy and contact charging of organic monolayers

Contact electrification, a surface property of bulk dielectric materials, has now been observed at the molecular scale using conducting atomic force microscopy (AFM). Conducting AFM measures the electrical properties of an organic film sandwiched between a conducting probe and a conducting substrate. This paper describes physical changes in the film caused by the application of a bias. Contact of the probe leads to direct mechanical stress and the applied electric field results in both Maxwell stresses and electrostriction. Additional forces arise from charge injection (contact charging). Electrostriction and contact charging act oppositely from the normal long-range Coulomb attraction and dominate when a charged tip touches an insulating film, causing the tip to deflect away from the film at high bias. A bias-induced repulsion observed in spin-coated PMMA films may be accounted for by either mechanism. In self-assembled monolayers, however, tunnel current signals show that the repulsion is dominated by contact charging.     

Developments for inverted atomic force microscopy

Atomic force microscopy (AFM) has been used to study a wide range of systems. Chemically and biologically modified probes have extended AFM by coupling chemical and biological information with the physical measurements. In an effort to further expand the capabilities of modified AFM probes, previous studies investigated the use of an inverted AFM design (i-AFM), wherein a microfabricated tip array is used to image a cantilever-supported sample. This report details developments in cantilever and tip array fabrication which are aimed at improving the applicability and performance of this i-AFM design. Using an epoxy-based procedure, commercial cantilevers were modified with a series of standard substrates, including template-stripped gold, highly oriented pyrolytic graphite, and mica. The samples on these cantilevers were imaged with i-AFM, and lateral force images are obtained. This paper demonstrates the first use of i-AFM for measuring friction.     

Quantitative current measurements using scanning magnetoresistance microscopy

We have demonstrated the capability of scanning magnetoresistance microscope (SMRM) to be used for quantitative current measurements. The SMRM is a magnetic microscope that is based on an atomic force microscope (AFM) and simultaneously measures the localized surface magnetic field distribution and surface topography. The proposed SMRM employs an in-house built AFM cantilever equipped with a miniaturized magnetoresistive (MR) sensor as a magnetic field sensor. In this study, a spin-valve type MR sensor with a width of 1 microm was used to measure the magnetic field distribution induced by a current carrying wire with a width of 5 microm and a spacing of 1.6 microm at room temperature and under ambient conditions. Simultaneous imaging of the magnetic field distribution and the topography was successfully performed in the DC current ranging from 500 microA to 8 mA. The characterized SV sensor, which has a linear response to magnetic fields, offers the quantitative analysis of a magnetic field and current. The measured magnetic field strength was in good agreement with the result simulated using Biot-Savart's law.