David L. Dickensheets - US grants

0079789
Costerton
This is a development proposal to build a miniature confocal laser scanning microscope for in situ imaging of bacterial biofilms in their natural environments.

Confocal microscopy is the dominant imaging modality for the study of bacterial adhesion onto surfaces, and their organization into communities called biofilms. Our understanding of these microbial communities is limited to those species that can be coaxed to grow in optically compliant environments such as flow cells, which are compatible with conventional microscopy tools. Extension of studies to films in their native environment, such as inside pipes and vessels or underground, are hindered because we do not have imaging tools that are compatible with those environments.

The applicant proposes to exploit recent advances in Silicon micromachining and microlens technologies to produce a confocal microscope smaller than 2 mm in diameter and only 10 mm long. Called the confocal microprobe, the instrument may be introduced through the working channel of a borescope or endoscope to permit in situ studies of biofilm formation and growth. The confocal microprobe will support brightfield imaging as well as fluorescent imaging with submicrometer resolution. It will interface to an existing commercial confocal optical microscope, and operation of the microprobe will be controlled via the user interface of our commercial instrument.

This new miniature instrument will be used extensively in the Center for Biofilm Engineering, where its impact will be felt on several programs. One of those programs is the study of biofilm formation in porous media such as soil, where biofilm research is targeting 'biobarriers" for isolation of pollutants or to improve secondary recovery of oil from injection wells. Another research direction in the Center that will benefit from this development is the microscopic study of fouling and microbially influenced corrosion in nuclear storage facilities, using an ultraminiature microscope delivered through the working channel of a borescope. The study of mixed species biofilms (such as subgingival plaque) in their natural environment is extremely important, and the confocal microprobe will offer tremendous advantage for those studies.

This instrument will be used collaboratively between the Center for Biofilm Engineering, the Department of Electrical and Computer Engineering and Microvision, Inc., the world leader in micromechanical laser beam scanning devices. Microvision is well positioned to capitalize on this development program and would be a willing partner to transition this technology from the university lab environment into the commercial arena.

The objective of this research is the advancement of microdevice technology for applications in the chemical, biological, electronic and optical sciences, and the fundamental and technological investigation of new nanomaterials. The approach is to acquire an inductively coupled plasma etcher that will be used, in concert with other existing shared equipment, to create the specialized micro- and nanostructures that are integral to this research.

Intellectual Merit: The Montana Microfabrication Facility (MMF) provides an excellent environment for research and training involving the fabrication and testing of microdevices and nanostructures. The requested equipment will facilitate productive, cross-disciplinary research in the areas of MEMS, MOEMS and micro-optics, integrated optics, microsensors, THz waveguide devices, neuro-electrical recording devices, magnetic thin film materials and nanowire research.

Broader Impacts: Access for regional researchers to the new equipment will be assured through its incorporation into the MMF. Because it is a user facility where the operational model is for students and scientists to do their own work, the MMF plays an important role in education and training. Undergraduate students, graduate students and postdoctoral scientists receive in-depth research training and experience with modern fabrication equipment. Undergraduate and graduate students taking courses in microfabrication and MEMS receive hands-on instruction on the same tools. Students and faculty in MSU's summer research experiences program, including students and faculty from Montana's tribal colleges, also have the opportunity to receive training on this new ICP etching tool. Additionally, the MMF is committed to supporting industrial users from our geographic region.

A grant has been awarded to Dr. David Dickensheets at Montana State University to develop a light microscope with agile electronic focus control. This project addresses a major technological barrier for in vivo microscopy: controlling the focus and managing aberrations when imaging through thick, unprepared specimens. We will investigate a method that adjusts the location of the focus in the sample under electronic control while maintaining the objective lens in a fixed position. This instrument will be compatible with CCD-based or scanned beam (confocal and two photon) types of microscopes, and will give the user fast, full-range focus control when imaging thick specimens at high numerical aperture, without requiring z-translation of the specimen stage.

Of the several possible technologies for beam focus control, MEMS deformable membrane mirrors are attractive for their precision, speed and potential low cost. However, previous implementations have suffered from limited range of motion, such that full range focus control at high NA was not possible. This project will develop new MEMS membrane mirrors capable of an order of magnitude more displacement than current devices and able to correct spherical aberration to maintain high Strehl ratio throughout the image volume. The improvement is achieved through the use of new membrane materials, accurate mechanical modeling and a new closed loop control technique for stable electrostatic actuation. This project will develop the new mirrors and control techniques, and demonstrate their use in a new instrument for two different confocal microscopy applications: microscopy of the developing chick embryo, and microscopy of human skin in vivo.

The scope of potential use for this technology is quite broad. The new instrument could support many sophisticated image acquisition schemes including oblique sectioning, enhanced depth of focus at high NA, and image feature tracking and stabilization. The technology is also critical for future miniaturized microscopes designed for laparoscope, endoscope or catheter platforms that presently have extremely limited ability to adjust focus in situ. Finally, advances made in this project will be relevant for a broad class of electrostatic MEMS actuators and the deformable mirrors will be of interest for applications in astronomy, photography, optical data storage and others. In addition to its scientific impact the project embraces integration of research and education, training two engineering Ph.D. students in cross disciplinary research and engaging 6 undergraduate students in engineering design teams. This project will also participate in an established summer internship program for students and faculty from Montana?s tribal colleges.

ECCS - 1002058
Wataru Nakagawa
Montana State University
Hybrid Micro/Nano-Optical Devices for High-Fidelity Imaging

ABSTRACT

The objective of this research is to develop next-generation optical micro-electromechanical systems (MEMS) through the use of nanostructured surfaces, leveraging their underlying compatibility to realize multifunctional hybrid micro/nano-optical devices. The result will be MEMS-based systems capable of optical performance commensurate with bulk optics, leading to new fully miniaturized high-fidelity optical imaging systems.

Intellectual merit: Optical nanostructures have emerged as a viable alternative to implement high-reflectivity and anti-reflection coatings as well as polarization control elements. A desirable characteristic of these devices is that the required optical properties can be engineered through the nanoscale structure, freeing the choice of materials (e.g. for manufacturability or compatibility). In this project, nanostructured devices using MEMS-compatible materials and manufacturing processes will be developed and integrated with a MEMS device. These multifunctional hybrid devices will be optimized for specific target applications in compact high-resolution imaging systems, and validated in applications-oriented optical microsystems.

Broader impacts: The synthesis of two rapidly advancing areas of optical technology - MEMS and nanostructures - will enable the development of high-performance, miniaturized, multifunctional optical devices. Such devices have potential applications ranging from medical imaging and environmental monitoring to consumer electronics such as cameras and display systems. This work will also offer a wealth of opportunities for the education and training of participating students in engineering research and systems development, and will include efforts to encourage undergraduate and pre-collegiate students to pursue education and careers in science and technology fields.

A grant has been awarded to Dr. David Dickensheets at Montana State University to develop a new instrument for electronic focus and aberration control in biological microscopy, with particular application to microscopy of thick living tissues or intact animals. A critical tool for studying living systems in their natural state, high-resolution microscopy of living and intact tissues remains a tremendous challenge. This project addresses a major technological barrier for in vivo microscopy: controlling the focus and managing aberrations when imaging through thick, unprepared specimens.
Most current instruments for vital microscopy rely on mechanically fixing the specimen in some manner and then translating the microscope objective lens in or out to adjust focus. A system has been developed based on a deformable MEMS mirror (MEMS is an acronym for micro-electromechanical systems) that adjusts the location of the beam focus in the sample while maintaining the objective lens in a fixed position. There is no mechanical translation of any lenses or of the sample, and therefore no vibration. Precise control of the mirror shape eliminates spherical aberration at every depth. Fast response time allows focus stepping or scanning, and when synchronized with lateral beam scanning allows sectioning of 3D samples along any oblique plane or even along convoluted surfaces such as a cell membrane. No competing technology offers the precision, speed and potential low cost afforded by MEMS deformable mirror technology. The underlying technology of electronic focus and aberration control using a MEMS deformable mirror has been demonstrated in our laboratory. The aim in this project is to bridge the gap between our first prototype demonstration and an instrument ready for commercialization and broad distribution. When fully developed, this technology will turn any laser scanning confocal or two-photon/multi-photon microscope into an electronically controlled 3D imaging instrument, capable of x-y, x-z or arbitrary trajectory scanning. With the proposed instrument and future software development, many sophisticated image acquisition schemes would become possible including multi-plane imaging, enhanced depth of focus at high NA, and image feature tracking and stabilization.
The scope of potential use for this new technology is extremely broad. The first module in development is for scanning laser microscopes, but future applications include fast, aberration-free electronic focus control for wide-field white light and fluorescence microscopes and miniaturized in-vivo microscopes designed for laparoscope, endoscope or catheter platforms that presently have extremely limited ability to adjust focus in situ. Advances made with the deformable mirrors will be of interest for applications in astronomy, photography and optical data storage. In addition to its scientific impact the project embraces integration of research and education, training one female engineering Ph.D. student and providing research experience for several undergraduate engineering students, who will participate directly in cross-disciplinary activities with biology researchers as they vet the technology. This project will also participate in an established summer internship program for students and faculty from tribal colleges in Montana.

An award is made to Montana State University to develop a scanning laser microscope that incorporates fast active focus control and adaptive aberration correction to provide high-quality three-dimensional microscopic images within thick living tissue or intact animals. Optical microscopy of intact tissues, coupled with the explosive advance of fluorescent markers that can be genetically encoded to specific cell types and structures, is an incredibly powerful tool for fundamental research into biological development and function. To realize the potential of this approach, however, it is necessary to improve the spatial and temporal resolution of deep-tissue imaging techniques. Microscopy of intact tissue suffers from low resolution, low contrast and poor efficiency due to deterioration of the beam focus deep in the specimen. The new microscope uses two types of deformable mirrors to control both the depth of focus and the quality of the images produced. In addition to the new microscope optics, the project also develops the necessary control algorithms and user interface to create an instrument that will be devoted to both research and research training in developmental biology and neuroscience.

The proposed instrument will facilitate research that is addressing fundamental questions about how nervous systems develop and function. While the project is specifically constructing a scanning laser microscope, the underlying technology can bring many of the same benefits to wide-field microscopes in the future. Active and adaptive optics have other potential applications, including small-format cameras and endoscopes and optical data storage read/write heads capable of high-speed focus and aberration correction with no moving lenses. The technology is therefore broadly relevant. In addition to its scientific impact the project embraces integration of research and education, training two engineering graduate students and providing cross-disciplinary research experience for several undergraduate engineering and bioscience students. A new course module will be developed that features the active/adaptive microscope as a platform to teach advanced biophotonic methods.

DESCRIPTION (provided by applicant): Reflectance confocal microscopy, a type of scanning laser microscopy, has shown early success for non- invasive screening and diagnosis of skin malignancies while reducing unnecessary biopsy of benign lesions, guiding surgery by detection of cancer margins without the need for biopsies, and by monitoring patients post-treatment without requiring further biopsies. Because the technique is non-invasive and immediate, the clinician can look at as many features as necessary to arrive at a diagnosis. This is contrasted with current practice, in which the clinician must guess at the best one or two locations for a painful, potentially disfiguring biopsy, and then wait several days for a definitive diagnosis froma pathologist. Care is improved and substantial cost savings accrue, because many unnecessary biopsies can be avoided (for skin cancer, as many as 80% of all biopsies show normal or benign tissue, at a cost of some $5 billion annually in the US). Cancer of organs other than skin can be similarly diagnosed. For example, initial success is being seen using this technology for the detection of oral cancer. However, large instrument size remains a barrier toward widespread use of this technique in any but the most accessible sites on the body. To translate the diagnostic potential of confocal reflectance microscopy to other locations in the body, we are developing a new instrument called the integrated scanning laser microscope, or iLSM, that will be small enough for use in a pencil probe or endoscope. This instrument will achieve high resolution, high contrast images showing cellular detail, and, significantly, simultaneously provide a wide-field color video image of the tissue surface, allowing the clinician see where the microscopic images are being taken in the context of the surface features of a lesion. This wide-field view is essential for accurate sampling of a suspicious lesion, and has been missing from all previous attempts to miniaturize scanning laser microscopes. Recent technological advances in two areas, MEMS active optics and miniaturized CMOS cameras, have made the iLSM possible. This project takes a novel approach to integrate a custom MEMS 3D laser scanner into a centimeter-scale, high NA objective lens. Microscopy sections can be en face, oblique, vertical, or fully 3D, with a field of view of 300 ?m. The microscope uses an annular pupil that devotes the outer 75% of the aperture area to high NA imaging, ensuring high resolution and efficient collection of scattered light. The inner 25% of the aperture area is reserved for a separate low NA, wide-field camera, also integrated into the lens, that will show several millimeters of tissue. This project will develop the custom laser scanner and validate the approach with in vivo imaging of the skin of 30 volunteers.

Nanotechnology, which gives us the ability to manipulate and interrogate physical systems on a length scale of nanometers to microns, has become pervasive in many fields of scientific inquiry and engineering. Access to basic nanotechnology tools has therefore become increasingly important, not only for so-called nanotechnologists but for scientists and engineers from many academic disciplines and from industry. The Montana Nanotechnology Facility (MONT), a NNCI site at Montana State University, promotes discovery, education and outreach related to nanotechnology by providing access to shared-use instruments, expert training on their safe and effective use, and broad-based education about nanoscale science and technology for learners at all levels who come from diverse communities. The MONT site serves both regional users in the northern Rocky Mountains and Great Plains and users from across the U.S. who need the specific expertise and equipment found at Montana State University. Those users are pursuing diverse objectives related to advances in health care diagnostics and surgical solutions, sources of clean energy, remediation strategies for contaminated soils, and technologies related to optical telecommunications, imaging systems and advanced computing. By enhancing our service to external users and building on its unique fabrication and characterization strengths MONT will help to meet a national need for access to nanotechnology, for training of the workforce that will develop the nanotechnology of the future, and for education and outreach that engages and informs students and teachers from kindergarten to graduate school, industrial users and the general public.

MONT helps meet the growing need faced by regional and national researchers for access to nanofabrication tools and processes at the interdisciplinary frontiers, with local expertise related to microelectromechanical systems (MEMS) and micro-opto-electromechanical systems (MOEMS), microfluidics, nanostructured materials with unique optical, mechanical or thermal properties, ceramic materials, bio-inspired and bio-derived nanostructures, and bacteria or bacterial biofilms incorporated into micro- or nano-engineered substrates. he goals of the MONT site are: (i) to increase the number of external users served; (ii) to increase the collective research output of MONT users; (iii) to enhance the MONT site's capabilities in the areas of its research strengths through heavily leveraged capital investment; and (iv) to create best-in-class educational opportunities for facility users, STEM educators and the general public. These goals are accomplished through specific initiatives that will add laboratory personnel to enhance training, assistance and advocacy for external users, establish a user grant program for external users to help address costs of facility use as well as local housing, invest in new tools and capabilities, and expand both on-site and web-based instructional and outreach activities related to nano-fabrication, nano-characterization and the ethics and societal impacts of nanotechnology. The project specifically improves access to nanotechnology infrastructure in the northern Rockies/Great Plains region, and it promotes discovery, education and outreach in emerging fields where nanotechnology is impacting the life sciences, health care, energy, the environment, and a number of important technology sectors.

Proposal ID: 2025391
PI: David Dickensheets
Institution: Montana State University
Title: NNCI: The Montana Nanotechnology (MONT) Facility

ABSTRACT
Non-Technical Description:
Nanotechnology, which gives us the ability to manipulate and interrogate physical systems on a length scale of nanometers to microns, has become pervasive in many fields of scientific inquiry and engineering. Access to basic nanotechnology tools has therefore become increasingly important for scientists and engineers from many academic disciplines and from industry. The Montana Nanotechnology Facility (MONT), an NNCI site at Montana State University, promotes discovery, education and outreach related to nanotechnology by providing access to shared-use instruments, expert training on their safe and effective use, and broad-based education about nanoscale science and technology for learners from diverse communities. The MONT site serves both regional users in the northern Rocky Mountains and Great Plains and users from across the U.S. who need the specific expertise and equipment found at Montana State University. Those users are pursuing diverse objectives that include advances in health care diagnostics and surgical solutions, sources of clean energy, remediation strategies for contaminated soils, and technologies related to optical telecommunications, imaging systems and advanced computing. Many users are developing technologies at the forefront where traditional disciplines such as life or earth sciences are converging with physics and engineering. By enhancing our service to external users and building on our unique fabrication and characterization strengths, MONT will help to meet a national need for access to nanotechnology, training the workforce that will develop the nanotechnology of the future, and education and outreach that engages and informs students and teachers from kindergarten to graduate school, facility users and the general public.

Technical Description:
MONT helps meet the growing need faced by regional and national researchers for access to nanofabrication tools and processes at the interdisciplinary frontiers, with local expertise in functional nanostructured materials, optical MEMS, microfluidics, quantum materials and biological and geological nanostructures and systems. The goals of the MONT site are: (i) to increase the number of external users served; (ii) to grow the local, regional and national impact of MONT and NNCI; (iii) to enhance MONT facility capabilities; (iv) to integrate local and NNCI best practices to offer ?best in class? educational opportunities to users, STEM educators and the public; and (v) to increase the diversity of our enterprise. These goals are accomplished through specific initiatives that enhance training, assistance and advocacy for external users; offer a research initiation user grant program; invest in new tools and capabilities; expand on-site and web-based instructional and outreach activities; connect our users to others across the NNCI network through topical research communities; create a regional network for nano-facilities in the northwest; and create a new research engagement program for students who are underrepresented in STEM. The project specifically improves access to nanotechnology infrastructure in the northern Rockies/Great Plains region, and it promotes discovery, education and outreach in emerging fields where nanotechnology is impacting the life sciences, health care, energy, earth sciences, the environment, and emerging technology sectors such as quantum science and engineering.

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.

1 Dermatologists rely on visual (clinical widefield) and dermoscopic examination of skin lesions to guide the need 2 for biopsy. With this approach, sensitivity is high, but specificity tends to be quite variable and lower, resulting 3 in millions of biopsies of benign lesions every year. To improve specificity, several optical technologies are 4 being developed to noninvasively detect skin cancer. Of these, reflectance confocal microscopy (RCM) is the 5 furthest advanced in clinical utility, proven for diagnosing skin cancers with high sensitivity and specificity. 6 RCM imaging, guided by dermoscopy, detects skin cancers with 2 times superior specificity, and reduces the 7 benign-to-malignant biopsy rate by 2 times, compared to that with dermoscopy alone. In 2016, the Centers for 8 Medicare and Medicaid Services granted current procedural terminology (CPT) reimbursement codes for RCM 9 imaging of skin. RCM imaging combined with dermoscopy is now advancing into clinical practice, sparing pa- 10 tients from unnecessary biopsies of benign lesions. However, toward widespread acceptance and adoption, a 11 key challenge is that clinical widefield examination, dermoscopy and RCM imaging are currently performed as 12 three separate procedures with separate devices. Clinicians do not precisely know the location of RCM imag- 13 es relative to the surrounding contextual lesion morphology that is seen with clinical widefield examination and 14 dermoscopy, resulting in lower and more variable diagnostic accuracy (particularly, sensitivity, positive and 15 negative predictive values). We propose a novel solution: (i) a new objective lens with an integrated micro- 16 camera, to deliver a concurrent widefield image of the skin surface surrounding the location of RCM imaging; 17 (ii) a new software algorithm for widefield image-based tracking of the location of RCM images within a dermoscopic 18 field of view; (iii) a new diagnostic approach that will proactively use widefield imaging to locate RCM images in 19 dermoscopic images. We intend to deliver this integrated widefield clinical, dermoscopic and RCM imaging ap- 20 proach into the clinic, toward a new standard for more accurate, consistent and faster RCM imaging to guide 21 patient care. Preliminary studies with a ?mock? objective lens and micro-camera on a bench-top set-up 22 demonstrated excellent optical sectioning (~2 µm) and resolution (~1 µm) for RCM imaging, and accurate and 23 repeatable location of RCM fields-of-view within the widefield image. RCM images showed excellent cellular 24 and morphologic detail in vivo. Our specific aims are (1) to develop a handheld reflectance confocal micro- 25 scope with integrated widefield camera; (2) to develop image processing algorithms for real-time widefield im- 26 aging-guided tracking of RCM image locations within dermoscopic fields; (3) to test and validate performance 27 on 100 patients. Although our proposition is for skin lesions, the research will surely have wider impact for 28 imaging in other settings, particularly, with miniaturized confocal microscopes and endoscopes, which have 29 very small fields-of-view. We are a highly synergistic team from Montana State University, Memorial Sloan 30 Kettering Cancer Center, Northeastern University and Caliber Imaging and Diagnostics (formerly, Lucid Inc.).