Molecular recognition imaging using tuning fork-based transverse dynamic force microscopy.

We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1-4nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1-2 degrees. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.

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.

An AFM/rotaxane molecular reading head for sequence-dependent DNA structures.

DNA secondary structure may prove to be a significant obstacle both for enzymes that process DNA through an orifice and for the passage of DNA through nanopores proposed for some novel sequencing schemes. A nanomechanical molecular "tape reader" is assembled by threading a nanopore over DNA and pulling it using an atomic force microscope. Its formation is confirmed by studying the force required to open hairpins under that geometry. Unfolding induced by this 0.7-nm-diameter aperture requires 40 times more force than that reported for pulling on the ends of the DNA. Kinetic analysis shows that much less strain is required to destabilize the double helix in this geometry. Consequently, much more force is required to provide the free energy needed for opening.

Application of biotin-4-fluorescein in homogeneous fluorescence assays for avidin, streptavidin, and biotin or biotin derivatives.

Biotin-4-fluorescein (B4F) is a convenient molecular probe for (strept)avidin and for unlabeled biotin in homogeneous fluorescence assays. The primary standard is a 16 microM working solution of d-biotin which is used to titrate an aliquot of a (strept)avidin stock solution while monitoring the tryptophane fluorescence of (strept)avidin. This serves to standardize the (strept)avidin stock solution, an aliquot of which is then titrated with a roughly 16 microM working solution of B4F while monitoring the fluorescence of B4F. Specific binding is accompanied by quenching, but after saturation of all binding sites, the appearance of free ligand causes a sharp rise of intense fluorescence, the beginning of which allows to calculate the effective concentration of B4F in the working solution. Measurement of avidin in a crude sample is exemplified by mixing 8 pmol of B4F with various amounts of diluted egg white in a volume of 1 mL. Hereby, the extent of fluorescence quenching linearly correlates with the concentration of functional avidin. Moreover, a sharp minimum of fluorescence is observed when exactly 2 pmol of avidin is present in the sample. The latter assay has been adapted to measure between 0.5 and 5 pmol of d-biotin in 1 mL of sample by adding 1.9 pmol of avidin and 8 pmol of B4F. This competitive assay correctly measures the small dose of d-biotin in multivitamin tablets (e.g., 150 microg in 5 g of solid) after subtracting the background fluorescence of the colored aqueous solution.

Dynamic force microscopy imaging of plasmid DNA and viral RNA.

Plasmid DNA and viral RNA were imaged in a liquid environment by dynamic force microscopy (DFM) and fine structures of DNA with heights of 1.82+/-0.66 nm were obtained in topographical images. In simultaneously acquired phase images, DNA could be imaged with better contrast at lower imaging forces. By splitting the cantilever oscillation signal into lower and upper parts, the contribution of the adhesion between tip and sample to the topographical images was eliminated, resulting in better signal-to-noise ratio. DFM of the single stranded RNA genome of a human rhinovirus showed loops protruding from a condensed RNA core, 20-50 nm in height. The mechanical rigidity of the RNA was determined by single molecule pulling experiments. From fitting RNA stretching curves to the Worm-Like-Chain (WLC) model a persistence length of 1.0+/-0.17 nm was obtained.

Age determination of blood spots in forensic medicine by force spectroscopy.

We present a new tool for the estimation of the age of bloodstains, which could probably be used during forensic casework. For this, we used atomic force microscopy (AFM) for high-resolution imaging of erythrocytes in a blood sample and the detection of elasticity changes on a nanometer scale. For the analytic procedure we applied a fresh blood spot on a glass slide and started the AFM detection after drying of the blood drop. In a first step, an overview image was generated showing the presence of several red blood cells, which could easily be detected due to their typical "doughnut-like" appearance. The consecutively morphological investigations in a timeframe of 4 weeks could not show any alterations. Secondly, AFM was used to test the elasticity by recording force-distance curves. The measurements were performed immediately after drying, 1.5 h, 30 h and 31 days. The conditions were kept constant at room temperature (20 degrees C) and a humidity of 30%. The obtained elasticity parameters were plotted against a timeline and repeated several times. The elasticity pattern showed a decrease over time, which are most probably influenced by the alteration of the blood spot during the drying and coagulation process. The preliminary data demonstrates the capacity of this method to use it for development of calibration curves, which can be used for estimation of bloodstain ages during forensic investigations.

A combined optical and atomic force microscope for live cell investigations.

We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ("receptor-positive sites") was significantly higher than that on sites lacking receptors.

Single molecule studies of antibody-antigen interaction strength versus intra-molecular antigen stability.

We investigated molecular recognition of antibodies to membrane-antigens and extraction of the antigens out of membranes at the single molecule level. Using dynamic force microscopy imaging and enzyme immunoassay, binding of anti-sendai antibodies to sendai-epitopes genetically fused into bacteriorhodopsin molecules from purple membranes were detected under physiological conditions. The antibody/antigen interaction strength of 70-170 pN at loading rates of 2-50 nN/second yielded a barrier width of x = 0.12 nm and a kinetic off-rate (corresponding to the barrier height) of k(off) = 6s(-1), respectively. Bacteriorhodopsin unfolding revealed a characteristic intra-molecular force pattern, in which wild-type and sendai-bacteriorhodopsin molecules were clearly distinguishable in their length distributions, originating from the additional 13 amino acid residues epitope in sendai purple membranes. The inter-molecular antibody/antigen unbinding force was significantly lower than the force required to mechanically extract the binding epitope-containing helix pair out of the membrane and unfold it (126 pN compared to 204 pN at the same loading rate), meeting the expectation that inter-molecular unbinding forces are weaker than intra-molecular unfolding forces responsible for stabilizing native conformations of proteins.

Dynamic force microscopy imaging of native membranes.

We employed magnetic ACmode atomic force microscopy (MACmode AFM) as a novel dynamic force microscopy method to image surfaces of biological membranes in their native environments. The lateral resolution achieved under optimized imaging conditions was in the nanometer range, even when the sample was only weakly attached to the support. Purple membranes (PM) from Halobacterium salinarum were used as a test standard for topographical imaging. The hexagonal arrangement of the bacteriorhodopsin trimers on the cytoplasmic side of PM was resolved with 1.5nm lateral accuracy, a resolution similar to images obtained in contact and tapping-mode AFM. Human rhinovirus 2 (HRV2) particles were attached to mica surfaces via nonspecific interactions. The capsid structure and 2nm sized protein loops of HRV2 were routinely obtained without any displacement of the virus. Globular and filamentous structures on living and fixed endothelial cells were observed with a resolution of 5-20nm. These examples show that MACmode AFM is a favorable method in studying the topography of soft and weakly attached biological samples with high resolution under physiological conditions.

Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4'-dithiodipyridine.

Since its introduction in 1959, Ellman's reagent (5,5'-dithio-bis(2-nitrobenzoic acid)) has been the favorite reagent for spectrophotometric measurement of protein sulfhydryls. Meanwhile however, evidence has accumulated that many protein sulfhydryls give an incomplete reaction with Ellman's reagent, even during prolonged assay times. In the present study, the kinetic problem was solved by including cystamine as a "mediator" between the protein sulfhydryl and Ellman's reagent, as previously applied in an enzymatic thiol assay [9]. As an alternative, 4,4'-dithiodipyridine (DTDP) was used in place of Ellman's reagent. Due to its small size, amphiphilic nature, and lack of charge, DTDP quickly reacts with poorly accessible protein sulfhydryls, without any catalysis by cystamine. The DTDP method and the Ellman/cystamine method were both optimized for maximal sensitivity, minimal sample consumption (detection limit 0.2 nmol mL(-1), determination limit 0.6 nmol mL(-1)), and minimal assay time (5 min). In validation experiments, both methods gave identical results and the measured sulfhydryls/protein matched the expected values. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00216-002-1347-2.