Volume determination of human metaphase chromosomes by scanning force microscopy

The scanning force microscopy (SFM) yields the topography of the investigated surface. A procedure was developed which starts from this three-dimensional information to estimate the volume of a biological specimen. The volume of spread human metaphase chromosomes was determined in air and rehydrated in aqueous buffer. A difference of the determined volume of a air-dried metaphase chromosome set was found compared to values from electron microscopic investigations, and could be correlated with differences in the hydration state of the chromosomes. SFM-based relative volumes of air-dried chromosomes resembles literature data regarding volume range and distribution. Possible application of SFM-based relative volume measurements for chromosome classification purposes is discussed.     

Deposition of supercoiled DNA on mica for scanning force microscopy imaging

The deposition of DNA molecules on mica is driven and controlled by the ionic densities around DNA and close to the surface of the substrate. Dramatic improvements in the efficiency and reproducibility of DNA depositions were due to the introduction of divalent cations in the deposition solutions. The ionic distributions on DNA and on mica determine the mobility of adsorbed DNA molecules, thus letting them assume thermodynamically equilibrated conformations, or alternatively trapping them in non-equilibrated conformations upon adsorption. With these prerequisites, mica does not seem like an inert substrate for DNA deposition for microscopy, and its properties greatly affect the efficiency of DNA deposition and the appearance of the molecules on the substrate. In our laboratory, we have some preliminary evidence that mica could also participate in DNA damage, most likely through its heavy metal impurities.     

The structure of a model pulmonary surfactant as revealed by scanning force microscopy

The structures formed by a pulmonary surfactant model system of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and recombinant surfactant-associated protein C (SP-C) were studied using scanning force microscopy (SFM) on Langmuir-Blodgett films. The films appeared to be phase separated, in agreement with earlier investigations by fluorescence light microscopy. There were smooth polygonal patches of mostly lipid, surrounded by a corrugated rim rich in SP-C. When the films were compressed beyond the equilibrium surface pressure, the protein-rich phase mediated the formation of layered protrusions. The height of these multilamellar structures embodied equidistant steps slightly higher than a DPPC double layer in the gel phase. At the air-water interface too, a high compressibility at low surface tension was indicative of the exclusion of matter. The exclusion process proved to be fully reversible. The present study demonstrates that some of the matter of the model pulmonary surfactant can move in and out of the active monolayer. The SFM images revealed a lipid-protein complex that was responsible for the reversible exclusion of double-layer structures. This mechanism may be important in the natural system too, to keep the surface tension of the alveolar air/water interface constantly low over the range of area encountered upon breathing.     

Bovine serum albumin conformation on methyl and amine functionalized surfaces compared by scanning force microscopy

We investigated the adsorption of albumin on chemically modified gold surfaces by scanning force microscopy operating both in contact and noncontact mode. The surface modification was performed with thiol-based self-assembling molecules carrying amine or methyl groups. The albumin on the aminoethanethiol-coated gold formed a uniform layer and single molecules could be distinguished. On the dodecanethiol-coated surface the protein adsorbed in aggregates or single isolated molecules depending on the incubation time. The width of the albumin molecule on both surface was similar, but the height was much lower on the amine than on the methyl surface. This was interpreted as a difference in the conformation of albumin depending on the substrate, and could explain the promotion of cell adhesion on amine-treated polymers coated with albumin.     

Visualizing life on biomembranes by atomic force microscopy

Since its invention in 1986, the atomic force microscope (AFM) has become one of the most widely used near-field microscopes. Surfaces of hard samples are imaged routinely with atomic resolution. Soft biological samples, however, are still challenging. In this brief review, the AFM technique is introduced to the experimental biologist. We discuss recent data on imaging molecular structures of biomembranes, and give detailed information on the application of the AFM with two representative examples. One is imaging plasma membrane turnover of transformed renal epithelial cells during migration in vivo, and the other is visualizing macromolecular pore complexes of the nuclear envelope of aldosterone-sensitive kidney cells.     

A constant compliance force modulation technique for scanning force microscopy (SFM) imaging of polymer surface elasticity

A new method of force modulation scanning force microscopy (SFM) imaging based on a constant compliance feedback loop is presented. The feedback adjusts the loading force applied by the SFM tip to the surface in order to maintain a constant compliance beneath the tip. The new method, constant compliance force modulation (CCFM), has the advantage of being able to quantify the loading force exerted by the tip onto the sample surface and thus to estimate the elastic modulus of the material probed by the SFM tip. Once the elastic modulus of one region is known, the elastic moduli of other surface regions can be estimated from the spatial map of loading forces using the Hertz model of deformation. Force vs. displacement measurements made on one surface locality could also be used to estimate the local modulus. Several model surfaces, including a rubber-toughened epoxy polymer blend which showed clearly resolved compliant rubber phases within the harder epoxy matrix, were analyzed with the CCFM technique to illustrate the method's application.     

Contributions of linker histones and histone H3 to chromatin structure: scanning force microscopy studies on trypsinized fibers

Little is known about the mechanisms that organize linear arrays of nucleosomes into the three-dimensional structures of extended and condensed chromatin fibers. We have earlier defined, from scanning force microscopy (SFM) and mathematical modeling, a set of simple structural determinants of extended fiber morphology, the critical parameters being the entry-exit angle between consecutive linkers and linker length. Here we study the contributions of the structural domains of the linker histones (LHs) and of the N-terminus of histone H3 to extended fiber morphology by SFM imaging of progressively trypsinized chromatin fibers. We find that cleavage of LH tails is associated with a lengthening of the internucleosomal center-to-center distance, and that the somewhat later cleavage of the N-terminus of histone H3 is associated with a flattening of the fiber. The persistence of the "zigzag" fiber morphology, even at the latest stages of trypsin digestion, can be attributed to the retention of the globular domain of LH in the fiber.