Eric O. Potma - US grants

This award is for developing improved contrast in Coherent Anti-Stokes Raman Scattering [CARS] Microscopy through Focus-Engineering [FE-CARS]. Coherent anti-Stokes Raman scattering (CARS) microscopy has rapidly ascended as a noninvasive tool for imaging cells and tissues with chemical selectivity. Based on its unique vibrational selectivity to lipids, proteins, and DNA, the full range of potential CARS applications to biological problems is vast. Nonetheless, due to the presence of a ubiquitous nonresonant background and the weak vibrational response of several molecular groups, applications have been limited to imaging of lipids so far. To open up a new range of applications for CARS microscopy in biology and biomedicine, background suppression and contrast improvement methods are imperative. This work introduces a new approach to CARS microscopy, which provides immediate contrast improvement. The approach is based on controlling the spatial interference of the CARS waves emanating from the focal volume. Emission control is achieved by spatial phase shaping, or focal engineering, of the incident laser pulses using a spatial light modulator (SLM). The use of appropriate phase masks allows controlled destructive interference of unwanted background contributions, while constructive interference accentuates vibrationally resonant objects. A focus-engineered (FE-) CARS microscope will be developed, with the following milestones: 1) Characterizing and optimizing contrast improvement with FE-CARS through computer-controlled phase shaping of the incident transverse beam profiles; 2) Combining FE-CARS with two-photon excited fluorescence (TPEF) and focus-engineered second harmonic generation (SE-SHG) for multimodal imaging of tissues and cells. FE-CARS multimodal imaging will be tested on a) lipid depositions and calcium hydroxyapapatite crystals; b) structural changes to collagen fibers under the influence of glycerol or dimethyl-sulfoxide.

The proposed microscope development is anticipated to have far reaching implications for biological imaging studies. The proposed instrument will be used for training graduate students, undergraduate students and pre-college students in the field of nonlinear microscopy. The instrument will be used in several outreach programs. Among these programs, the PI organized the Minority Science Program of the School of Biological Sciences at the university and a 4-week Summer School for high school students.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5). Eric Olaf Potma of the University of California, Irvine, is supported by an award from the Experimental Physical Chemistry program to develop a coherent anti-Stokes Raman scattering (CARS) microscope with super-resolution. Development of this microscope will enable chemically selective imaging with a resolution down to the 50 nm length scale, opening up new areas in optical imaging of biological samples and engineered materials. The instrument will be used to study the nonlinear optical response of metallic nanowires, and to examine the distribution of sub-micrometer sized lipids droplets in breast cancer cells.

This work may lead to the development of methods that to better examine samples with microscopic and nanoscopic details. The investigator is also committed to utilize advanced instruments for the purpose of fostering enthusiasm for physical chemistry among high school students. An annual 4-week Summer School will be held, in which high school students have the opportunity to attend college-style lectures and participate in high-end research experiments.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In this project, the hydrodynamic radius of several dextrans of various length are determined. The hydrodynamic radius is relevant to diffusion studies of labeled dextrans in neuronal systems, where the molecular radius is a determining factor in gap junction permeability.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recent developments in optical imaging have revolutionized the way in which we can examine cells and tissue in vivo. The number of modern optical imaging technique is continuously expanding, each offering a unique contrast mechanism and sensitivity to important bio-compounds in the system under study. For instance, tissue morphology can be non-invasively examined with optical coherence tomography (OCT). Over the past decade, the development of nonlinear methods such as two photon excited fluorescence (TPEF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy has given the biomedical researcher convenient tools for selectively visualizing endogenous structures without the need of labeling. The multi-dimensional approach to microscopy integrates various imaging techniques and contrast mechanisms to better assess the biological specimen. Combinations of the TPEF, SHG, CARS and OCT techniques have previously been realized into optical microscopes, however, a full integration of these imaging modalities has not been accomplished. In this proposal we push the envelope of multi-dimensional imaging in the following way: a)Combining the TPEF, SHG, CARS and OCT into a single imaging platform. b)Optimizing the image contrast and penetration depth by controlling the spectral phase. The proposed multi-dimensional imaging platform uniquely permits the three-dimensional mapping of key tissue and cell components such as flavin metabolites, elastin networks, collagen filaments and lipid pools along with the detailed tissue morphology. Such an optical assessment of the biological specimen would provide one of the most detailed microscopic views of the sample currently possible.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

With this award from the Major Research Instrumentation (MRI) program Professor Vartkess A. Apkarian and colleagues Rachel Martin, Reginald M. Penner and Eric Potma from the Department of Chemistry at the University of California-Irvine (UCI) will acquire a confocal Raman microscope to facilitate research at the NSF Center for Chemical Innovation on Chemistry at the Space-Time Limit and the UCI Center for Solar Energy. The instrument will be broadly utilized for the molecular characterization of fabricated novel materials, nano-structured devices for solar cells, single molecule devices based on nano-tube and nano-wire platforms, nano-catalysts, polycrystalline hydrates and protein aggregates. These are all examples of heterogeneous specimens in which molecular composition and function greatly vary with space.

A confocal Raman microscope is a highly flexible instrument, principally used for the non-destructive chemical characterization of heterogeneous specimens. Raman spectra allow the identification of molecular structure and environment. The confocal Raman microscope produces images of specimens, consisting of spectra obtained with a spatial resolution of 1 micrometer. The instrument will be an integral part teaching and research activities of two research centers and will in addition serve the broader research communities at UCI.

An award is made to the University of California, Irvine (UCI), to develop a new type of microscope that can be used to study the properties of cell membranes. The SCRM instrument provides a unique solution for cell membrane studies where the use of fluorescent labels is questionable, thus enabling new discoveries in cell biology and membrane biophysics not previously possible with current technologies. The development and application of the SCRM technology forms a thorough training opportunity for two graduate students and one undergraduate student in the areas of engineering, optical physics and biology. An important component of this program is to bring the new SCRM technology into the classroom, which is achieved by using the prototype instrument in a graduate level course on Molecular Biophysics. The new microscope will also be actively used in the Laboratory Experiments & Activities in the Physical Sciences (LEAPS) program, which brings underrepresented students from the Santa Ana School District to the UCI campus.

The special feature of this microscope is its capability to interrogate membranes of live cells with high contrast, but without the use of fluorescent labels. This new mode of inquisition, called surface-sensitive coherent Raman microscopy (SCRM), is achieved through merging the imaging properties of two existing techniques: coherent Raman scattering (CRS) microscopy and total internal reflection illumination. Compared to conventional CRS microscopy, SCRM offers a ten times higher axial resolution at imaging speeds of more than 10 frames per second.

This Major Research Instrumentation award supports the acquisition of an ultrafast multimodal spectroscopy system. This system enables advanced time-resolved spectroscopy experiments, a key capability in material and molecular research. The system will be placed in the Laser Spectroscopy Facility (LSF), housed in the Department of Chemistry of the University of California Irvine. Traditionally, ultrafast lasers and spectroscopy have not been part of user facilities because former ultrafast laser technologies were too complex and unreliable to support many users. Consequently, the user community of ultrafast technologies has remained small, and the unique research capabilities of ultrafast optical techniques have remained inaccessible to an audience interested in such capabilities. In this Program, a state-of-the-art ultrafast laser system is made accessible through a three-pronged model based on optimized facility conditions, strong user support and a partnership with industry. This model addresses past limitations to make ultrafast laser technology and its research capabilities accessible to a much broader community of researchers. In making the technology available to a much larger user base, the impact of ultrafast spectroscopy is significantly amplified. The Program ensures broad exposure and dissemination of ultrafast spectroscopy capabilities. On the UCI campus alone, the Laser Spectroscopy Facility (LSF) in which the system will be housed supports more than 300 users, and serves 13 departments on campus. By leveraging strong connections with institutions and companies neighboring UCI, a large community of researchers will have access to the ultrafast spectroscopy capabilities offered through the facility. The impact of the requested technology is further fortified by a strong user training program, established channels of dissemination, and an outreach program designed around the ultrafast spectroscopy instrument

The most fundamental processes in matter evolve on ultrafast time scales. Examples include the making and breaking of chemical bonds, conformational motions of molecules, evolution of optical excitations, and electron transfer processes. These phenomena form the mechanistic basis of scientific challenges in chemistry, biology and materials science: designing efficient catalysts, understanding nature's solution to light harvesting materials, and optimizing the efficiency of solar cells, to name a few. Ultrafast laser technologies have proven indispensable for meeting these challenges, as they provide direct recordings of such fast fundamental processes. Consequently, the demand for ultrafast spectroscopy is growing, as an increasingly expanding pool of chemists, chemical engineers, physicists, and biologists come to rely on this technology. The goal of this Program is to bring the unique capabilities of ultrafast laser science to a broad community of researchers. This goal is achieved by implementing a three-pronged model: 1) Acquisitions of a commercial ultrafast laser light source and nonlinear optical spectrometer, with unprecedented performance, versatility, stability, and simplicity of operation. The system is made available through a staffed user facility at the University of California, Irvine (UCI); 2) A partnership with Newport Optics, the supplier of the instrument. The partnership establishes a push-pull mechanism between technology and application for impacting a broad community beyond the users at UCI; 3) The Ultrafast Consultation Board (UCB), a team of experimental and theoretical ultrafast spectroscopists who provide general assistance, technical guidance, mentoring and help with the interpretation of data. By providing depth to the analysis of acquired spectroscopic data, the UCB bolsters the scientific impact of measurements made by non-experts, expands the user base, and magnifies the breadth of research topics supported by this Program.

In this project funded by the Chemical Measurement and Imaging program of the Chemistry Division, Professor Eric Potma of the University of California, Irvine, is developing a fast laser-scanning nonlinear optical (NLO) microscope platform that includes a new chemically selective imaging modality: sum-frequency generation (SFG). This advance offers researchers active in the fields of biology and materials research a new form of chemical imaging contrast, which was previously not available at practical acquisition speeds and resolution. The addition of the SFG modality onto the optical microscope relies on new achromatic optical components, which are especially designed and developed in this project, and which may find important new applications in other forms of infrared imaging and microscopy. Further broader impacts of this work include the use of the SFG imaging platform as a training tool in a biophysics graduate course and the development of community-oriented, interactive walking tours through local wildlands that illuminate biophysical effects in nature.

This project integrates the unique imaging capabilities of SFG into a laser-scanning optical microscope. SFG makes it possible to visualize a special class of materials, namely those that exhibit non-centrosymmetry on a molecular level. Because SFG is also sensitive to molecular vibrations, the resulting images can differentiate non-centrosymmetric materials based on their chemical bond vibrations. As an imaging modality, this contrast mechanism enables researchers to perform detailed studies on a myriad of materials, including biological fibers and chiral microcrystals, at image acquisition speeds that have hitherto remained out of reach.

This PFI: AIR Technology Translation project enables the translation of an optical lens that provides achromatic focusing of light over an extended wavelength range, from the visible to the mid-infrared range. Achromatic focusing allows different wavelengths of light, for example red and blue, to be brought into focus at the same focal length, enabling a clearer image. It is useful for a wide range of optics applications, from microscopy to photography to hyperspectral imaging. Previous achromatic lenses have been limited in their wavelength range, whereas other broadband achromatic lenses use materials and production techniques that are expensive. The product to be developed in this project offers a combination of materials that are not only affordable but also production friendly, resulting in the fabrication of a relatively inexpensive optic with superior achromatic properties. The project will result in a prototype lens that can be readily used in optical microscopy applications, providing imaging opportunities from the visible to the mid-infrared.

Discovered during a research-oriented project, the proposed achromat is composed of calcium fluoride and sapphire. Initial simulations have confirmed that the combination of these two materials produces achromatic lenses over an extended range. The project will further fine-tune the material parameters before the components are fabricated and the lenses are assembled with high precision. The prototype will be extensively tested before copies are disseminated for purchase to users in the field.

The project will form an extensive training experience for a graduate and an undergraduate student, who will be involved with the engineering, testing and commercial decision making process. The project also engages engineers and business development personnel from the Zygo, a market leader in custom optics fabrication, to drive this technology translation effort from research discovery toward commercial reality.

Advances in laser-scanning optical microscopy, a high-resolution imaging technique, have made it possible to image the chemical and structural details of living cellular and tissue systems at high resolution. Unfortunately, microscopic variations in tissue composition scatter the laser light, causing degradation of the image quality and limits in imaging depth. In fact, light scattering is the main limitation of using laser-scanning microscopy for tissue imaging. A promising approach to improve image quality and depth is to use adaptive optics (e.g. deformable mirrors) to shape the laser beam in a way that counteracts the effects of light scattering in the tissue. While promising, adaptive optics techniques currently use optimization schemes that do not account for the mechanisms of scattering. This leaves the method prone to artifacts, especially as scattering becomes more severe at greater depths. Thus, a mechanistic (physical) insight into the scattering would be very helpful to guide and fundamentally improve adaptive optics optimization schemes. The goal of this project is to develop an integrated modeling, computational, and experimental approach--a Virtual Microscopy Simulator (VMS)--to determine the adaptive optics signal corrections necessary to fully correct for scattering induced distortions relevant to laser scanning microscopy. The outcomes of this work improve adaptive optics technics and allow imaging at greater depths in tissue materials, which will dramatically improve the impacts of optical microscopy in the biomedical sciences. Beyond making the VMS platform available to the science community, Education and Outreach plans include participating in a UC-HBCU summer research program and providing courses and mentorship within an NSF IGERT program in Biophotonics.

This goal of this project is to fundamentally improve optical imaging of tissues by delivering new insights in counteracting the effects of light scattering in tissues using a model-based approach to adaptive optics that will improve image quality and penetration depth. This approach addresses many of the limitations encountered by current technologies, including: a) constraint to superficial layers due to light scattering, b) non-unique solutions and artifacts when deeper layers are accessed via wavefront shaping based on empirically based optimization schemes, c) lack of beacon sensors that could provide information to generate a compensating phase pattern, d) production of distorted focal volumes, e) iterative optimization methods that make imaging slow and, most important, f) absence of methods to model wave-based light propagation in tissue materials of meaningful volumes, leaving no model-based support for adaptive optics in tissue imaging. The new framework will be used to compute the adaptive optics signal corrections necessary to fully correct for scattering induced distortions relevant to laser scanning microscopy, and nonlinear optical microscopy in particular. The Research Plan is organized under three objectives. OBJECTIVE 1 is to develop a Virtual Microscopy Simulator (VMS) to model focused beam propagation, (linear and nonlinear) signal generation, and signal detection in turbid tissues. The input module will allow users to provide input data such as microscopy type, lens data, incident beam parameters, scattering data, nonlinear susceptibility data, detector specifications and adaptive optics data (Deformable Mirrors or Spatial Light Modulators settings). The input data will be fed into the computational engine to execute the computations. The computational module will be designed to handle all computations: a) Signal Generation, b) Signal Emission and Detection, and c) Adaptive Optics Computations. The Output module will consist of text data files of 3D focal volume data, far-field radiation data, and image data. OBJECTIVE 2 is to perform experimental validation of the VMS in tissue-mimicking phantom systems with well-defined scattering and signal generation elements. Four classes of phantoms with nonlinear optical targets will be prepared to validate the VMS: a) agarose or Sylgard only (Non-scattering), b) agarose or Sylgard with microspheres, c) Collagen Hydrogel with and w/out microspheres, and d) hybrid specimens with a layer of agarose or Sylgard matrix and a layer of collagen hydrogel with microspheres. OBJECTIVE 3 is to establish model-based adaptive optics design principles through usage of the VMS to predict the input wavefronts necessary to counteract the dispersive effects of scattering media that impede diffraction-limited focal volume formation and signal generation in scattering. The aims of the objective are to a) examine potential differences between the corrected focal volume and a theoretical, undistorted volume, b) study the performance of the algorithms from the perspective of the ideal wavefront needed to correct the image, and c) develop an adaptive algorithm that reproduces the undistorted image, even in the presence of scatterers situated between the focal volume and the collecting lens.

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.

Project Summary A genuine label-free imaging technology, vibrational microscopy provides maps of cells and tissues with exceptionally high chemical contrast as it directly probes the fundamental vibrational modes of samples. Vibrational imaging approaches include IR-absorption micro-spectroscopy and confocal Raman microscopy, methods that have been successfully commercialized (a growing 500 million dollar market) and are now common tools of inquiry found in analytical and biological laboratories. Over the past four decades, these techniques have had a measurable impact in the fields of biology and biomedicine, offering a spatially resolved assessment of healthy and diseased tissues from a molecular perspective. This proposal aims to significantly improve the capabilities of vibrational microscopy. We propose a new imaging approach that merges the desirable properties of IR absorption microscopy with some of the unique properties of coherent, nonlinear optical (NLO) excitation of molecules. This novel IR-NLO technique improves the spatial resolution of IR absorption microscopy by tenfold, while offering higher sensitivity to fingerprint molecular vibrations relative to Raman-based microscopy methods. Our team is comprised of experts in coherent Raman scattering microscopy and IR microspectroscopic imaging. Our innovation makes it possible to rapidly acquire IR absorption images of fingerprint vibrational modes with a resolution of 0.5 micrometer or better. The preliminary data shows that the IR-NLO approach can be successfully adopted in a rapid laser-scanning microscope, allowing convenient vibrational imaging of tissue specimens. In our proposal we develop, test, and improve the new IR-NLO technology. The validation of the technology is achieved through extensive biomedical imaging studies and comparison with the state of the art IR microscopy available today. The proposed program tackles a major challenge in IR spectroscopic microscopy, namely the improvement of imaging resolution. This new capability is significant, as the higher resolution enables the identification of sub-micrometer intra- and extra-cellular structures in the tissue, which hitherto have remained invisible in IR-imaging. The high-resolution imaging property thus dramatically improves the diagnostic capabilities of the technique. By setting a new resolution standard for fingerprint vibrational imaging, the IR-NLO technology is likely to have a significant impact in tissue imaging and can enable its use in both research and clinical domains for pathology.

This project supports fundamental research on an emerging method for the fabrication of microstructures, called two-photon polymerization (TPP). In TPP, a focused laser beam writes three-dimensional polymer structures of any configuration, enabling the fabrication of intricate objects and devices at a length scale many times smaller than the thickness of a human hair. Developments in TPP have been hampered by the inability to improve the repeatability, structural integrity, mechanical properties and further downsizing of the written structures. These limitations are chemical in nature, but deciphering the chemical composition at such small length scales has remained a significant challenge. In this project, this hurdle is overcome by using a new approach capable of generating detailed chemical maps of TTP microstructures. Using a light-based analytical method called tip-enhanced Raman scattering, a close-up view of evolving chemical changes in TTP microstructures is obtained with nanometer precision. By unraveling the hitherto hidden chemical and structural changes at the nanoscale, TTP procedures can be much improved, making a large-scale implementation of TTP technologies possible. This project thus accelerates the translation of TPP from academic labs towards large-scale industrial applications. The project also provides training to a specialist and two undergraduate students who have the opportunity to acquire sought-after skills in high-end instrumentation. In addition, this project includes an outreach effort to middle school students in the community, in which students have the chance to produce micrometer-sized versions of 3D objects of their choosing.

Two-photon polymerization (TPP) is a popular technology for the fabrication of micro-structures, yet the industrial use of TPP has been hindered by poor consistency between written structures limiting a large-scale implementation of this additive manufacturing technique. Variations in the degree of conversion, solvent permeation, voxel overlap, and material inhomogeneities are not well understood, giving rise to a low overall repeatability. The root of these problems is insufficient knowledge of the polymerization process at the nanoscale. This project addresses several longstanding questions regarding the photo- physics and photo-chemistry of TPP that have limited a broader implementation of the technique. The project focuses on outstanding questions about the time-sequenced process of photo- initiation followed by polymerization as well as the resulting nanoscale morphology and chemistry. The excitation dynamics, radical formation and resin interactions of photoinitiators are studied by ultrafast spectroscopy experiments, and the degree of cross-linking, material inhomogeneities and solvent permeation at the nanoscale are studied by tip-enhanced Raman scattering. The insights acquired in this work enable an optimization of key TPP parameters, producing new guidelines for photoinitiator development, directions for controlling the degree of polymerization, new laser illumination strategies of improving resolution and management of the pyrolysis process at the nanoscale. Consequently, the deliverables in this project accelerate the translation of TPP from academic labs towards large-scale industrial applications.

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.

Project Summary/Abstract Nonlinear optical (NLO) microscopy techniques have become important tools of inquiry for understanding both tissue biology and tissue pathology. Other than more conventional confocal fluorescence microscopy approaches, NLO microscopy enables label-free probing of tissue structures and components, at depths beyond what can be achieved with standard optical imaging techniques. NLO microscopy has become the method of choice for studying glycolysis and lipid metabolism a wide variety of tissues, studying myelin degeneration in nervous tissues, detecting migrating melanocytes in skin, mapping disease-induced changes to the extracellular matrix, and more. Novel advances in NLO microscopy are intimately linked to new scientific inquiries and discoveries in tissue biology. Since the 1990s, the Beckman Laser Institute (BLI) has played a leading role in developing NLO imaging technologies and applying these methods to solving outstanding problems in biology and biomedicine. To continue its pioneering role in advancing NLO imaging techniques, through this proposal the BLI is requesting a replacement of a laser-scanning NLO microscope, an ailing 12-year old user instrument. While this microscope has served more than 160 users, its vendor no longer services the instrument because of age, and its capabilities are incompatible with the evolving imaging needs of our user base. The requested replacement is a Leica SP8 multiphoton microscope, which is configured for high- resolution, meso-scale tissue imaging based on a wide variety of NLO contrast mechanisms: two- photon excited fluorescence (TPEF), second-harmonic generation (SHG) and third-harmonic generation (THG). In addition, we have worked with Leica engineers to enable imaging based on coherent anti-Stokes Raman scattering (CARS), a modality never before offered in combination with other femtosecond NLO modalities on a commercial laser-scanning microscope. The merger of all these NLO techniques in one instrument makes it possible to perform label-free imaging of lipids, protein density, carbohydrates, nucleic acids, collagen, NADH, elastin, melanin and more. Equipped with five sensitive detectors, fluorescence lifetime detection technology, resonant scanners, rapid mosaic-style image acquisition, enhanced spectral tuning of excitation and detection windows and an upright configuration with an open sample staging area, this unique instrument offers the advanced tissue imaging capabilities needed to propel the science of our user base into the next decade.

This project develops a portfolio of molecular tags that emit strong signals when stimulated by mid-infrared (MIR) light. These molecular MIR tags are designed to light up selected parts in the cell when illuminated with an MIR microscope. The MIR tags developed by the investigators enable the visualization of molecular diffusion and chemical conversion, including numerous metabolic processes in cells and tissues, processes that have hitherto remained out of reach for MIR microscopy. This development transforms the MIR microscope from an instrument for inspecting static images of cells and tissue to a technology for studying dynamic processes such as cellular cholesterol uptake, protein synthesis and lipogenesis. This capability opens up new opportunities for investigating cellular processes that are difficult to study with standard optical microscopy methods. The investigators are committed to broaden the participation of a diverse pool of students by providing summer research training to students from HBCUs through the Access to Careers in Engineering and Sciences (ACES) program.

MIR imaging, typically in the form of Fourier transform infrared (FTIR) microscopy, is a label-free imaging tool based on molecular vibrational contrast. MIR labels or tags can significantly improve the specificity of MIR imaging, yet MIR labels have so far not been used for imaging purposes. The use of MIR labels would expand the complementary vibrational palette in IR microscopy, opening up a new catalogue of biorthogonal molecular probes based on IR transitions, and offering strategies for super-multiplex imaging. In this project, the PI and co-PI develop a portfolio of chemical motifs that exhibit an exceptionally strong IR-response. They will use these motifs as vibrational tags of small molecules, including cholesterol, glucose, nucleic acids, amino acids and other metabolites. In addition, the team will design probes suitable for fluorescence encoded infrared (FEIR) excitation and detection, enabling multiplex labeling studies in the MIR microscope with sensitivities that reach the single molecule limit.

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.

ABSTRACT A genuine label-free imaging technology, vibrational microscopy provides maps of cells and tissues with exceptionally high chemical contrast as it directly probes the fundamental vibrationals modes of samples. Vibrational imaging approaches include IR-absorption micro-spectroscopy and confocal Raman microscopy, methods that have been successfully commercialized (a growing 500 million dollar market) and are now common tools of inquiry found in analytical and biological laboratories. Given the much stronger IR light-matter interaction, IR microscopy has a particularly high potential to make a measurable impact in the fields of biology and biomedicine. At the same time, the mid-IR (MIR) imaging technology has remained stagnant, as MIR technology still relies on cooled cameras with low pixel density, preventing practical applications in efficient mapping of cultured cells and tissue sections. This proposal aims to introduce a radically new MIR detection approach that overcomes the fundamental hurdles that have plagued a broader implementation of traditional MIR cameras. We propose that direct, on-chip MIR detection can be achieved in an sCMOS camera through the process of non-degenerate two-photon absorption (NTA) enabled by a near-infrared gate pulse. By replacing cryogenically cooled MIR arrayed detectors with a modern sCMOS camera, the proposed work overcomes a key limitation in MIR microscopy and represents an important step toward a more practical implementation of MIR imaging in the biomedical sciences. Our team includes experts in biomedical vibrational imaging and nonlinear optics, and our preliminary data underlines the feasibility of the NTA method for MIR detection. In the proposed work we push NTA for use with modern sCMOS cameras, determine its utility for MIR microscopy and compare its performance for biomedical imaging applications with established MIR microspectroscopy methods.