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 ~70 nm 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.

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.

Complexity in biological systems requires coordinated research efforts using ­techniques 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 ­discoveries in biological sciences have usually been preceded by major discoveries in physicochemical sciences and engineering. Our understanding of biological ­systems 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 ­systems at nanoscale in both structural and temporal domains.

IMPORTANCE OF THE FIELD:Engineering of cell culture substrates provides a unique opportunity for precise control of the cellular microenvironment with both spatial as well as temporal resolutions. This greatly enhances studies of cell-cell, cell-matrix and cell-factor interaction studies in vitro. AREAS COVERED IN THIS REVIEW:The technologies used for micropatterning in the biological field over the last decade and new applications in the last few years for dynamic control of surfaces, tissue engineering, drug discovery, cell-cell interactions and stem cell studies are presented. WHAT THE READER WILL GAIN:The reader will gain knowledge on the state of the art in micropatterning and its wide ranging applications in cell patterning, with new pathways to control the cell environment. TAKE HOME MESSAGE:Micropatterning of cells has been studied and developed enough to be widely applied ranging from single cell assays to tissue engineering. Techniques have evolved from many-step processes to direct writing of biologically selective patterns.

Without understanding the property of stickiness there are limits as to how far we can use it and how sticky we can make an object. Understanding of what affects stickiness is critical. Are surface roughness and stickiness related? What is the difference between the sticky and non-sticky objects at a molecular level? We decided to look at the difference between the sticky and non-sticky objects. We reasoned that if we collect sticky and non-sticky objects and compare them through the naked eye, a high powered microscope, and an atomic force microscope (AFM), then the objects that are stickier will have more surface roughness than the objects that are less sticky. Results from our imaging of and analysis of the force of adhesion (which gives a measure of stickiness) between non-sticky objects and sticky objects through the AFM have shown us a different relationship between the surface roughness and stickiness than we had reasoned – the relationship that we have discovered is that stickiness is inversely related to the surface roughness of the materials. Our findings could be used to design new adhesives with different materials that are stronger, lighter and more cost effective that the adhesives used today.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

Alzheimer's disease (AD) is a protein misfolding disease. Early hypothesis of AD pathology posits that 39-43 AA long misfolded amyloid beta (Abeta) peptide forms a fibrillar structure and induces pathophysiological response by destabilizing cellular ionic homeostasis. Loss of cell ionic homeostasis is believed to be either indirectly due to amyloid beta-induced oxidative stress or directly by its interaction with the cell membrane and/or activating pathways for ion exchange. Significantly though, no Abeta specific cell membrane receptors are known and oxidative stress mediated pathology is only partial and indirect. Most importantly, recent studies strongly indicate that amyloid fibrils may not by themselves cause AD pathology. Subsequently, a competing hypothesis has been proposed wherein amyloid derived diffusible ligands (ADDLs) that are large Abeta oligomers (approximately >60 kDa), mediate AD pathology. No structural details, however, of these large globular units exist nor is there any known suitable mechanism by which they would induce AD pathology. Experimental data indicate that they alter cell viability by non-specifically changing the plasma membrane stability and increasing the overall ionic leakiness. The relevance of this non-specific mechanism for AD-specific pathology seems limited. Here, we provide a viable new paradigm: AD pathology mediated by amyloid ion channels made of small Abeta oligomers (trimers to octamers). This review is focused to 3D structural analysis of the Abeta channel. The presence of amyloid channels is consistent with electrophysiological and cell biology studies summarized in companion reviews in this special issue. They show ion channel-like activity and channel-mediated cell toxicity. Amyloid ion channels with defined gating and pharmacological agents would provide a tangible target for designing therapeutics for AD pathology.

We describe a silicon chip-based supported bilayer system to detect the presence of ion channels and their electrical conductance in lipid bilayers. Nanopores were produced in microfabricated silicon membranes by electron beam lithography as well as by using a finely focused ion beam. Thermal oxide was used to shrink pore sizes, if necessary, and to create an insulating surface. The chips with well-defined pores were easily mounted on a double-chamber plastic cell recording system, allowing for controlling the buffer conditions both above and below the window. The double-chamber system allowed using an atomic force microscopy (AFM) tip as one electrode and inserting a platinum wire as the second electrode under the membrane window, to measure electrical current across lipid bilayers that are suspended over the pores. Atomic force imaging, stiffness measurement, and electrical capacitance measurement show the feasibility of supporting lipid bilayers over defined nanopores: a key requirement to use any such technique for structure-function study of ion channels. Online addition of gramicidin, an ion-channel-forming peptide, resulted in electrical current flow across the bilayer, and the I-V curve that was measured using the conducting AFM tip indicates the presence of many conducting gramicidin ion channels.

Cerebral amyloid diseases are considered to be part of an emerging complex group of chronic and progressive entities collectively known as Disorders of Protein Misfolding that include, among many others, Alzheimer's disease, polyglutamine–repeat disorders, cataracts, amyotrophic lateral sclerosis, Parkinson's disease and other synucleinopathies, systemic and cerebral amyloidosis, tauopathies, prion diseases, and type–II diabetes. In these disorders, soluble proteins normally found in biological fluids change their conformation and form either insoluble structures that accumulate in the form of intra– and extra–cellular aggregates or fibrillar lesions usually associated with cell toxicity, complement activation and the local release of inflammatory mediators and oxidative stress products. Recent studies have shown that pre–fibrillar conformations of amyloid proteins are sufficient to induce cellular toxicity. However, the 3D structural conformations of these globular structures, a key missing link in designing effective prevention and treatment approaches, still remain undefined. To test the hypothesis that different amyloid molecules form ion channel–like structures when incorporated in lipid bilayers. Recombinant or synthetic Aβ1–40, α–synuclein, ABri, ADan, serum amyloid A, and amylin were inserted in reconstituted membranes. Structural and functional parameters were monitored by atomic force microscopy (AFM), circular dichroism spectrometry, gel electrophoresis, and electrophysiological recordings. All these unrelated proteins were initially monomeric or monomeric/dimeric, exhibited different secondary structures in solution and similar globular configurations under AFM. When reconstituted in lipid bilayers these small globular assemblies underwent supramolecular conformational changes. AFM images of the resulting membrane structures were morphologically compatible with ion–channel–like structures. Electrophysiological recordings indicated that all these molecules, when inserted in lipid bilayers, elicit single ion–channel conductances. Proteins re–extracted from the membranes showed higher degree of oligomerization when compared with the freshly solubilized material (tetramers to octamers). In reconstituted membranes, amyloids form morphologically compatible ion channel–like structures and elicit single ion–channel currents consistent with electrophysiological studies showing amyloid channel activity in cell membranes. Thus, by triggering destabilization of cellular ionic homeostasis, amyloids may directly induce cell dysfunction and degeneration in a variety of protein misfolding disorders.

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.

Microfluidic channels are microreactors with a wide range of applications, including molecular separations based upon micro/nanoscale physicochemical properties, targeting and delivery of small amount of fluids and molecules, and patterned/directed growth. Their successful applications would require a detailed understanding of phenomena associated with the microscale flow of liquids through these channels, including velocity, viscosity and miscibility. Here we demonstrate a highly sensitive piezoresistive cantilever to measure flow properties in microfluidic channels. By milling down the legs of the piezoresistive cantilevers, we have achieved significantly higher mechanical sensitivity and a smaller spring constant, as determined by AFM. These cantilevers were used in microchannels to measure the viscosity and flow rate of ethylene glycol mixtures in water over a range of concentrations, as well as of low viscosity biologically relevant buffers with different serum levels. The sensor can be used alone or can be integrated in AFM systems for multidimensional study in micro and nanochannels.

Cisplatin is the most effective cytotoxic agent against many cancers. Its usage, however, is limited due to inefficient uptake by the target cells. A liposomal formulation of cisplatin is reported to partly overcome this limitation. Physicochemical characteristics of the liposome-cisplatin preparation, including its size, stability, encapsulation efficiency, and cytoplasmic internalization efficiency, play a significant role in an effective usage of liposomal formulations. We have used atomic force microscopy (AFM) to determine physicochemical characteristics of cisplatin-encapsulated liposomes, AFM and fluorescence microscopy to examine their cytoplasmic internalization, and Live/Dead assay to examine their cell toxicity. Nonencapsulated cisplatin is globular and 10-50 nm in size. AFM force-dissection and stiffness measurements show that cisplatin-encapsulated liposomes are significantly stiffer ( approximately 100%) and more stable than liposomes without encapsulated cisplatin. Cisplatin-encapsulated liposomes of approximately 250 nm diameter (nanoliposomes) are most efficiently internalized and induce cell toxicity in a time-dependent manner. Liposomes without cisplatin of similar dimensions, although internalized in the cell cytoplasm, do not induce cell toxicity.

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.

Extract HTML view is not available for this content. However, as you have access to this content, a full PDF is available via the ‘Save PDF’ action button. Extended abstract of a paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, USA, July 31--August 4, 2005