High-Throughput Nonlinear Optical Microscopy

High-resolution microscopy methods based on different nonlinear optical (NLO) contrast mechanisms are finding numerous applications in biology and medicine. While the basic implementations of these microscopy methods are relatively mature, an important direction of continuing technological innovation lies in improving the throughput of these systems. Throughput improvement is expected to be important for studying fast kinetic processes, for enabling clinical diagnosis and treatment, and for extending the field of image informatics. This review will provide an overview of the fundamental limitations on NLO microscopy throughput. We will further cover several important classes of high-throughput NLO microscope designs with discussions on their strengths and weaknesses and their key biomedical applications. Finally, this review will close with a perspective of potential future technological improvements in this field.National Institutes of Health (U.S.) (9P41EB015871-26A1)National Institutes of Health (U.S.) (R01-EX017656)National Institutes of Health (U.S.) (5 R01 NS051320)National Institutes of Health (U.S.) (4R44EB012415-02)National Science Foundation (U.S.) (CBET-0939511)Singapore-MIT AllianceSkolkovo Institute of Science and TechnologySingapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology)Wellcome Trust (London, England) (Massachusetts Institute of Technology. Postdoctoral Fellowship 093831/Z/10/Z

Proceedings of the 2018 Joint Workshop of Fraunhofer IOSB and Institute for Anthropomatics, Vision and Fusion Laboratory

The Proceeding of the annual joint workshop of the Fraunhofer IOSB and the Vision and Fusion Laboratory (IES) 2018 of the KIT contain technical reports of the PhD-stundents on the status of their research. The discussed topics ranging from computer vision and optical metrology to network security and machine learning. This volume provides a comprehensive and up-to-date overview of the research program of the IES Laboratory and the Fraunhofer IOSB

Polarization Sensor Design for Biomedical Applications

Advances in fabrication technology have enabled the development of compact, rigid polarization image sensors by integrating pixelated polarization filters with standard image sensing arrays. These compact sensors have the capability for allowing new applications across a variety of disciplines, however their design and use may be influenced by many factors. The underlying image sensor, the pixelated polarization filters, and the incident lighting conditions all directly impact how the sensor performs. In this research endeavor, I illustrate how a complete understanding of these factors can lead to both new technologies and applications in polarization sensing. To investigate the performance of the underlying image sensor, I present a new CMOS image sensor architecture with a pixel capable of operation using either measured voltages or currents. I show a detailed noise analysis of both modes, and that, as designed, voltage mode operates with lower noise than current mode. Further, I integrated aluminum nanowires with this sensor post fabrication, realizing the design of a compact CMOS sensor with polarization sensitivity. I describe a full set of experiments designed as a benchmark to evaluate the performance of compact, integrated polarization sensors. I use these tests to evaluate for incident intensity, wavelength, focus, and polarization state, demonstrating the accuracy and limitations of polarization measurements with such a compact sensor. Using these as guides, I present two novel biomedical applications that rely on the compact, real-time nature of compact integrated polarimeters. I first demonstrate how these sensors can be used to measure the dynamics of soft tissue in real-time, with no moving parts or complex optical alignment. I used a 2 megapixel integrated polarization sensor to measure the direction and strength of alignment in a bovine flexor tendon at over 20 frames per second, with results that match the current method of rotating polarizers. Secondly, I present a new technique for optical neural recording that uses intrinsic polarization reflectance and requires no fluorescent dyes or electrodes. Exposing the antennal lobe of the locust Schistocerca americana, I was able to measure a change in the polarization reflectance during the introduction of the odors hexanol and octanol with the integrated CMOS polarization sensor

Real-time tissue viability assessment using near-infrared light

Despite significant advances in medical imaging technologies, there currently exist no tools to effectively assist healthcare professionals during surgical procedures. In turn, procedures remain subjective and dependent on experience, resulting in avoidable failure and significant quality of care disparities across hospitals. Optical techniques are gaining popularity in clinical research because they are low cost, non-invasive, portable, and can retrieve both fluorescence and endogenous contrast information, providing physiological information relative to perfusion, oxygenation, metabolism, hydration, and sub-cellular content. Near-infrared (NIR) light is especially well suited for biological tissue and does not cause tissue damage from ionizing radiation or heat. My dissertation has been focused on developing rapid imaging techniques for mapping endogenous tissue constituents to aid surgical guidance. These techniques allow, for the first time, video-rate quantitative acquisition over a large field of view (> 100 cm2) in widefield and endoscopic implementations. The optical system analysis has been focused on the spatial-frequency domain for its ease of quantitative measurements over large fields of view and for its recent development in real-time acquisition, single snapshot of optical properties (SSOP) imaging. Using these methods, this dissertation provides novel improvements and implementations to SSOP, including both widefield and endoscopic instrumentations capable of video-rate acquisition of optical properties and sample surface profile maps. In turn, these measures generate profile-corrected maps of hemoglobin concentration that are highly beneficial for perfusion and overall tissue viability. Also utilizing optical property maps, a novel technique for quantitative fluorescence imaging was also demonstrated, showing large improvement over standard and ratiometric methods. To enable real-time feedback, rapid processing algorithms were designed using lookup tables that provide a 100x improvement in processing speed. Finally, these techniques were demonstrated in vivo to investigate their ability for early detection of tissue failure due to ischemia. Both pre-clinical studies show endogenous contrast imaging can provide early measures of future tissue viability. The goal of this work has been to provide the foundation for real-time imaging systems that provide tissue constituent quantification for tissue viability assessments.2018-01-09T00:00:00

Roadmap on structured light

Structured light refers to the generation and application of custom light fields. As the tools and technology to create and detect structured light have evolved, steadily the applications have begun to emerge. This roadmap touches on the key fields within structured light from the perspective of experts in those areas, providing insight into the current state and the challenges their respective fields face. Collectively the roadmap outlines the venerable nature of structured light research and the exciting prospects for the future that are yet to be realized.Peer ReviewedPostprint (published version

Development and Application of Two-Photon Excitation Stimulated Emission Depletion Microscopy for Superresolution Fluorescence Imaging in Thick Tissue

Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of 1-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular features of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality which overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. In this thesis, I describe the development of 2PLSM combined with STED microscopy for superresolution fluorescence imaging of neurons embedded in thick tissue. Furthermore, I describe the application of this method to studying the biophysics connecting synaptic structure and function in dendritic spines

Development and Application of a Synthetic Near Infrared Fluorescent Probe for Imaging Modulatory Neurotransmitters

Dopamine neurotransmission plays critical roles in brain function in both health anddisease and aberrations in dopamine neurotransmission are implicated in severalpsychiatric and neurological disorders, including schizophrenia, depression, anxiety, andParkinson’s disease. Until recently, measuring the dynamics of dopamine and otherneurotransmitters of this class could not be achieved at spatiotemporal resolutionsnecessary to study how dopamine regulates the plasticity and function of neurons and neuralcircuits, and how dysfunctions in this regulation lead to disease. Probes that satisfy criticalattributes in spatiotemporal resolution and chemical selectivity are needed to facilitateinvestigations of dopamine neurochemistry.To address this need, this dissertation describes the synthesis and implementation ofan ultrasensitive near-infrared “turn-on” nanosensor (nIRCat) for the catecholamineneuromodulators dopamine and norepinephrine. To guide probe development, we presentresults from a computational model that offers insight into the spatiotemporal dynamics ofdopamine in the striatum, a subcortical structure that is enriched in dopamine. With thismodel, we elucidated the kinetic requirements for a prototypical optical indicator as well asoptimal imaging frame rates needed for measuring dopamine neurochemical dynamics.Stochastic modeling of dopamine dynamics, driven by kinetic phenomena of vesicularrelease, diffusion and clearance, provide a platform to evaluate dopaminergic volumetransmission arising from a single terminal or ensemble terminal activity. With this work,we illustrate that only probes with kinetic parameters in a particular range are feasible fordopamine imaging at spatiotemporal scales likely to be encountered in brain tissue.In two subsequent chapters, we describe the development and in vitrocharacterization of nIRCats, synthesized from functionalized single wall carbon nanotubes(SWCNT) that fluoresce in the near infrared range of the spectrum. We show that nIRCatsexhibit maximal relative change in fluorescence intensity (ΔF/F0) of up to 35-fold inresponse to catecholamines and have optimal dynamic range that span physiologicalconcentrations of their target brain analytes. Through a combination of experimental andmolecular dynamics approaches, we elucidate the photophysical principles and intermolecularinteractions that govern the molecular recognition and fluorescence modulation of nIRCats by dopamine.Finally, we demonstrate that nIRCat can be used to measure electrically andoptogenetically evoked release of dopamine in striatal brain slices, revealing hotspots ofactivity with a median size of 2 μm, and exhibiting a log-normal size distribution that extendsup to 10 μm. Moreover, nIRCats are shown to be compatible with dopamine pharmacologyand permit studies of how receptor-targeting drugs modulate evoked dopamine release. Ourresults suggest nIRCats may uniquely support similar explorations of processes that regulatedopamine neuromodulation at the level of individual synapses, and exploration of the effectsof receptor agonists and antagonists that are commonly used as psychiatric drugs andpsychoactive molecules that modulate the release and clearance profiles of dopamine. Weconclude that nIRCats and other nanosensors of this class can serve as versatile syntheticoptical tools to monitor interneuronal chemical signaling in the brain extracellular space atspatial and temporal scales pertinent to the encoded information

Time-Resolved Photoemission Electron Microscopy: Development and Applications

Time-resolved photoemission electron microscopy (TR-PEEM) belongs to a class of experimental techniquescombining the spatial resolution of electron-based microscopy with the time resolution of ultrafast opticalspectroscopy. This combination provides insight into fundamental processes on the nanometer spatial andfemto/picosecond time scale, such as charge carrier transport in semiconductors or collective excitations ofconduction band electrons at metal surfaces. The high spatiotemporal resolution also offers a detailed view of therelationship between local structure and ultrafast photoexcitation dynamics in nanostructures and nanostructuredmaterials, which is beneficial in exploring new materials and applications in opto-electronics and nano-optics.This thesis describes the investigation of ultrafast photoexcitation dynamics in metal- and III-V semiconductornanostructures using TR-PEEM. We investigate hot carrier cooling in individual InAs nanowires where we findevidence that electron-hole scattering strongly contributes to the intra-band energy relaxation of photoexcitedelectrons on a sub-picosecond time scale and we observe ultrafast hot electron transport towards the nanowiresurface due to an in-built electric field. We demonstrate the combination of TR-PEEM with optical time-domainspectroscopy to enable time- and excitation frequency-resolved PEEM imaging. The technique is applied to GaAssubstrates and nanowires. TR-PEEM is further used to investigate localized and propagating surface plasmonpolaritons. We explore the optical properties of disordered, porous gold nano-particles (nanosponges). Using TRPEEM,we can resolve several plasmonic hotspots with different resonance frequencies and lifetimes within singlenanosponges. We also explore excitation and temporal control of surface plasmon polaritons by means of singlelayeredcrystals of the transition metal dichalcogenide WSe2.In addition, this thesis includes developments in ultrafast optics, aiming to expand the capabilities of the TR-PEEMsetup. We present a setup for generating tunable broadband ultraviolet (UV) laser pulses via achromatic secondharmonic generation. The setup is suitable for operation at high repetition rates and low pulse energies due to its highconversion efficiency. Further, we describe a transmission grating-based interferometer for the generation of stable,phase-locked pulse pairs. Pulse shaping based on liquid crystal technology allows accurate control over the temporalshape of femtosecond laser pulses. We characterize Fabry-Perot interferences affecting the accuracy of such pulseshapers, and we demonstrate a calibration scheme to compensate for these interference effects

Coupling qualitative and quantitative analyses of pharmaceutical materials enabled by second harmonic generation microscopy

The detection and characterization of crystallinity is critical throughout the drug development process. From the initial establishment of an active pharmaceutical ingredient’s (API) crystal structure to stability testing and quality control, the phase of an API affects the solubility, bioavailability, stability, and efficacy of a drug product. Second harmonic generation (SHG) microscopy has recently been developed as a selective and rapid method for imaging crystallinity in drug formulations. While SHG microscopy can enable the high signal-to-noise (SNR) detection of crystallinity, the intrinsic chemical information content within SHG images is relatively low. In cases of trace crystallinity and/ or small crystal volumes, new tools capable of rapid, qualitative crystal characterization are needed to fill this measurement gap. Several strategies for increasing the chemical information content of SHG microscopy were developed. Following combined computational and experimental studies to help determine the body of crystalline API structures amenable to imaging by SHG microscopy, measurements by confocal Raman spectroscopy and synchrotron X-ray diffraction were performed on regions of interest (ROI) identified by SHG. In both cases, spatial restriction of the spectroscopic technique to these regions of interest lowered the detection limits of Raman and synchrotron X-ray diffraction by several orders of magnitude. To further expand the capabilities of SHG microscopy, nonlinear optical Stokes ellipsometric (NOSE) microscopy was developed to assess crystal structure characteristics through the polarization dependence of SHG. Rapid (8 MHz) polarization modulation enabled NOSE microscopies at video rates (up to 15 Hz). Following development and validation, NOSE microscopy was used in conjunction with an iterative, nonlinear least-squares fitting algorithm to discriminate polymorphic crystal forms of the small molecule D-mannitol. Finally, to extend the linear dynamic range of photon counting measurements as described here-in, a novel digital filter derived from linear discriminant analysis (LDA) was developed and validated via theoretical and experimental nonlinear optical (NLO) measurements