Michael Lee Roukes - US grants

Many important analytical studies of biomolecules are not feasible because current technology is both too insensitive and at a scale too gross for experimentation with extremely small sample volumes. The overall objective of this proposal is to explore the potential of micro- and nanometer scale technology to overcome these limitations. Two underlying principles motivate this work: molecular detection capability can be greatly enhanced in micro/nanometer scale devices. small volumes, from micro- to pico-liters or less, can be more appropriately handled in small devices. The ultimate objective in these endeavors is to realize integrated instruments that permit in situ extraction and analysis of targeted molecular components from an individual cell. In these initial efforts, the immediate goal is to study a specific class of molecules involved in the process of sea urchin embryogenesis using prototypical microdevices specifically designed for this research. Transcription factor (TF) proteins regulate differential gene expression in temporally and spatially restricted regions of the embryo by binding with high affinity to specific DNA sequences. With existing technology, functional TF/DNA binding studies can only be achieved using hundreds of thousands of embryos. To study TF/DNA binding from small regions within an embryo, or from individual cells, microdevices that provide the following functions will be sequentially constructed, evaluated, and optimized in this research pro~ram: Manipulation and transport of cells, small tissue fragments, and solutions. Lysis of cells/organelles to open their contents for further analysis. Reagent mixing, to combine synthetic, fluorescent oligonucleotides and unlabeled, non-specific DNA with cell extracts. Electrical conductivity measurements/optical absorption spectroscopy upon picoliter solutions. Electrophoretic separation and detection of fluorescent molecules. Many materials systems widely employed in contemporary nanostructure research are chosen to optimize electronic or photonic properties, but these are not necessarily ideally suited, nor perhaps even compatible, with biological systems. An important thrust in this initial program is the development of materials/processing combinations, especially highly-anisotropic dry etching procedures, which permit high resolution microfabrication while retaining biological compatibility. Devices will be constructed using both high resolution optical and electron beam lithography, dry and wet etching processes, and both thermallyand sputter- deposited thin film materials. At each step of their development, the instruments created will be applied to the aforementioned TF/DNA binding assays. This iterative and interactive research, carried out on both nanofabrication and biological fronts, will drive the practical attainment of new classes of high resolution instruments for biological science. It is anticipated that this work will spawn new research efforts at the nanometer scale in both the science of biological phenomena and the technology of bioinstrumentation.

w:\awards\awards96\num.doc 9705411 Roukes Calorimetry and Thermal Transport at the Quantum Limit. This new research program focuses on reduced-dimensional heat flow and thermal equilibration in nanostructures. The investigators have developed new techniques for the surface nanomachining of suspended semiconductor structures that enable the construction of miniature, thermally-isolated devices. These possess integral transducers permitting the local introduction of heat, and local temperature measurements. Coupling these with high sensitivity dc SQUID-based methods for electron thermometry at millikelvin temperatures is ultimately expected to yield sensitivity sufficient for exploration of energy exchange processes involving individual quanta. Single- phonon phenomena, with analogs in classical and quantum optics, should become observable. Intriguing possibilities include phonon shot noise, phonon bunching, anticorrelated electron- phonon relaxation, and the phonon-by-phonon energy decay of a quasi-isolated thermal reservoir. With this level of sensitivity, calorimetry experiments elucidating processes involving individual atoms and molecules are also possible. %%% Calorimetry and Thermal Transport at the Quantum Limit. This new research program focuses on the question of how heat flows in extremely small objects, i.e. nanostructures. Ultrasmall systems can behave as if they are reduced- dimensional (i.e., less than 3D). At low temperatures, for systems that are small enough, heat flow and thermal equilibration will ultimately occur by the exchange of individual energy quanta. This domain, where heat transfer is "granular" (delivered quanta-by-quanta rather than as a steady flow) remains completely unexplored. A complete understanding of these issues will become crucial with the continued miniaturi zation of devices leading to nanotechnology. These studies are now possible through new nanomachining techniques developed by the investigators, allowing the creation of suspended, ultrasmall semiconductor structures with three- dimensional relief -- with on-board electronic measurement circuitry. Among possible applications of this research, these experiments should enable direct observation of the heat evolved during chemical reactions involving *individual* atoms and molecules. The devices developed should also offer unprecedented sensitivity for measurement of optical and infrared radiation. ***

In 1991 it was proposed that nuclear magnetic resonance (NMR) spectrometry with sensitivity at the level of a single proton might be achievable through mechanical detection using scanned probe techniques similar to AFM. Achieving this degree of sensitivity would constitute a truly revolutionary advance; it would permit three-dimensional atomic-scale imaging, with chemical specificity. Mechanically-detected MRI, often called Magnetic Resonance Force Microscopy (MRFM) is now many orders of magnitude more sensitive than conventional MRI. The mechanical detection technique has clearly provided staggering advances, and it is clear that significant additional gains are on the horizon for MRFM. We project that MRI with atomic resolution will be attained in just a few years.
The PI and co-PI propose as an ultimate goal MRI instruments with single-nucleus resolution, will require a radically new approach, one that involves single-chip integration of hybrid electro/magneto/opto/mechanical devices. Realizing such devices would be an important departure from the path being taken by all current workers, worldwide, in this new field of MRFM. This new research direction is what has been proposed in this submission. This grant, if awarded, will allow the PI to leverage his current efforts in, and facilities for, Nano Electro Mechanical Systems (NEMS) toward making important headway on the engineering and science of novel integrated systems (nanochips) for MRFM. When fully implemented, the magnetic resonance force microscope will become a unique scanned probe instrument offering the high spatial resolution of atomic force microscopy (AFM), while simultaneously offering three dimensional visualization capabilities of MRI within the engineering sciences it will enable new, high resolution studies of the microscopic subsurface properties for a broad range of new materials and electronic devices based upon buried interfaces. It is a technique that is both non-destructive and chemically specific.

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Roukes

Major advances have recently been made at California Institute of Technology (Caltech) in developing and employing what are, largely, individual nanometer-scale structures for applications ranging from fundamental science to technological applications. Within the research groups of the co--P1's, Professor Scherer and Professor Roukes, who have worked together on nanofabrication for the past fifteen years, electron beam lithography techniques have been developed and used for the construction of a wide range of functional nanometer-scale devices. Lateral dimensions below 10 nm are routinely obtained, and students in these groups have developed both expertise in the requisite electron-beam-control code and an in-depth understanding of the specialized resist processing and pattern transfer techniques enabling ultrahigh resolution. The time is now ripe to exploit these advances by creating nanosystems - i.e. advanced structures that comprise coherently coupled arrays of the individual nanoscale elements the authors are perfecting. This equipment acquisition proposal, if funded, would enable such research.

Nanodevice arrays are emerging as a priority in nanoscale science and technology. As detailed in this proposal (and its accompanying letters of support), nanoscale arrays will find immediate applications within the proposers' research programs. These currently involve 13 Caltech professors, in disciplines spanning fundamental physics, chemistry, biology, and engineering and materials science. Among the specific topics currently being pursued are: quantum optics, quantum computation, nanophotonics, spin electronics, nanomechanics, neurophysiology, biotechnology, electrochemistry and molecular electronics. These applications require fabrication of structures spanning a hierarchy of size scales -from the smallest dimensions accessible via state-of-the-art nanofabrication techniques, to the millimeter to centimeter domain of integrated, chip-based systems. Fabrication of these complex nanoscale arrays requires multiple, successively-aligned steps of large-field electron beam lithography over the wafer scale.

A second important research thrust would be enabled by the proposed instrumentation. This focuses upon future technological applications requiring nanometer-scale features produced lithographically en masse. This scale is far below the dimensions currently accessible via deep ultraviolet lithography, the current industry standard for state-of-the-art commercial production lines. To address this technological need, much recent effort world-wide has focused upon development of new, high-resolution, high-throughput lithographic methods. Projection x-ray lithography, shaped electron beam lithography, and mechanical transfer methods (embossing, molding, or stamping) all have evolved as principle contenders for the definition of sub-I 100nm structures over large areas. All of these techniques, however, have in common the need for wafer-scale high-resolution masks. These are normally generated by vector-scanned electron beam lithography. There are currently no alternative lithographic tools which offer comparable flexibility, resolution and placement accuracy for this purpose as state-of-the-au commercial electron beam writers. Student access to such an instrument would greatly enhance research and training in the proposers' university setting.

An entirely new level of instrumentation is required to successfully initiate these proposed endeavors. Specifically, the capability of writing large (wafer scale) fields of features at the sub-5Onm scale is absolutely crucial. This can only be done with a state-of-the-art electron beam writer; however the acquisition of such an instrument is significantly beyond the scope of most funding programs. Here the PIs propose to purchase an electron-beam lithography system for this laboratory. The cost for this instrument will be shared by Caltech ($l.5M), the NSF ($1.OM), and DARPA/DURINT ($l.OM). The laboratory established with these funds will constitute an interactive, "expert" facility within the larger efforts of the PI's. This select, focused group of researchers will include undergraduate and graduate students, staff and faculty members. This group will be collectively dedicated to establishing routes to next-generation structures involving large arrays of nanoscale elements.

In mesoscopic systems at low temperatures, heat transport and thermal equilibration occur in a very different manner from macroscopic systems at room temperature. This is due to the small heat capacities involved, and very long thermal relaxation times to reach equilibrium with a heat reservoir, the environment. At the ultimate limit, thermal transport involves exchange of a single energy channel between a system and the environment. During the preceding phase of this project, investigators observed, for the first time, this predicted quantization of thermal conductance. This places an important, hard upper bound on the thermal conductance available through future molecular electronic devices. The current project continues the investigation of heat capacities of nanomachined mesoscopic systems: Suspended semiconductor nanostructures that are thermally-isolated and have integral transducers that permit the localized introduction of heat and local temperature measurements. Heat capacity measurements on minute samples with unprecedented sensitivity should be possible. This should provide data relevant to the engineering of miniaturized thermal detectors, and will provide crucial information relating to limits of power dissipation in molecular-scale and ultrasmall electronic devices. With this level of sensitivity, calorimetry experiments that elucidate processes involving individual atoms and molecules should also become possible for the first time. The effort will introduce undergraduates, graduate students, and postdoctoral researchers to advanced techniques in nanofabrication and in techniques and principles of ultrasensitive measurements.
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Future electronics will likely be based upon molecular scale devices. Active electronic devices, at any scale, require power to operate and this must ultimately be dissipated to their surroundings. However at the molecular scale the processes that govern power dissipation become very weak; hence it can be problematic. This domain had remained largely unexplored until 1999, when, in a previous NSF-funded research program, investigators observed the quantization of thermal conductance -- a fundamental limit to the rate at which power can be conducted from a small system to its surroundings. In their current proposal, the authors propose to continue with research in this realm, turning now to the heat capacity of very small systems, i.e. their ability to "store" energy. Their approach involves suspended semiconductor nanostructures, fabricated by new surface nanomachining processes they have developed. These enable the construction of complex exploratory devices at the nanometer-scale, with internal components allowing quantitative and precise measurements on their properties to be carried out. In the proposed research program these will be utilized to obtain a more complete understanding of heat transport and the heat capacity of nanometer-scale structures. They should also prove to be extremely useful for the engineering of miniaturized thermal detectors, and will provide crucial information relating to limits of power dissipation in molecular-scale and ultrasmall electronic devices. With this level of sensitivity, experiments that elucidate processes involving heat flow between individual atoms and molecules should also become possible for the first time. The effort will introduce undergraduates, graduate students, and postdoctoral researchers to advanced techniques in nanofabrication and in techniques and principles of ultrasensitive measurements.

This proposal was received in response to NSE, NSF-0019. This NIRT project focuses on the study of charge transport in molecular systems. An interdisciplinary team from physics and chemistry, working in the areas of nanoscale synthesis, high-precision structural characterization, electrochemistry, DNA chemistry, microfluidic techniques, and the use of novel nanoscale materials will be engaged in the effort. There are two principal experimental thrusts to the work: 1) the systematic exploration of electronic tunneling through solvents and other small, well-characterized molecules and 2) a rigorous study of charge transport through DNA. The award is jointly funded by the Divisions of Physics and Chemistry.

ABSTRACT


This award provides partial support for travel of young researchers (advanced graduate students, post doctoral researchers and very new faculty) to attend the First International School and Conference on Nanoscale/Molecular Mechanics to be held in Maui, Hawaii from May 12-17, 2002. The school will be held two days prior to the conference. A panel of distingushed lecturers has been selected to make presentations. Many of the pioneers in nanoscale mechanics will make presentations at the conference.

DESCRIPTION (provided by applicant): Mass spectrometry (MS) is emerging as one of the most important tools for proteomics. Modern MS protocols, recently developed for identification of low concentration analytes in the presence of very large backgrounds, have been central to many important advances in systems biology. However, these protocols are accompanied with important limitations, especially in terms of their sensitivity and ease of methodology. Advances emerging from nanoscience may offer new approaches to MS. Recently, using nanoelectromechanical systems (NEMS) in vacuo, the proposers have demonstrated mass sensing at the 7 zeptogram level -- the mass of an individual 4 kilodalton molecule. This represents a million-fold improvement over the most recent state-of-the-art laboratory demonstrations using microscale devices, and a billion-fold improvement over commercial devices. Yet the work is still in its infancy; significant further improvements with NEMS are imminent. In this initial (R21) effort, the proposers will make the crucial initial steps to transform this advance in nanoscience into a real technology that is directly applicable to biological mass spectrometry. A significant amount of research will now be required to transform this unprecedented mass sensitivity into a usable and efficient new form of MS. Realizing this transformation is the principal focus and intent of the proposed work. NEMS-MS offers the promise of sensitivity down to the single-molecule level. A new single-molecule, NEMS-based mass spectrometry (NEMS-MS) would enable powerful new assays utilizing extremely minute amounts of precious/rare analyte. In contrast to genomics research -- where gene amplification can produce large amounts of identical analytes enabling more "conventional" MS protocols -- extreme sensitivity is crucial for proteomics. If successful, this exploratory program will provide benchmark demonstrations of single-molecule mass sensing, chart a methodical course toward the realization of single-molecule mass spectrometry for proteomics, and assemble a next-phase effort (R01) for the full realization of this vision. The collaborative team includes some of the forefront practitioners of proteomic-based mass spectrometry, of nanodevice physics and microfluidics, and of novel surface chemistry approaches to MS.

A grant has been awarded to California Institute of Technology under the supervision of Professor Michael Roukes to develop instrumentation for next-generation mass spectrometry based on nanoelectromechanical systems (NEMS). NEMS devices, given their extremely minute size, are exquisitely sensitive to added mass -- even down to the addition of individual molecules. These sensors now form the basis for a new approach to molecular identification, NEMS-based mass spectrometry (NEMS-MS), for resolving vast numbers of molecules, one-by-one. This project will culminate in assembly of benchtop NEMS-MS systems and their automated control electronics. In domestic and international collaborations with leaders in proteomics mass spectrometry, these systems will enable pioneering, benchmarking studies of NEMS-MS proteomics, ultimately carried out on individual cells.

Research in the life sciences and medicine now requires significant technological innovation to proceed from the post-genomics era to new frontiers in single-molecule proteomics. Especially important, and especially challenging, is research in ?systems biology?, which strives to elucidate the deterministic biochemical ?circuit diagrams? for living systems. These ?circuits? hierarchically encompass the entire organism -- from organs, to individual cells, down to cascades of individual molecular processes within cells that give them their function. Through advanced and, of necessity, massively-detailed measurements it will ultimately become possible to assemble complex blueprints for living systems, down to their fine details, routinely. This will provide an unparalleled window into how living systems function and, thus, provide unprecedented predictive power. Such deterministic research on biological systems will ultimately help engender predictive and personalized medicine based upon a person?s genetic predispositions and present physiological state. NEMS-MS promises a new paradigm that is essential for such research: high-throughput biological mass spectrometry with single-molecule resolution.

DESCRIPTION (provided by applicant): Mass spectrometry is one of the most important tools for proteomics. Especially important are new protocols recently implemented for the identification of low- concentration analytes in the presence of very large backgrounds. NEMS-based mass spectrometry (NEMS-MS) can provide complementary and powerful new assays for extremely rare or dilute analytes. If successful, the proposed program will culminate in prototype demonstrations of new methods for biological mass spectrometry providing unprecedented resolution - carried out in close collaboration with the world's preeminent leaders in the field of proteomic mass spectrometry. The application below charts a methodical course toward the realization of single-molecule NEMS-MS. We have shown theoretically that the intrinsic resolution of nanoelectromechanical systems (NEMS) -based mass detectors is well below 1 Da. For the field of biological mass spectrometry (MS) the implications of this are profound. To follow-on from our successful R21-funded pilot program - in which we have achieved the first demonstration of single-molecule NEMS mass detection in real time - we propose a 5- year research and development program (R01) to develop compact, next-generation, high-throughput mass spectrometers with single-molecule resolution. These will be based on the large-scale integration of microfluidic-interfaced NEMS. PUBLIC HEALTH RELEVANCE: Proteomics is the study of the biological machinery that underlies all life processes - and it is key to biomedical and pharmaceutical research. The paramount tool for protein identification is mass spectrometry (MS), but existing tools typically work only if hundreds of millions of identical molecules can be extracted from cell cultures for analysis. This project proposes to develop a new technique from nanoscience, already validated in prototype form, allowing MS studies at the single-molecule level that will unambiguously elucidate the details of biochemical networks that give cells their function.

This work will advance our understanding of how cells process of mechanical signals. The effort is based upon Single-Cell-Pico-Force-Microscopy (SCPFM), a new instrument poised to become widely applicable to many studies in cell biology. SCPFM integrates near-single-molecule force measurement capabilities of Nano Electro Mechanical Systems (NEMS) with integrated microfluidics enabling precision fluid control, and thereby precision pharmacological stimulation, of individual cells. Understanding cellular force generation and regulation is critical to understanding many basic aspects of cell function; among these are: cell motility, cell division, and cell organization; many aspects of organism function: wound healing, inflammation, and embryogenesis; and numerous diseases, including many cancers and cardiovascular disease. Specifically, this work will investigate the compliance sensing and force response of the Extra-Cellular-Matrix (ECM) as a model system for studying the role of mechanics in cell regulation. SCPFM enables, for example, detailed and systematic study of the molecular-mechanical responses of individual lamellipodia to pharmacological and mechanical stimulation. Force-time records of compliance sensing and response events will be acquired in this work, and these will be used to assemble a library of molecular-mechanical force signatures with which to analyze the force-time records.



SCPFM combines device physics, nano-fabrication, and electrical engineering with surface chemistry, cell biology and biochemistry in a complex, interconnected system. This presents an extraordinary opportunity for graduate students and post-docs to become interdisciplinary scientists capable of leading and successfully integrating interdisciplinary teams and projects. Undergraduate and minority students will have the opportunity to be involved through Caltech?s SURF and MURF programs (Summer/Minority Undergraduate Research Fellowship). The impact of this research will be carried into the local community by the graduate students and post-docs involved in cooperation with Caltech?s Classroom Connection, which partners researchers with local high school science classes where they serve as role models for pre-college students.

DESCRIPTION Abstract: New tools have enabled some of the most important advances in biology and medicine. We are at the threshold of an exciting new technological era in which deciphering deep levels of biological complexity will be routine. It will become possible to tackle biological and medical problems at what were once thought to be unimaginably large hierarchical scales, all the while observing and coordinating unprecedented levels of detail down to the molecular scale. And it is plausible that this will all be possible in real time - ultimately providing a continuous window into the evolving systems biology of organisms. This effort seeks to hasten the realization of this vision by leveraging recent advances nanosystems technology, an approach that coordinates vast numbers of individual nanodevices into a coherent whole with emergent functionality. The goal is development of biomedical tools that simultaneously enable new physical windows of observation, while amassing the requisite sophistication to address complex problems. Four initial projects are proposed from a realm of many: (i) fast typing of individual bacteria without culturing;(ii) obtaining physiological "fingerprints" from exhaled breath;(iii) using cell mechanics and motility as a new tool in cancer research;and (iv) following the metabolism of individual cells to provide early screening of libraries of therapeutic drug candidates. Each example illustrates how existing nanosystems technology can be leveraged to realize new biomedical tools. Each harnesses the complementarity of scale between individual, unit nanosensors and their targets. Using the well-validated approach of state-of-the-art microelectronic foundry production, a realistic plan is outlined for producing robust tools in sufficient quantities to enable biological and medical research continuity. This research and production paradigm will enable groundbreaking, collaborative systems research in biomedical sciences though realization of tools ca

In this project, the PIs will directly measure the metabolism and thermogenesis of nematode soil worm Caenorhabditis elegans (C. elegans) and other model organisms by recording the heat production of the organisms by a microfluidic calorimeter. The calorimeter is based on the microfluidic calorimeter platform developed at Caltech and will have superior performance, including a power resolution at 2% of single worm power generation, a response time of 0.5s and on-chip worm incubation enabling continuous monitoring of a worms activity for over 48 hours. C. elegans will serve as the major target because it is an important model organism that has been extensively used in biological research. In this program thermogenesis in these manipulated worms will be employed to differentiate metabolic pathways and study longevity. First, a number of small molecule inhibitors specific to different biochemical pathways, such as Cyclohexamine (targets Ribosomes) and Actinomycin D (targets RN Polymerases I, II & III), will be used to investigate the contribution of these pathways to metabolism, which will provide a clearer picture of the relative contribution of several important biochemical pathways to overall metabolism in C. elegans. Next, the effect of diet and starvation on the metabolism of adult worms will be studied during their development. Finally, the relation between metabolism and longevity for worms with life-extending genetic mutations will be investigated. The PI and co-PI are committed to dissemination of the technology, outreach and education. The Kavli Nanoscience Institute at Caltech is capable of moderate-scale production of devices. The PI and Co-PI have actively participated in Caltech's Summer Undergraduate Research Program (SURF) and Minority Undergraduate Research Program (MURF) for the past two decades. The co-PI has also has a track record of mentoring high school students, a number of whom have been included as co-authors on papers in top journals based on their summer and after-school research.

ABSTRACT
The proposed effort will use modern integrated wavelength division multiplexing to minimize the
amount of external points of interface needed by optical probes while maximizing the total points of optical stimulation on an augmented electrophysiological neural recording probe. Array waveguide grating demultiplexers will be fabricated on neural probes and will be used to segregate multiple optical signals passed over a common fiber. The improvement of optical channel density will allow for more precise stimulation of local nuclei, enable high spatial and temporal multiplexing of selective stimulation of neurons and will allow for more flexibility when implanting neural probes. To allow for individual or multiple channels to be activated, the authors will build a ?polychromator? which will utilize a grating to separate spectral components from a broadband light source and project the spectrum onto a digital mirror device (DMD), similar to those found in digital projectors. Since the spectral information will be encoded spatially, spectral bands can be selected using the DMD by activating or deactivating columns of pixels. The resulting spectral bands will be collimated and used to provide a source with carefully controlled and manipulated spectral bands, allowing for direct spatial control of the stimulus through on-probe demultiplexing.

DESCRIPTION (provided by applicant): Our understanding of the properties of individual neurons and their role in brain computations has advanced significantly during the last few decades. However, we are still very far from understanding how large assemblies of cells interact to process information. Electrophysiology is the gold standard with unmatched temporal resolution, but is currently limited in terms of its ability record from every single neuron withina volume with cell-type specificity. Optical imaging provides a powerful alternative method, which enables localization of neurons in anatomical space and cell-type specificity via genetically encoded fluorescent markers. The current state-of-the-art in functional brain imaging is two-photon fluorescence laser-scanning microscopy. But this approach works best only on the surface of the brain, or transparent tissues and is not easily scalable. More generally, light scattering and absorption in tissue impose significant fundamental limits: in mammalian brains, accessible depths in vivo are restricted to superficial cortical regions, d1mm. Endoscopic methods developed to circumvent such restrictions impart significant damage to tissue above the imaging site given the large probe diameter (0.3 to >1 mm) and thus are quite limited (e.g. cannot be used to study cortical columns). Here we propose a novel paradigm for functional optical imaging that surmounts these limitations. It permits function- al imaging with cellular resolution in highly scattering brain tissue, enables complete coverage of all neurons within a target volume, and has long-term prospects for human applications. Our approach, which we term integrated neurophotonics, is based on distributing a dense 3-D lattice of emitter and detector pixels within the brain itself. These pixel arrays are embedded onto neurophotonic probes, realized as implantable, ultra narrow shanks that leverage recent advances in both integrated nanophotonics. Used with functional optical reporters (e.g. GCaMP6), one 25-shank probe module will be capable of recording the activity of all neurons within a 1- mm3 volume (~100,000 neurons) with single cell resolution. The methodology is scalable; multiple modules can be tiled to densely cover extended regions deep within the brain. It will ultimately permit simultaneous recording from millions of neurons at arbitrary positions and depths in the brain, to unveil the dynamics of complete neural networks - with single-cell resolution and cell-type specificity. Ultra-narrow neurophotonic probes will perturb brain tissue minimally, imposing negligible tissue displacement and minute local power dissipation. Importantly, they are readily producible though existing wafer-scale foundry (factory) based methods and thus will be widely available for use by the community. They will transform studies of circuit- level mechanisms of brain computation and neuropsychiatric disorders, and will accelerate drug discovery via high throughput in vivo screening. Our multi-disciplinary team spans all requisite expertise: nanotechnology and large-scale-integration for development of neurophotonic probe arrays (Roukes, Shepard), and in vivo testing and computational analysis (Tolias, Siapas).

Proposal Number: 1403817
P.I.: Roukes, Michael L.
Title: Biophotonic neural probes for studying the brain's immune response

Significance and Importance: An award is made to Caltech to engender new tools for studying the immune response to a brain-machine interface in small mammals. To develop the next generation of neural probes for massively parallel stimulation and recording, and facilitate advanced brain-machine interfaces, we must attain a better understanding of the brain's immune response to chronic neural implants. The existing state-of-the-art generally only enables such studies to be performed after an animal has been sacrificed. The technology developed through this research will merge the latest advances in nanobiophotonics, implantable neural probes, and state-of-the-art microfluidics to develop, fabricate, and test next-generation technology that will enable real-time monitoring of the immunogenic response of the brain to implanted neural probes, including the ability to perform real-time monitoring of the use of drug regimens to temper the immune response. The project provides an exceptional opportunity for training interdisciplinary scientists and engineers. In broader outreach, the researchers will collaborate with the Community Science Academy at Caltech to develop an iPAD-facilitated learning module for K-12 use.

Technical description: The overarching goal of the 3-year project will be the design and development neurochemical probes targeted to the detection of cytokines and chemokines in mice and other small animals. Year 1 will focus on demonstration of detection of targets of interest at concentrations of physiological relevance (100pg/mL to 10ng/mL) with photonic micro-ring resonators. Year 2 will focus on the integration of photonic micro-ring resonators with etched microfluidics with dialysate membranes appropriate for the detection of specific chemokine and cytokine targets (a 100kDa molecular weight cut-off will be targeted). Year 3 will focus on the development, fabrication, and calibration of neurochemical probes for use in vivo. Evaluation and calibration of probes will be performed in 'tissue phantoms' made from agarose with dissolved chemokines and cytokines. These will include both targets of interest and potential interferents. We anticipate that by the end of this effort we will be able to provide at least 10 calibrated probes to each of the Siapas (Caltech), Tolias (Baylor College of Medicine) and Laurent (Max Planck Institute for Brain Research) neuroscience research groups for use in vivo. In this third phase we will also begin design discussions in the Alliance for Nanosystems VLSI (very-large-scale integration; co-founded by the PI in 2007) to transfer our Caltech-based fabrication processes to standardized production en masse within our partner's (CEA/LETI) micro-/nano-electronics foundry. The graduate student funded by this effort will learn to employ optical engineering, microfabrication techniques, and neuroscience in order to produce and employ advanced experimental measurement systems. In addition to graduate student education, undergraduate students will contribute to the project through the SURF and MURF (summer- and minority- undergraduate research) programs at Caltech. The K-12 learning module developed as an outreach effort in collaboration with the Community Science Academy at Caltech will include a low-cost, portable, iPAD-interfaced refractometer and demonstration of total internal reflection for use in Pasadena Unified School District and Los Angeles Unified School District classrooms.

This award is being made jointly by two Programs- (1) Biophotonics, in the Division of Chemical, Bioengineering, Environmental and Transport Systems (Engineering Directorate), and (2) Instrument Development for Biological Research, in the Division of Biological Infrastructure (Biological Sciences Directorate).

DESCRIPTION (provided by applicant): Mapping spatiotemporal electrochemical responses in vivo, especially within the brains of awake vertebrates, can elucidate the role of neuromodulation in brain activity. However, present tools in neuroscience are insufficient for the task. In the past decade, advances in the technology of multiplexed neural probes for electrophysiology has improved both the complexity and the spatial resolution of simultaneous electrical recordings that are now possible within brain tissue. The state-of-the-art is represented by silicon-based neural nanoprobes that we are developing to enable simultaneous in vivo electrical recording from 1000 sites. Probes that enable local electrochemical sensing in vivo, by comparison, have not kept apace with these advances in electrophysiology. Nonetheless, improvements have been made to electrochemical sensors for in vivo detection of individual neuromodulators of interest, such as dopamine and acetylcholine -- and local sensing at physiologically relevant levels is now possible with spatial and temporal resolution of ~400?m and ~1 second, respectively. We propose to leverage the expertise we've gained in engineering highly multiplexed nanoprobes for electrophysiology, and to build upon the recent improvements of in vivo electrochemical sensing, to develop a new generation of highly multiplexed, multi-site neural nanoprobes for simultaneous electrochemical sensing of multiple neuromodulator targets in vivo. The probes will be fabricated as long (~5mm), narrow (~50?m) silicon shanks that prove optimal for brain recording; onto these will be integrated a multiplicity of chemical sensing triads. Each triad will comprise three distinct sites for amperometric sensing of neurochemical targets -- for example, dopamine, acetylcholine, and choline -- and linear arrays of these triads, separated with less than 100¿m pitch, will be assembled along the probe shanks. These sensor arrays will enable simultaneous detection of the spatiotemporal variation of multiple different neuromodulator targets across extended regions in the brain. An important application is sampling neuromodulator variations along the multiple cortical and hippocampal layers of the brains of rats, mice and other small animals, where individual layer thicknesses can be ~100?m. The advanced, probe-based electrochemical sensing technology we will develop in this effort will open a new window into the spatiotemporal evolution of functional neurochemical heterogeneities across distributed brain regions.

Understanding how the brain works is arguably one of the most significant scientific challenges of our time and the focus of the BRAIN initiative. It is widely believed that neural circuit function is emergent, the result of complex interactions between constituents with individual neurons forming synaptic connections with thousands of other neurons. Mapping of these complex circuits has been virtually impossible because of the reliance on electrophysiological recordings which sample these networks extremely sparsely. These tools for extracellular spike recordings are only able to simultaneously record from several tens to a few hundred neurons. Raw signals from these recording electrodes are first filtered to remove out-of-band signals. Putative spike events are then detected and extracted. Finally, these snippets of time-series event are sorted, typically on the basis of waveform shapes, into clusters. Even at the very modest bandwidths for these systems, computing systems struggle to save the data and process the resulting data sets. Scalability of these measurement techniques by many orders of magnitude in recording density and channels will be essential to future progress in understanding neuron circuits.

This project is exploiting emerging electrophysiological recording systems in which the electrode (and channel) count is increased by almost three orders of magnitude over conventional systems with data bandwidths exceeding 1GB/sec. To handle these data bandwidths and resulting data volumes and deliver scalability, this project will develop dedicated hardware and associated algorithms for spike detection and sorting that allow these tasks to be performed in real-time in close proximity to the recording system. Compression by more than three orders of magnitude is possible by these means by taking advantage of the special spatiotemporal local structure in these data sets; by exploiting strong prior information about the spiking signal and reducing the dimensionality of the problem accordingly; and by adapting and extending modern scalable nonparametric Bayesian inference methods. In addition to providing important new tools for neuroscience, the tools developed here for scalable real-time event detection and annotation have broad applicability to other spatiotemporal data sets (or more generally, any data set comprising multiple streams of data, in which the streams could involve different data modalities) in which objects of interest are spatially and temporally localized with fixed spatial footprints. Examples abound in cell and molecular biology, particle and solid-state physics, financial monitoring, monitoring of power networks, and sensor networks.

The objective of the proposed three-year research project is to develop high-speed, high-spatial- resolution, deep-penetration photoacoustic computed tomography (PACT) for real-time imaging of neuro-activities in mouse brains in vivo. The proposed hardware imaging system will be unprecedented in the field of PACT in terms of its volumetric rate and spatial resolving power, which benefit from the use of the largest ever number of sensing elements with one-to-one mapped digitization channels. In comparison to existing high-resolution optical neuroimaging modalities such as two-photon microscopy, the proposed system will provide deeper penetration, and higher volumetric imaging speed for whole mouse brain imaging. The timing for such an exciting project is perfect because of two events. (1) Our unpublished ongoing work has shown for the first time that PACT has imaged through the entire mouse brain, with abundant vessels resolved (see the images in the Aim 4 section). (2) High-frequency ultrasonic transducer elements with omnidirectional sensitivity can be massively integrated by the Shepard lab to accomplish three-dimensional (3D) acoustic detection (discussed in the Aim 3 section). By using an unprecedented 1536 ultrasonic transducer elements one-to-one mapped to data acquisition channels, we will perform real-time 3D PACT at single neuron resolution and 2 kHz volumetric rate, which has never been achieved before. The specific aims include: 1. Develop implantable photonic probes with arrays of photonic emitters. 2. Develop high-frequency ultrasonic transducer arrays for PACT. 3. Develop deep brain high-frequency PACT. 4. Use PACT to image neuro-activities in mouse brains in vivo.

This Major Research Instrumentation (MRI) grant will enable the acquisition of a three-ion-beam microscope, the ORION NanoFab system, from Carl Zeiss Microscopy. This tri-beam system can provide unprecedented resolution, precision and versatility for the fabrication and characterization of materials and devices all the way down to the nanometer scale (roughly a few times the atomic spacing). The ORION NanoFab system is expected to make a significant impact on interdisciplinary nanoscience research, particularly in the areas of quantum matter and technology, medical and bio-engineering, photonic and optoelectronic research, meta-materials, and renewable energy science. The tri-beam system will be located at the Kavli Nanoscience Institute (KNI) of the California Institute of Technology (Caltech), which provides laboratories with state-of-the-art infrastructure and houses centralized nanofabrication and nano-characterization facilities for researchers at Caltech, the Jet Propulsion Laboratory (JPL), and corporations and other institutes in the greater area of Southern California. As this form of tri-beam microscopy is only in its infancy, Caltech will also be partnering with Zeiss in a technical outreach effort to bring experts together to advance new ideas and applications of the tri-beam tool. This collaborative outreach plan includes: hosting annual workshops at Caltech with industrial and global research-community users of the ORION NanoFab to exchange information on research highlights, technical challenges, and new technical developments and applications. As part of outreach effort there is also a plan to offer nanoscience "demo days" for K-12 students in which the advanced instrumentation of the ORION NanoFab and other tools in the KNI can be used to explore the nanoscopic world, as well as lectures and lab tours at Caltech to local high school students and teachers on topics of nano-science and technology (nano-S&T) and applications of modern microscopy.

This Major Research Instrumentation (MRI) grant will enable the acquisition of a three-ion-beam microscope, the ORION NanoFab system, from Carl Zeiss Microscopy. The ORION NanoFab is a three-ion-beam nano fabrication and microscopy system capable of an imaging resolution of 0.5 nm and a cutting resolution of ≲2nm, virtually independent of material. The system is designed to seamlessly switch between gallium, neon and helium beams, so that one has the option of employing the gallium focused ion beam (FIB) to pattern materials at the micro-scale, taking advantage of the powerful yet gentle neon beam for precision nano-machining with speed, or using the helium beam to fabricate delicate sub-10 nm structures that demand extremely high machining fidelity and/or cutting of delicate materials (such as graphene) that are prone to damage by high-energy electrons or heavy-element ion beams. Its capability of maskless nano-patterning also minimizes possible contamination due to the multiple steps required in processing and removing masks. The ORION NanoFab system is expected to make a significant impact on interdisciplinary nanoscience research, particularly in the areas of quantum matter and technology, medical and bio-engineering, photonic and optoelectronic research, meta-materials, and renewable energy science. The tri-beam system will be located at the Kavli Nanoscience Institute (KNI) of the California Institute of Technology (Caltech), which provides laboratories with state-of-the-art infrastructure and houses centralized nanofabrication and nano-characterization facilities for researchers at Caltech, the Jet Propulsion Laboratory (JPL), and corporations and other institutes in the greater area of Southern California. In partnership with Zeiss we also plan to bring together the industrial and global research-communities in a series of annual workshops at Caltech designed to help advance the technology and applications of multi-beam microscopy and nanofabrication.

This project will place into the hands of many experimental neuroscientists validated, massively-multiplexed tools for recording of neuronal activity, for chemical sensing of neuromodulators, and for highly-patterned optogenetic stimulation with concurrent electrical recording ? in any region of the brain. This will be accomplished by making use of both PIs' decades-long working relationship with microchip foundries, to enable mass production of neural nanoprobes, of VLSI application-specific integrated circuits (?microchips?), and of supporting instrumentation for read-out and control. Our paramount objective in technology development is to optimize usefulness for end- users. We will achieve this by a highly-interactive program that: 1) solicits user needs; 2) assembles and validates neural nanoprobe systems in vivo; 3) deploys complete systems to neuroscientists; 4) provides technical support to enhance the end-users' success with the new neurotechnology; and, subsequently, 5) solicits feedback to enable the design of successive generations of neurotechnology. The technology to be produced and disseminated is based upon the PI's validated neural nanoprobes and advanced, custom microchips for their readout and control. Our existing 256-channel nanoprobe layers modules (assembled into 1,024 channel 3D arrays) and microchips were fabricated by the foundries that will be used in this effort. These systems have been validated in vivo. In Y1, nanoprobe layer modules will be fabricated with 1,024 channels, and will be stackable into composite 3D systems with 10,240 full time/full bandwidth channels. In Y2 nanoprobe layer modules with 8,192 channels will be mass produced; these will be stackable to configure dense, composite 3D systems with ~100,000 full time/full bandwidth channels. These first two production runs enable systems for electrophysiological stimulation, recording, and neurochemical sensing. A third production run will integrate optogenetic stimulation with proximal multisite electrophysiological recording. These hybrid nanoprobes will contain 512 e-pixels for optogenetic stimulation and 512 proximal recording electrodes. This new technology will have lasting impact by incorporating diverse needs of the community at the outset. Using these electrophysiological, neurochemical, and optogenetic probes, eight enthusiastic ?alpha adopters? will investigate cortical and subcortical circuitry underlying movement and mood disorders such as Parkinson's disease in rat models (Gradinaru Lab); the behavioral and computational roles of cortical layers and circuits in the mouse whisker system (Bruno Lab), visual systems in the mouse (Yuste Lab) and primates (Tolias Lab); speech representations in human patients (Yvert Lab); the role of sleep in memory consolidation (Laurent Lab); coupling between neuronal activity and energy supply (Magistretti Lab); and the thirst nucleus of the mouse hypothalamus (Oka Lab). These users will provide direct feedback to enable probe refinement early in the effort. Interested ?beta? end-users, beyond these alpha adopters, will be recruited through solicitations in our publications, postings on our website, short talks at neuroscience conferences and by directly contact.

This project will place into the hands of many experimental neuroscientists validated, massively-multiplexed tools for recording of neuronal activity, for chemical sensing of neuromodulators, and for highly-patterned optogenetic stimulation with concurrent electrical recording ? in any region of the brain. This will be accomplished by making use of both PIs' decades-long working relationship with microchip foundries, to enable mass production of neural nanoprobes, of VLSI application-specific integrated circuits (?microchips?), and of supporting instrumentation for read-out and control. Our paramount objective in technology development is to optimize usefulness for end- users. We will achieve this by a highly-interactive program that: 1) solicits user needs; 2) assembles and validates neural nanoprobe systems in vivo; 3) deploys complete systems to neuroscientists; 4) provides technical support to enhance the end-users' success with the new neurotechnology; and, subsequently, 5) solicits feedback to enable the design of successive generations of neurotechnology. The technology to be produced and disseminated is based upon the PI's validated neural nanoprobes and advanced, custom microchips for their readout and control. Our existing 256-channel nanoprobe layers modules (assembled into 1,024 channel 3D arrays) and microchips were fabricated by the foundries that will be used in this effort. These systems have been validated in vivo. In Y1, nanoprobe layer modules will be fabricated with 1,024 channels, and will be stackable into composite 3D systems with 10,240 full time/full bandwidth channels. In Y2 nanoprobe layer modules with 8,192 channels will be mass produced; these will be stackable to configure dense, composite 3D systems with ~100,000 full time/full bandwidth channels. These first two production runs enable systems for electrophysiological stimulation, recording, and neurochemical sensing. A third production run will integrate optogenetic stimulation with proximal multisite electrophysiological recording. These hybrid nanoprobes will contain 512 e-pixels for optogenetic stimulation and 512 proximal recording electrodes. This new technology will have lasting impact by incorporating diverse needs of the community at the outset. Using these electrophysiological, neurochemical, and optogenetic probes, eight enthusiastic ?alpha adopters? will investigate cortical and subcortical circuitry underlying movement and mood disorders such as Parkinson's disease in rat models (Gradinaru Lab); the behavioral and computational roles of cortical layers and circuits in the mouse whisker system (Bruno Lab), visual systems in the mouse (Yuste Lab) and primates (Tolias Lab); speech representations in human patients (Yvert Lab); the role of sleep in memory consolidation (Laurent Lab); coupling between neuronal activity and energy supply (Magistretti Lab); and the thirst nucleus of the mouse hypothalamus (Oka Lab). These users will provide direct feedback to enable probe refinement early in the effort. Interested ?beta? end-users, beyond these alpha adopters, will be recruited through solicitations in our publications, postings on our website, short talks at neuroscience conferences and by directly contact.

This project is to develop an unprecedented instrument for studying proteomics - the collection of proteins constituting the molecular machinery underlying all life forms. Genes are the molecular precursors of this cellular machinery; genes are the templates encoding how such proteins are constructed within cells. A profound technological revolution has recently enabled detailed studies of genomic templates; indeed, it has permitted decoding the human genome itself. Similar advances in technology for proteomics has not occurred. Underlying this is the fact that genomic studies are enabled by making billions of identical copies of individual genes. This gene amplification then enables their straightforward analysis en masse. No similar molecular amplification process exists for proteins. In fact, critical processes in health and disease are often determined by only a few copies of a protein molecule within a cell. Fundamental advances in biology and medicine can therefore only be made if proteins are studied molecule-by-molecule. A practical means for accomplishing this is identified in this effort, and assembly of novel instrumentation for such analyses is proposed.

The research team has identified a unique technological path toward these ends that concatenates three key elements. First, single-molecule analysis of intact proteins and protein complexes. This is based on two novel approaches previously invented by this team - nanomechanical mass spectrometry and inertial imaging. Second, microwave-frequency cavity optomechanics. This enables ultrasensitive measurements upon the key nanomechanical devices, down to the quantum-mechanical limits of detection. Third, state-of-the-art high-resolution native mass spectrometry. This enables studies of intact (unfragmented) proteins and protein complexes. These three building-blocks will be assembled into a singular hybrid instrument to enable a new multi-physical approach for single-protein analyses that surmounts the limitations of all current methodologies. It offers realistic prospects for automated, high-throughput protein purification, and for identification of intact protein species. Further it is technology that could ultimately be widely disseminated. Deep proteomic profiling of individual cells will be transformational for biological research, clinical medicine, and pharmaceutical development. Surprisingly, no other technology is poised to enable this. The proposed work will be highly cross-disciplinary in nature, bringing together efforts of researchers spanning physics, engineering, chemistry, biology, and mathematics. This project's highly-collaborative and rich research environment will provide unparalleled opportunities for graduate students and postdocs involved in its broader efforts. The team and the collaborators are committed to providing long-term access to this instrument for biological and medical research - both in academia and industry.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

The Second International Workshop on the Frontiers of Nanomechanical Systems (FNS/2019) will be held in Palm Springs, California, 10 to 14 February 2019. This workshop will serve to bring together the international research community engaged in studying nanomechanical systems and is intended to promote exchange of ideas, approaches, and techniques. This should advance collaborative, cross-disciplinary research on nanomechanics and further accelerate the progress in the field, both in terms of fundamental studies and applications. Graduate students and research associates are especially welcome at the conference. To attract them, information about the conference was broadly disseminated, and colleagues were asked to encourage their students and postdocs to attend. There are already 32 graduate students participating in the workshop, which is roughly one-third of the entire participant cohort. The participants are limited to 110 scientists to ensure lively interaction and full participation throughout. The remaining two-thirds of the participants are roughly divided equally between postdocs and senior scientists (either professorial faculty, or professional scientific researchers). The scholarships expected from this grant are intended to be provided to prospective students and postdocs. This should significantly increase and enhance the participation of students and postdocs at the workshop. Steps have been taken to encourage members of the underrepresented groups to attend as well. A measure of success of this effort is that, along with female invited speakers, a female graduate student has been chosen to give an oral contributed talk, which is an exceptional opportunity for a student given the level of the conference.

The workshop FNS/2019 comes from the challenges that are posed by nanomechanical systems. They include, but are not limited to, (i) the occurrence of comparatively strong quantum and classical fluctuations and (ii) the vibration nonlinearity. Along with the invariably present thermal fluctuations, NEMS are subject to nonequilibrium fluctuations. Characterizing and understanding these fluctuations, as well as revealing their consequences is complicated and requires developing new techniques. At the same time, because of the small system size, vibrations with even comparatively small amplitudes become nonlinear, with the nonlinearity often coming into play already for thermal fluctuations. Addressing these challenges requires concerted international effort.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

The broader impact/commercial potential of this Partnerships for Innovation - Technology Translation (PFI-TT) project is to develop instrumentation that will enable deep profiling of cellular proteins. The methods will be made broadly applicable in fundamental biological research. The project?s central goal is to develop and validate instrumentation for studying proteomics ? the collection of proteins constituting the molecular machinery underlying all life forms. Genes, the molecular precursors of cellular machinery, are templates encoding how proteins are constructed within cells. The genomics revolution has recently enabled decoding of the human genome. Similar advances in proteomics technology has not occurred since genomic studies rely upon gene amplification to making billions of identical copies to enable analysis en masse. No similar molecular amplification process exists for proteins, hence single-molecule analyses are key. The proposed research relies upon the collaborative efforts of researchers spanning physics, engineering, chemistry, biology, and mathematics ? from both academia and industry. Component technologies for a feasible, commercializable instrument will be pursued. This project?s highly collaborative and rich research environment provides an opportunity for the graduate student who will be involved in this effort to gain important career skills.

The proposed project will develop instrumentation to unravel the staggering complexity of protein biology. An individual mammalian cell comprises roughly 3B proteins, spanning about 7,000 unique types, with concentrations spanning 8 orders of magnitude ? ranging from cellular proteins present with just a few copies, to those with expression levels of order 100M. In human blood serum this range is a 1000X greater, spanning 11 orders. Resolving the full spectrum of protein constituents is essential to fundamental questions in biology and medicine. Detailed analyses ? at the population level, yet with single-molecule resolution ? will stratify fine details lost in existing consensus-type approaches to proteomics that ?average over? the protein population, thus providing information only about the most prevalent species. Critical processes in health and disease are often determined by only a few copies of a protein molecule within a cell. Accordingly, single-molecule resolution of proteins and protein complexes in the presence of an immense overabundance of the most prevalent species is essential.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.