Fast and accurate: high-speed metrological large range AFM for surface and nanometrology

Low measurement speed remains as a major shortcoming of the scanning probe microscopic techniques. It leads not only to a low measurement throughput, but also to a significant measurement drift over the long measurement time needed (up to hours or even days). In this paper, development of a high speed metrological large range atomic force microscope (HS Met. LR-AFM) with a capable measurement speed up to 1 mm/s is presented. In its design, a high accurate nanopositioning and nanomeasuring machine (NMM) is combined with a high dynamic flexure hinge piezo stage to move sample. The AFM output signal is combined with the position readouts of the piezo stage and the NMM to derive the surface topography. This design has a remarkable advantage that it well combines different bandwidth and amplitude of different stages/sensors, which is required for high speed measurements. While the HS Met. LR-AFM significantly reduces the measurement time while maintains (or even improves) the metrological performance than the previous Met. LR-AFM, its application capabilities are extended significantly. Two application examples, the realisation of reference areal surface metrology and the calibration of a kind 3D nano standards, have been demonstrated in the paper in detail

System dynamics approach to user independence in high speed AFM

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 135-146).As progress in molecular biology and nanotechnology continues, demand for rapid and high quality image acquisition has increased to the point where the limitations of atomic force microscopes (AFM) become impediments to further discovery. Many biological processes of interest occur on time scales faster than the observation capability of conventional AFMs, which are typically limited to imaging rates on the order of minutes. Imaging at faster scan rates excite resonances in the mechanical scanner that can distort the image, thereby preventing higher speed imaging. Although traditional robust feedforward controllers and input shaping have proven effective at minimizing the influence of scanner distortions, the lack of direct measurement and use of model-based controllers has required disassembling the microscope to access lateral motion with external sensors in order to perform a full system identification experiment, which places excessive demands on routine microscope operators. This work represents a new way to characterize the lateral scanner dynamics without addition of lateral sensors, and shape the commanded input signals in such a way that disturbing dynamics are not excited in an automatic and user-independent manner. Scanner coupling between the lateral and out-of-plane directions is exploited and used to build a minimal model of the scanner that is also sufficient to describe the source of the disturbances. This model informs the design of an online input shaper used to suppress components of the high speed command signals. The method presented is distinct from alternate approaches in that neither an information-complete system identification experiment, nor microscope modification are required. This approach has enabled an increase in the scan rates of unmodified commercial AFMs from 1-4 lines/second to over 100 lines/second and has been successfully applied to a custom-built high speed AFM, unlocking scan rates of over 1,600 lines/second. Images from this high speed AFM have been taken at more than 10 frames/second. Additionally, bulky optical components for sensing cantilever deflection and low bandwidth actuators constrain the AFM's potential observations, and the increasing instrument complexity requires operators skilled in optical alignment and controller tuning. Recent progress in MEMS fabrication has allowed the development of a new type of AFM cantilever with an integrated sensor and actuator. Such a fully instrumented cantilever enables direct measurement and actuation of the cantilever motion and interaction with the sample, eliminating the need for microscope operators to align the bulky optical components. This technology is expected to not only allow for high speed imaging but also the miniaturization of AFMs and expand their use to new experimental environments. Based on the complexity of these integrated MEMS devices, a thorough understanding of their behavior and a specialized controls approach is needed to guide non-expert users in their operation and extract high performance. The intrinsic properties of such MEMS cantilevers are investigated, and a combined approach is developed for sensing and control, optimized for high speed detection and actuation.by Daniel J. Burns.Ph.D

Nanoscale characterization of biopolymers using atomic force microscopy and plasmon-enhanced raman spectroscopy

Proteins are large, complex macromolecules found in every cell and cell component and play a variety of roles essential to the body's functioning. There are four levels of protein structures, which can be classified as follows: primary, secondary, tertiary, and quaternary. Understanding the structure of proteins is often necessary to understand their biological processes. Several analytical techniques are commonly used to characterize the different structures of proteins. For example, X-ray crystallography, nuclear magnetic resonance (NMR), and electron microscopy (EM) are established techniques. Atomic force microscopy (AFM) is another imaging technique that can be used to characterize the nanoscale surface properties of materials. AFM's ability to produce images up to atomic-level resolution is one of its most significant advantages. It can also be used to examine a wide variety of materials, such as metals, semiconductors, polymers, and biological samples, and it can be used in a variety of environments, including air, liquid, and vacuum. Raman spectroscopy is a powerful analytical technique that identifies and studies the vibrational modes of molecules. Nonetheless, this Raman effect is a rare event, and proteins in the cell are present at extremely low concentrations, which contributes to the weak Raman signal. In order to improve the sensitivity and resolution of the method, scientists employ a technique based on the surface plasmon resonance effect, which increases the local electric field of the incident light and, consequently, the Raman signal (plasmon-enhanced Raman spectroscopy). When combined with the AFM technique, both high-resolution images and detailed chemical information can be retrieved, making this technique a highly effective method for characterizing protein structures. In this thesis, three levels of protein structures were characterized using AFM and plasmon-enhanced Raman spectroscopy

Functional analysis of biological matter across dimensions by atomic force microscopy (AFM): from tissues to molecules and, ultimately, atoms

For a detailed understanding of biological tissues and proteins and their dynamical processes the 3D structures of the components involved must be known. Most of the structural data have been obtained through the combination of three major techniques: X-ray crystallography, NMR and TEM. These three methods enable the determination of the structure of biological macromolecules at near atomic resolution and each of those was developed over many years to perfection. Nevertheless each one has its advantage and limitations and all of them may be considered today as useful and complementary tools (Schabert and Engel 1995). In addition to those established techniques, probably the most spectacular advances have been achieved with the AFM, which is the first imaging device allowing direct correlation between structural and functional states of biomolecules in their physiological environment over a range of scaling very much comparable to the capabilities of EM. Moreover, the AFM has the striking possibility to observe biological matter with high resolution in space, time and applied force and with a striking signal-to-noise ratio that typically allows to directly using unprocessed data. Using time-lapse AFM, it is possible to monitor the structural and conformational changes with various biological processes such as the assembly/ disassembly mechanisms of proteins. The AFM tip can also be used to manipulate and control single molecules with forces in the piconewton range. Two interesting examples are the mechanical unfolding of proteins and the measurement of the actin-myosin interaction force. Theoretically the AFM enables to image, probe and manipulate biomolecules in a fully native state at submolecular resolution. However, at the moment this is only feasible for reconstituted transmembrane proteins that are forming 2D crystals in vitro. Imaging of a huge variety of other interesting specimens, e.g. DNA, motor proteins, living cells or the sponge like nuclear pore complex, only reveal macromolecular or even a poorer resolution and protocols for investigating and analyzing other types of biological samples, e.g. connective tissues (cartilage, tendon, bone) or the plaque particles in the coronary arteries, so far are not worked out. In this doctoral thesis I refined or developed several new preparation protocols applicable for the investigation on a variety of biological or medical relevant samples: In a first project we applied high resolution AFM imaging to the luminal and cytoplasmic face of the asymmetric unit membrane (AUM) of the urine bladder epithelium that forms 2D crystalline plaques in vivo. Those urothelial plaques are meant to be involved in a variety of pathological processes, like bladder cancer that ranges among the five most frequent cancer diseases. Moreover, the investigation of AUM in the AFM could offer a basic understanding of the plaque forming process at the innermost part of the bladder, which potentially might also give new insights into a wide variety of structural and dynamical changes occurring in a lipid membrane in situ. In a second project we succeeded to establish a protocol for the robust immobilization of different assemblies of the water soluble protein actin, a 43-kDa protein that plays a fundamental role in muscle contraction, cytoskeletal processes and motility. We achieved high-resolution AFM images of the G- and F-actin arrays that hold promise for detailed structural analyses at the molecular level of morphological changes induced by chemical effectors. This project aims ultimately to trace the muscle-dynamics in its different conformational steps throughout the ATPase/ cross-bridge cycle and at the level of single molecules, i.e. to trace the mechanical interaction of the molecular motor myosin with actin filaments. In a third project we developed new preparation and evaluation protocols that should enable us to directly measure the critical biomechanical parameters of soft biological tissue at the micrometer to nanometer scale. Therefore AFM imaging and indentation-based AFM have enabled us to trace alterations of the tissue architecture at all relevant scales. As a first clinically relevant sample we imaged and mechanically tested articular cartilage in its normal state and compared it to a disease state caused by osteoarthritis. These results are paving new vistas for monitoring disease processes at the scale they are actually occurring, i.e. at the cellular to molecular level. Elasticity measurements at different scales on native articular cartilage provided us with a better understanding of cartilage biomechanics and offer new possibilities in mapping cartilage mechanical properties, which will also include the viscoelastic properties in further measurements. In a next step, individual building blocks were specifically removed out of the tissue architecture by enzyme action. Those digestion experiments helped us to model or mimic alterations caused by pathological degradation processes. At this stage sufficiently smooth samples for high resolution imaging can only be prepared from frozen samples by employing cryo-microtomy. For a refinement of the procedure a protocol has to be developed for combining high resolution imaging with the mechanical probing at the specific sites of interest. Of course the final goal would be that all preparation steps could be done under near physiological conditions. Concerning clinically relevant applications, this work opens the possibility of an in vivo analysis of cartilage consistence in the knee or the investigation of plaque particles in the coronary arteries. This directly leads to the ultimate goal of in situ force mapping by a minimally invasive procedure, i.e. by incorporating the AFM into an arthroscope or into a heart catheter. In summary: In the context of structural research in biology and medicine the AFM is a sensitive microscopical technique with exciting new possibilities to explore new areas of revealing interactions of molecules and merging them with those obtained in the related fields of biochemistry and genetics or by different techniques to gain a comprehensive understanding of cell function: motility, organelle dynamics, membrane transport, and regulation. Moreover, it might be very useful to integrate AFM and AFM-based techniques rather than separating the different techniques and disciplines. Structural, genetical and biochemical data might eventually be composed with the intermediate resolution of EM and AFM and atomic resolution achieved by X-ray crystallography and NMR. In a truly integrated approach the AFM should be complementary to – and not competing with – other microscopical techniques, diffraction methods and spectroscopies. The power of AFM is rooted in its unique capabilities of an extremely high dimensional resolution capability and its resolution in applying and/ or measuring forces that potentially allows the tracing of the fundamental biological processes of life, for example, the translocation of mRNA into protein by the ribosome, the production of molecular “fuel” (e.g., ATP) in mitochondria, and the translocation of ions and small molecules across membranes by channels and pumps. Further developments in sample preparation and instrumentation will open up new AFM capabilities in terms of high-resolution imaging, a better temporal resolution in time-lapse imaging, therefore leading to a real-time imaging and a more time efficient force-mapping and maybe a direct data processing. A higher spatial resolution could be achieved by employing a more sensitive feedback system that requires lower loading forces in combination or in addition to novel and more efficient operational modes, like an improved and more sensitive tapping mode or even better, a real non-contact mode in liquid. Currently new AFMs are designed that are specialized for faster recording of data (Ando et al. 2001; Viani et al. 1999). These instruments operate shorter cantilevers with low-noise characteristics that respond much faster than those currently in use. The new design allows much higher image speed for tapping mode in liquid and also drastically reduces the time for force mapping. Recently a scan rate of up to one frame per second and one to a few minutes for the force mapping have been achieved respectively. This is an important step to trace conformational changes of biological matter in their physiological states and studying the dynamic processes in real time. In combination with more specific software this eventually allows the recording frames at video speed in the future. As stated in the introduction, AFM does not only provide the “eyes’’ for high resolution imaging but it also provides the “fingers’’ to measure and manipulate matter at the atomic and molecular level. This is of great relevance in the life sciences: Recently it has been demonstrated by dissecting DNA from a particular part of the chromosome that the probe can also be used as a nanoscalpel to dissect fragile biological samples (Thalhammer et al. 1997). In this context, force-spectroscopy is another dedicated new methodology, which continuously is under development for investigation, the dynamical processes occurring during the unfolding of a biomolecule (Oesterhelt et al. 2000; Rief et al. 2000; Rief et al. 1997). It might be possible to determine forces generated by a biomolecule on the microsecond time scale. A faster and more sensitive force-spectroscopy based on AFM might allow studying biological processes with greater resolution in time and applied force. By employing IT AFM the tip can be placed directly atop individual structural features to measure its biomechanical properties. In this doctoral thesis I have demonstrated that biomechanical information such as the elastic modulus can be strongly dependent on the experimental scale which is given by the size of the indenter. This could be especially interesting for putative clinical applications, such as obtaining clinical relevant information at fundamentally different levels of tissues architecture. The attainable spatial resolution is directly related to the shape and sharpness of the scanning probe tip. The apex of commercially available tips typically has typical radii of curvature of some nanometers (Czajkowsky et al. 2000) and will improve in the future in terms of sharpness and in reduction of cantilever noise. There may be a practical advantage in using other tips grown in situ onto cantilevers or carbon nanotubes using an electron microscope. Both types of tips possess many unique properties that make them ideal AFM probes. Their high aspect ratio provides faithful imaging of deep trenches, while good resolution is retained due to their nanometer-scale diameter. These geometrical factors also lead to reduce tip-sample adhesion, which therefore might allow a gentler imaging. New types of cantilevers might be more specific in terms of their physical or chemical functioning of the probe or on the other hand biochemically compatible for biomedical applications. Multifunctional cantilevers will provide the extension to a whole set of new experimental opportunities of monitoring possibly a wide range of multiple signals, like voltage, currents of ions or electrons and fluorescently labeled molecules (Engel et al. 1999). Nanotubes are electrically conductive, which allow their use for the measurements of (surface-) potentials, and they can be modified at their ends with specific chemical or biological groups for high resolution functional imaging. Nanotubes could also be used as nanopipettes for drug delivery or as an electrical conductive tip for measuring local potentials, currents or trafficking of biomolecules across membranes. Arrays of cantilevers (millipede) are under development that could be used for mechanically probing simultaneously neighboring regions of the specimen. This could allow mapping mechanical properties or imaging of larger regions within a short time or a more reliable diagnosis, i.e. different tissues or the plaques in the coronal arteries involved in heart diseases, all of them with an extremely high interest for medical research and treatment of diseases. Combined AFM-SNOM (scanning near-field optical microscopy) - probe tips are potentially of great use and can be handled almost like the commonly used AFM probes. SNOM allows the detection of a conventional AFM height image and additionally an optical image with an improved optical resolution of more than one order of magnitude compared to conventional LM. SNOM therefore offers in addition to the topography also corresponding optical information of fluorescently labeled biological features beyond the diffraction limit of conventional light microscopy. One potential application might be the detection of the green fluorescent protein expressed by some cell strains. These tips can either be used as a detector to follow for example the specimen of green fluorescent proteins or as an effector to induce photoactive processes at the single molecule level. One of our future projects is to detect the bidirectional trafficking through the nuclear pore complex by fluorescently labeled cargo. To achieve this we are developing a protocol to tightly seal the nuclear envelope of Xenopus oozytes over microfabricated pores. The goal is to measure in their complete functional state the transport rates of these nuclear gates at the level of single pores. In a comparison with genetically modified pores we aim to understand the regulation of cargoes between the nucleus and cytoplasm. The impacts of nano science in medically relevant applications foster the development of new devices and analytical tools for clinical practice. Due to the very high signal-to-noise ratio, AFM-based techniques offer the observation, probing and characterization of the chemical and mechanical properties of single molecules within a tissue in a physiologically relevant environment. Therefore pathological changes in human tissue can be registered and hopefully cured at an early stage of the disease progression. On the other hand by expanding our ability to characterize and control biological tissue in much more detail diagnostics and therapeutics could be revolutionized in terms of delivery of drugs in small amounts with a high resolution to the target. The new possibilities opened by the AFM will also have an important impact for engineering and (quality-) control of surface properties of new biocompatible implants, tissue-engineered organs or novel biodegradable products that can be resorbed by the body after use. Progress in nanobiotechnology will promote the healing of natural tissue and will stimulate mimicking high-performance biocompatible nanosystems such as artificial active components or biological implants for organ and joint replacement. Thus new implants will be designed with nanoscale features that will replace degenerated, diseased or damaged tissues, e.g. arthritic cartilage or bone. Since its invention the AFM has opened new avenues for a wide range of exciting applications ranging from basic structural research up to new developments for in situ applications in a clinical environment. However, diseases are often caused largely by damages either at the molecular or the cellular scale. For a local assessment today’s surgical tools are simply too big. In the future we should get tools that are molecular, both in their size and their precision; and then we will be able to intervene directly at the level where the damage occurs and correct it. Ultimately the dream of Richard Feynman and Albert R. Hibbs might become true that once nanoscale machines can be injected into the bloodstream for dialysis or drug delivery

AFM-STED correlative nanoscopy provides a new view on the formation process of misfolded protein aggregates

The main part of my PhD work focused on the application of an advanced integrated technique, based on the coupling of an atomic force microscope (AFM) and a stimulated emission depletion (STED) microscope in the study of amyloid fibrils formation. This coupled system allows the acquisition of super-resolution fluorescence images, perfectly overlapped with AFM topography. Exploiting the extended capability offered by this technique, I highlighted some important features on the mechanisms followed by the labeled and unlabeled proteins through their aggregation pathway. The results demonstrates that labeled molecules are involved only in selected pathways of aggregation, among the multiple that are present in the aggregation reaction. In a second part of my work, I investigated the process of interaction between Alpha-synuclein (\u3b1-Syn), the pathological peptide associated to the Parkinson\u2019s disease, and model lipid membranes. The aim of this study was to identify molecular mechanisms that are indicated as the base of neurodegeneration, not only in Parkinson\u2019s disease, but also in a large class of disorders, indicated as protein misfolding diseases

Single-Chip Scanning Probe Microscopes

Scanning probe microscopes (SPMs) are the highest resolution imaging instruments available today and are among the most important tools in nanoscience. Conventional SPMs suffer from several drawbacks owing to their large and bulky construction and to the use of piezoelectric materials. Large scanners have low resonant frequencies that limit their achievable imaging bandwidth and render them susceptible to disturbance from ambient vibrations. Array approaches have been used to alleviate the bandwidth bottleneck; however as arrays are scaled upwards, the scanning speed must decline to accommodate larger payloads. In addition, the long mechanical path from the tip to the sample contributes thermal drift. Furthermore, intrinsic properties of piezoelectric materials result in creep and hysteresis, which contribute to image distortion. The tip-sample interaction signals are often measured with optical configurations that require large free-space paths, are cumbersome to align, and add to the high cost of state-of-the-art SPM systems. These shortcomings have stifled the widespread adoption of SPMs by the nanometrology community. Tiny, inexpensive, fast, stable and independent SPMs that do not incur bandwidth penalties upon array scaling would therefore be most welcome. The present research demonstrates, for the first time, that all of the mechanical and electrical components that are required for the SPM to capture an image can be scaled and integrated onto a single CMOS chip. Principles of microsystem design are applied to produce single-chip instruments that acquire images of underlying samples on their own, without the need for off-chip scanners or sensors. Furthermore, it is shown that the instruments enjoy a multitude of performance benefits that stem from CMOS-MEMS integration and volumetric scaling of scanners by a factor of 1 million. This dissertation details the design, fabrication and imaging results of the first single-chip contact-mode AFMs, with integrated piezoresistive strain sensing cantilevers and scanning in three degrees-of-freedom (DOFs). Static AFMs and quasi-static AFMs are both reported. This work also includes the development, fabrication and imaging results of the first single-chip dynamic AFMs, with integrated flexural resonant cantilevers and 3 DOF scanning. Single-chip Amplitude Modulation AFMs (AM-AFMs) and Frequency Modulation AFMs (FM-AFMs) are both shown to be capable of imaging samples without the need for any off-chip sensors or actuators. A method to increase the quality factor (Q-factor) of flexural resonators is introduced. The method relies on an internal energy pumping mechanism that is based on the interplay between electrical, mechanical, and thermal effects. To the best of the author’s knowledge, the devices that are designed to harness these effects possess the highest electromechanical Qs reported for flexural resonators operating in air; electrically measured Q is enhanced from ~50 to ~50,000 in one exemplary device. A physical explanation for the underlying mechanism is proposed. The design, fabrication, imaging, and tip-based lithographic patterning with the first single-chip Scanning Thermal Microscopes (SThMs) are also presented. In addition to 3 DOF scanning, these devices possess integrated, thermally isolated temperature sensors to detect heat transfer in the tip-sample region. Imaging is reported with thermocouple-based devices and patterning is reported with resistive heater/sensors. An “isothermal electrothermal scanner” is designed and fabricated, and a method to operate it is detailed. The mechanism, based on electrothermal actuation, maintains a constant temperature in a central location while positioning a payload over a range of >35μm, thereby suppressing the deleterious thermal crosstalk effects that have thus far plagued thermally actuated devices with integrated sensors. In the thesis, models are developed to guide the design of single-chip SPMs and to provide an interpretation of experimental results. The modelling efforts include lumped element model development for each component of single-chip SPMs in the electrical, thermal and mechanical domains. In addition, noise models are developed for various components of the instruments, including temperature-based position sensors, piezoresistive cantilevers, and digitally controlled positioning devices

Advanced Microscopy Techniques for the Molecular Scale Analysis and Physical Characterization of \u3cem\u3eEscherichia coli\u3c/em\u3e Spheroplasts

Atomic force microscopy (AFM) holds a unique position in microbiology because of its potential for nanometer (nm) scale imaging and piconewton (pN) force detection. These features can be exploited to characterize bacteria from the cellular down to the molecular level. In order to pursue such characterization studies, reliable sample preparation techniques must be developed. Spheroplasts are bacteria which have been treated with enzymes to remove cell wall components. Because the cytoplasmic membrane is exposed in spheroplasts, they are suitable for localizing transporters and other membrane proteins using AFM techniques. Constituents on the surface of intact bacteria are responsible for their adhesion to various substrates in vivo. The absence of these constituents in spheroplasts necessitates specialized immobilization strategies. This study presents a technique in which spheroplasts are immobilized by cross linking them with glutaraldehyde to mica surfaces pretreated with aminopropyltriethoxysilane. As suggested by the AFM images, this approach facilitates stable imaging in appropriate buffers. Because the sample preparation strategies presented are compatible with optical and atomic force microscopies, investigations in which molecular system components are monitored can be targeted. Evidence that the cells retain membrane integrity, continue glucose uptake, increase in diameter and initiate protein synthesis after immobilization is also presented. Based on this data, it is concluded that metabolic processes continue in immobilized spheroplasts. As a result of this finding, spheroplasts are proposed to be a platform for various imaging-based investigations. Elasticity and indentation measurements on intact bacteria and spheroplasts revealed significant differences between the two forms. They also provided the justification for using glutaraldehyde fixed spheroplasts for molecular recognition experiments designed to locate the glucose transporter on the surface of spheroplasts. An avidin-biotin system was used in which biotin was tethered to the AFM tip using a polyethylene glycol linker, When this functionalized tip probed spheroplast surfaces previously immunolabeled with a biotinylated antibody and avidin, molecular recognition was demonstrated. The fact that the biotin functionalized tips can be used in multiple applications is an attractive feature of this strategy. That results from AFM experiments can be validated with optical microscopy techniques is also an advantage

Scanning probe microscopy investigations of (1) arrays of cysteine-coated CdS nanoparticles and (2) structures formed during the early stages of the corrosion of copper surfaces

Scanning probe microscopy (SPM) characterizations are becoming more prevalent for surface investigations due to their capabilities for obtaining structural information and physical measurements. New capabilities of SPM for studying and controlling nanoscale processes are emerging as valuable assets in research. A fundamental understanding of the interactions of surface reactions provides essential information for developing workable applications for nanotechnology. Two applications of SPM are discussed in this dissertation. The first investigation uses atomic force microscopy (AFM) for the characterization of nanostructures produced with a newly developed lithographic technique called “two-particle” lithography. This new technique is based on particle lithography for the patterning of nanoparticles. Structural templates of either latex or silica guide the deposition of nanoparticles to generate 2D arrays of nanopatterns. The surface coverage, size and periodicity of the nanoparticle structures can be controlled by changing the particle size of the templating sphere. Particle lithography provides test platforms to enable multiple reproducible SPM measurements for nanostructures which have well-defined geometries and surface arrangements. The second part of the dissertation discusses the results from using AFM to study the earliest stages of the onset of water corrosion of copper surfaces with nanoscale resolution. Within a few hours of exposure to water of varying chemistries, dramatic differences in the morphology of copper surfaces were observed by ex situ AFM topography imaging. Surface characterizations of the treated copper samples were used systematically to evaluate changes for copper surfaces with various chemical treatments and to investigate mechanisms of passivation and corrosion

Biomarker Sensors and Method for Multi-Color Imaging and Processing of Single-Molecule Life Signatures

The invention is a device including array of active regions for use in reacting one or more species in at least two of the active regions in a sequential process, e.g., sequential reactions. The device has a transparent substrate member, which has a surface region and a silane material overlying the surface region. A first active region overlies a first portion of the silane material. The first region has a first dimension of less than 1 micron in size and has first molecules capable of binding to the first portion of the silane material. A second active region overlies a second portion of the silane material. The second region has a second dimension of less than 1 micron in size, second molecules capable of binding to the second portion of the active region, and a spatial distance separates the first active region and the second active region