Arjan Quist

AMYLOID BETA PROTEIN-(1-42) FORMS CALCIUM-PERMEABLE, ZN2+-SENSITIVE CHANNEL

Amyloid β protein (AβP) forms senile plaques in the brain of the patients with Alzheimer’s disease. The early-onset AD has been correlated with an increased level of 42-residue AβP (AβP1–42). However, very little is known about the role of AβP1–42 in such pathology. We have examined the activity of AβP1–42 reconstituted in phospholipid vesicles. Vesicles reconstituted with AβP show strong immunofluorescence labeling with an antibody raised against an extracellular domain of AβP suggesting the incorporation of AβP peptide in the vesicular membrane. Vesicles reconstituted with AβP showed a significant level of 45Ca2+ uptake. The 45Ca2+ uptake was inhibited by (i) a monoclonal antibody raised against the N-terminal region of AβP, (ii) Tris, and (iii) Zn2+. However, reducing agents Trolox and dithiothreitol did not inhibit the 45Ca2+uptake, indicating that the oxidation of AβP or its surrounding lipid molecules is not directly involved in the AβP-mediated Ca2+ uptake. An atomic force microscope was used to image the structure and physical properties of these vesicles. Vesicles ranged from 0.5 to 1 μm in diameter. The stiffness of the AβP-containing vesicles was significantly higher in the presence of calcium. The stiffness change was prevented in the presence of zinc, Tris, and anti-AβP antibody but not in the presence of Trolox and dithiothreitol. Thus the stiffness change is consistent with the vesicular uptake of Ca2+. These findings provide biochemical and structural evidence that AβP1–42 forms calcium-permeable channels and thus may induce cellular toxicity by regulating the calcium homeostasis in Alzheimer’s disease.

Extremely sharp carbon nanocone probes for atomic force microscopy imaging

A simple and reliable catalyst patterning technique combined with electric-field-guided growth is utilized to synthesize a sharp and high-aspect-ratio carbon nanocone probe on a tipless cantilever for atomic force microscopy. A single carbon nanodot produced by an electron-beam-induced deposition serves as a convenient chemical etch mask for catalyst patterning, thus eliminating the need for complicated, resist-based, electron-beam lithography for a nanoprobe fabrication. A gradual, sputtering-induced size reduction and eventual removal of the catalyst particle at the probe tip during electric-field-guided growth creates a sharp probe with a tip radius of only a few nanometers. These fabrication processes are amenable for the wafer-scale synthesis of multiple probes. High resolution imaging of three-dimensional features and deep trenches, and mechanical durability enabling continuous operation for many hours without noticeable image deterioration have been demonstrated.

Fabrication of high-aspect-ratio carbon nanocone probes by electron beam induced deposition patterning

A high-aspect-ratio cone-shaped carbon nanotube (CNT), which we refer to as a carbon nanocone (CNC), was fabricated for scanning probe microscopy (SPM) by a novel and reliable patterning technique and dc plasma chemical vapour deposition. Carbon dots from electron beam induced deposition (EBID) were utilized as convenient chemical-etch masks to create catalyst patterns for the growth of a single CNC probe on a tipless cantilever and an array of CNC probes on a silicon substrate. This resist-free EBID process is an efficient way of preparing a patterned catalyst and resultant nanoprobe on the specific edge location of the cantilever. The CNC probe produces high-resolution images of specimens in air as well as in liquid. No degradation in imaging performance was observed after a period of continuous scanning. The CNC bed-of-nails array imaged in contact mode by a commercial Si3N4 probe demonstrates the mechanical toughness/sturdiness of the CNC tip. This also indicates the possibility of using the CNC bed-of-nails as a convenient means for the characterization of SPM tips.

High performance, LED powered, waveguide based total internal reflection microscopy

Total internal reflection fluorescence (TIRF) microscopy is a rapidly expanding optical technique with excellent surface sensitivity and limited background fluorescence. Commercially available TIRF systems are either objective based that employ expensive special high numerical aperture (NA) objectives or prism based that restrict integrating other modalities of investigation for structure-function analysis. Both techniques result in uneven illumination of the field of view and require training and experience in optics. Here we describe a novel, inexpensive, LED powered, waveguide based TIRF system that could be used as an add-on module to any standard fluorescence microscope even with low NA objectives. This system requires no alignment, illuminates the entire field evenly, and allows switching between epifluorescence/TIRF/bright field modes without adjustments or objective replacements. The simple design allows integration with other imaging systems, including atomic force microscopy (AFM), for probing complex biological systems at their native nanoscale regimes.

Structure and Permeability of Ion-channels by Integrated AFM and Waveguide TIRF Microscopy

Membrane ion channels regulate key cellular functions and their activity is dependent on their 3D structure. Atomic force microscopy (AFM) images 3D structure of membrane channels placed on a solid substrate. Solid substrate prevents molecular transport through ion channels thus hindering any direct structure-function relationship analysis. Here we designed a ~70u2005nm nanopore to suspend a membrane, allowing fluidic access to both sides. We used these nanopores with AFM and total internal reflection fluorescence microscopy (TIRFM) for high resolution imaging and molecular transport measurement. Significantly, membranes over the nanopore were stable for repeated AFM imaging. We studied structure-activity relationship of gap junction hemichannels reconstituted in lipid bilayers. Individual hemichannels in the membrane overlying the nanopore were resolved and transport of hemichannel-permeant LY dye was visualized when the hemichannel was opened by lowering calcium in the medium. This integrated technique will allow direct structure-permeability relationship of many ion channels and receptors.

Characterization of Nanoscale Biological Systems: Multimodal Atomic Force Microscopy for Nanoimaging, Nanomechanics, and Biomolecular Interactions

Complexity in biological systems requires coordinated research efforts using xadtechniques and approaches that are amenable to multiscale (from nano to micro and beyond) and multidimensional (including their structure, activity, and function) microsystems (e.g., cell membrane, cell organelles, biomacromolecules, and their individual as well as integrated functioning). Major experimental designs and xaddiscoveries in biological sciences have usually been preceded by major discoveries in physicochemical sciences and engineering. Our understanding of biological xadsystems at the time and length scale of micrometer and above is reasonable. Scaling down those systems at nanometer level is challenging and mostly unexplored as yet. The classic correspondence theory of nanoscale and microscale phenomena in physical systems is not likely to be valid for biological systems given the layers of complexity in the biological systems. Hence, one needs to examine biological xadsystems at nanoscale in both structural and temporal domains.