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We have investigated the fine-scale spatial genetic structure in a managed Scots pine forest. For this purpose, we perform a Bayesian genetic-cluster analysis of 96 geographically mapped individual seed trees of Swedish Scots pine based on 14 microsatellite loci. The analysis was carried out with the recently developed program GENECLUST (François et al., 2006), which provides the facility to jointly incorporate both spatial information from a geographical neighborhood structure through a Potts-Dirichlet model and account for variable degrees of inbreeding within the clusters. To evaluate whether inbreeding and spatial interaction should be included in the best-fitting statistical model for our data, we used the deviance information criterion (DIC), a weighted measure of model fit that accounts for an effective number of free parameters in a model. Analysis shows that a model with a single estimated cluster, with high levels of inbreeding (0.25) and with a moderate amount of spatial dependency within the unique cluster (Psi=0.2-0.4), best explains the data. We also carried out Bayesian parentage analysis, which enabled us to exclude the possibility that the sample constitutes one single full-sib family.

Covalent binding of bioligands to atomic force microscope (AFM) tips converts them into monomolecular biosensors by which cognate receptors can be localized on the sample surface and fine details of ligand-receptor interaction can be studied. Tethering of the bioligand to the AFM tip via a approximately 6 nm long, flexible poly(ethylene glycol) linker (PEG) allows the bioligand to freely reorient and to rapidly "scan" a large surface area while the tip is at or near the sample surface. In the standard coupling scheme, amino groups are first generated on the AFM tip. In the second step, these amino groups react with the amino-reactive ends of heterobifunctional PEG linkers. In the third step, the 2-pyridyl-S-S groups on the free ends of the PEG chains react with protein thiol groups to give stable disulfide bonds. In the present study, this standard coupling scheme has been critically examined, using biotinylated IgG with free thiols as the bioligand. AFM tips with PEG-tethered biotin-IgG were specifically recognized by avidin molecules that had been adsorbed to mica surfaces. The unbinding force distribution showed three maxima that reflected simultaneous unbinding of 1, 2, or 3 IgG-linked biotin residues from the avidin monolayer. The coupling scheme was well-reproduced on amino-functionalized silicon nitride chips, and the number of covalently bound biotin-IgG per microm2 was estimated by the amount of specifically bound ExtrAvidin-peroxidase conjugate. Coupling was evidently via disulfide bonds, since only biotin-IgG with free thiol groups was bound to the chips. The mechanism of protein thiol coupling to 2-pyridyl-S-S-PEG linkers on AFM tips was further examined by staging the coupling step in bulk solution and monitoring turnover by release of 2-pyridyl-SH which tautomerizes to 2-thiopyridone and absorbs light at 343 nm. These experiments predicted 10(3)-fold slower rates for the disulfide coupling step than actually observed on AFM tips and silicon nitride chips. The discrepancy was reconciled by assuming 10(3)-fold enrichment of protein on AFM tips via preadsorption, as is known to occur on comparable inorganic surfaces.

Recent developments in single molecule force spectroscopy have allowed investigating the interaction between two redox partners, Azurin and Cytochrome C 551. Azurin has been directly chemisorbed on a gold electrode whereas cytochrome c has been linked to the atomic force microscopy tip by means of a heterobifunctional flexible cross-linker. When recording force-distance cycles, molecular recognition events could be observed, displaying unbinding forces of approximately 95 pN for an applied loading rate of 10 nN/s. The specificity of molecular recognition was confirmed by the significant decrease of unbinding probability observed in control block experiments performed adding free azurin solution in the fluid cell. In addition, the complex dissociation kinetics has been here investigated by monitoring the unbinding forces as a function of the loading rate: the thermal off-rate was estimated to be approximately 14 s(-1), much higher than values commonly estimated for complexes more stable than electron transfer complexes. Results here discussed represent the first studies on molecular recognition between two redox partners by atomic force microscopy.

Simultaneous topography and recognition imaging (TREC), 2] a recent development in dynamic force microscopy, has proven to be a powerful technique in biophysical research. In contrast to the common force mapping mode, the slow imaging speed and the low lateral resolution are overcome by oscillating a functionalized tip close to its resonance frequency during the scan across the surface. In these studies, the topographical imaging of receptor molecules is combined with molecular recognition by their cognate ligands bound to the atomic force microscope (AFM) tip via a distensible tether. The binding sites are evident from the reduction in the oscillation amplitude, as a result of specific recognition during the lateral scan. The receptors are recognized by the ligand on the scanning tip with a lateral resolution of a few nanometers, yielding a topographic image and a separate map of recognition sites from a single scan. Because this new technique opens a broad range of biological applications, a stable and easy-to-use setup is of key interest for new users. Using well-characterized, high-affinity biological binding partners, such as avidin–biotin, for TREC imaging enables a better understanding of the key factors for optimizing the scanning parameters. The robust and well-described avidin–biotin interaction offers an ideal pair of binding partners. Avidin–biotin is 1) robust and reliable, 2) well-known in terms of binding properties, 3) easy to prepare with commercially available components. Moreover, 4) avidin can simply be adsorbed to mica, and mica is an ideal support for AFM imaging. Finally, 5) the biotin–PEG (poly(ethylene glycol)) tether is commercially available and can be attached to amino-functionalized AFM tips in one step. Single avidin molecules can easily be immobilized onto mica via electrostatic adsorption by virtue of their positive net charge at neutral pH. Using different concentrations of avidin in the adsorption buffer, the surface coverage of avidin on mica can be adjusted easily. In addition, a biotin residue was coupled to the AFM tip via a distensible PEG chain. More specifically, biotin was covalently tethered to the amino-functionalized tip in a single coupling step, using “biotin-PEG-NHS” which consists of a PEG chain with a biotin on one end and an amino-reactive N-hydroxysuccinimide ester function (NHS group) on the other. With this configuration, two independent maps were simultaneously acquired, that is, a topography image of the immobilized avidin molecules and a lateral map of the corresponding recognition sites, both recorded at experimental times comparable to normal AFM imaging. Before being applied to TREC, the tips were examined for a functional biotin residue by force spectroscopy experiments on a mica surface densely covered with avidin molecules. Force spectroscopy with an oscillating tip offers an important tool for adjusting the scanning parameters in TREC microscopy. In simultaneously recorded amplitude–distance and force–distance cycles (Figure 1), the oscillation amplitude and the cantilever bending force, respectively, were investigated.

We present a method for simultaneously recording topography images and localizing specific binding sites with nm positional accuracy by combining dynamic force microscopy with single molecule recognition force spectroscopy. For this we used lysozyme adsorbed to mica, the functionality of which was characterized by enzyme immunoassays. The topography and recognition images were acquired using tips that were magnetically oscillated during scanning and contained antibodies directed against lysozyme. For cantilevers with low Q-factor (approximately 1 in liquid) driven at frequencies below resonance, the surface contact only affected the downward deflections (minima) of the oscillations, whereas binding of the antibody on the tip to lysozyme on the surface only affected the upwards deflections (maxima) of the oscillations. The recognition signals were therefore well separated from the topographic signals, both in space (Delta z approximately 5 nm) and time (approximately 0.1 ms). Topography and recognition images were simultaneously recorded using a specially designed electronic circuit with which the maxima (U(up)) and the minima (U(down)) of each sinusoidal cantilever deflection period were depicted. U(down) was used for driving the feedback loop to record the height (topography) image, and U(up) provided the data for the recognition image.

Functionalization of atomic force microscope (AFM) tips with bioligands converts them into monomolecular biosensors which can detect complementary receptor molecules on the sample surface. Flexible PEG tethers are preferred because the bioligand can freely reorient and locally palpate the sample surface while the AFM tip is moved along. In a well-established coupling scheme [Hinterdorfer et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 3477-3481], a heterobifunctional PEG linker is used to tether thiol-containing bioligands to amino-functionalized AFM tips. Since antibodies contain no free thiol residues, prederivatization with N-succinimidyl 3-(acetylthio)propionate (SATP) is needed which causes a relatively high demand for antibody. The present study offers a convenient alternative with minimal protein consumption (e.g., 5 microg of protein in 50 microL of buffer) and no prederivatization, using a new heterobifunctional cross-linker that has two different amino-reactive functions. One end is an activated carboxyl (N-hydroxysuccinimide ester) which is much faster to react with the amino groups of the tips than the benzaldehyde function on its other end. The reactivity of the latter is sufficient, however, to covalently bind lysine residues of proteins via Schiff base formation. The method has been critically examined, using biotinylated IgG as bioligand on the tip and mica-bound avidin as complementary receptor. These experiments were well reproduced on amino-functionalized silicon nitride chips where the number of specifically bound IgG molecules (approximately 2000 per microm2) was estimated from the amount of specifically bound ExtrAvidin-peroxidase conjugate. For a bioscientific application, human rhinovirus particles were tethered to the tip, very-low-density lipoprotein receptor fragments were tethered to mica, and the specific interaction was studied by force microscopy.

The surface topography of red blood cells (RBCs) was investigated under near-physiological conditions using atomic force microscopy (AFM). An immobilization protocol was established where RBCs are coupled via molecular bonds of the membrane glycoproteins to wheat germ agglutinin (WGA), which is covalently and flexibly tethered to the support. This results in a tight but non-invasive attachment of the cells. Using tapping-mode AFM, which is known as gentle imaging mode and therefore most appropriate for soft biological samples like erythrocytes, it was possible to resolve membrane skeleton structures without major distortions or deformations of the cell surface. Significant differences in the morphology of RBCs from healthy humans and patients with systemic lupus erythematosus (SLE) were observed on topographical images. The surface of RBCs from SLE patients showed characteristic circular-shaped holes with approx. 200 nm in diameter under physiological conditions, a possible morphological correlate to previously published changes in the SLE erythrocyte membrane.