Imaging of biological macromolecules on mica in humid air by scanning electrochemical microscopy

Imaging of DNA, keyhole limpet hemocyanin, mouse monoclonal IgG, and glucose oxidase on a mica substrate has been accomplished by scanning electrochemical microscopy with a tungsten tip. The technique requires the use of a high relative humidity to form a thin film of water on the mica surface that allows electrochemical reactions to take place at the tip and produce a faradaic current (≈1 pA) that can be used to control tip position. The effect of relative humidity and surface pretreatment with buffer solutions on the ionic conductivity of a mica surface was investigated to find appropriate conditions for imaging. Resolution of the order of 1 nm was obtained.

Scanning electrochemical microscopy of living cells: Different redox activities of nonmetastatic and metastatic human breast cells

Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Cα), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators.

A novel assay for drug-DNA binding mode, affinity, and exclusion number: scanning force microscopy.

Determining the mode-of-binding of a DNA ligand is not always straightforward. Here, we establish a scanning force microscopic assay for mode-of-binding that is (i) direct: lengths of individual DNA-ligand complexes are directly measured; (ii) rapid: there are no requirements for staining or elaborate sample preparation; and (iii) unambiguous: an observed increase in DNA length upon addition of a ligand is definitive evidence for an intercalative mode-of-binding. Mode-of-binding, binding affinity, and site-exclusion number are readily determined from scanning force microscopy measurements of the changes in length of individual drug-DNA complexes as a function of drug concentration. With this assay, we resolve the ambiguity surrounding the mode of binding of 2,5-bis(4-amidinophenyl) furan (APF) to DNA and show that it binds to DNA by nonintercalative modes. APF is a member of an important class of aromatic dicationic drugs that show significant activity in the treatment of Pneumocystis carinii pneumonia, an opportunistic infection that is the leading cause of death in AIDS patients.

Direct observation of iron-induced conformational changes of mitochondrial DNA by high-resolution field-emission in-lens scanning electron microscopy.

When respiring rat liver mitochondria are incubated in the presence of Fe(III) gluconate, their DNA (mtDNA) relaxes from the supercoiled to the open circular form dependent on the iron dose. Anaerobiosis or antioxidants fail to completely inhibit the unwinding. High-resolution field-emission in-lens scanning electron microscopy imaging, in concert with backscattered electron detection, pinpoints nanometer-range iron colloids bound to mtDNA isolated from iron-exposed mitochondria. High-resolution field-emission in-lens scanning electron microscopy with backscattered electron detection imaging permits simultaneous detailed visual analysis of DNA topology, iron dose-dependent mtDNA unwinding, and assessment of iron colloid formation on mtDNA strands.

Large secretory structures at the cell surface imaged with scanning force microscopy.

Scanning force microscopy was used to image rat basophilic leukemia (RBL-2H3) cell surfaces under different stimulation conditions that either permit or inhibit secretion. Cross-linking the surface IgE receptors with dinitrophenol-conjugated bovine serum albumin initiates secretion in RBL cells with concomitant spreading of the cell body. Structures at the cell surface approximately 1.5 microns in diameter relate to secretion both spatially and temporally. The position of these surface pits and their sizes suggest that they may be related to the dense-core granules positioned along the cytoskeletal filaments in detergent-extracted, unactivated RBL cell processes. Topographic scanning force microscopy images of RBL cell surfaces at 2, 5, and 35 min after activation show that these structures persist and change in cross-sectional profile with time after activation. These structures may be related to the membrane retrieval mechanism of cells after intense stimulation.

Visualization of Cavitated Vessels in Winter and Refilled Vessels in Spring in Diffuse-Porous Trees by Cryo-Scanning Electron Microscopy1

Xylem cavitation in winter and recovery from cavitation in the spring were visualized in two species of diffuse-porous trees, Betula platyphylla var. japonica Hara and Salix sachalinensis Fr. Schm., by cryo-scanning electron microscopy after freeze-fixation of living twigs. Water in the vessel lumina of the outer three annual rings of twigs of B. platyphylla var. japonica and of S. sachalinensis gradually disappeared during the period from January to March, an indication that cavitation occurs gradually in these species during the winter. In April, when no leaves had yet expanded, the lumina of most of the vessels of both species were filled with water. Many vessel lumina in twigs of both species were filled with water during the period from the subsequent growth season to the beginning of the next winter. These observations indicate that recovery in spring occurs before the onset of transpiration and that water transport through twigs occurs during the subsequent growing season. We found, moreover, that vessels repeat an annual cycle of winter cavitation and spring recovery from cavitation for several years until irreversible cavitation occurs.

Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.

Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's palette of chemical indicators, fluorescent markers, and native biological fluorophores, including NADH, flavins, and green fluorescent proteins, that are applicable to living biological preparations. More than 25 two-photon excitation spectra of ultraviolet and visible absorbing molecules reveal useful cross sections, some conveniently blue-shifted, for near-infrared absorption. Measurements of three-photon fluorophore excitation spectra now define alternative windows at relatively benign wavelengths to excite deeper ultraviolet fluorophores. The inherent optical sectioning capability of nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background and photodamage. Here, the measured nonlinear excitation spectra and their photophysical characteristics that empower nonlinear laser microscopy for biological imaging are described.