Fluorescence hyperspectral imaging (fHSI) using a spectrally resolved detector array

The ability to resolve multiple fluorescent emissions from different biological targets in video rate applications, such as endoscopy and intraoperative imaging, has traditionally been limited by the use of filter-based imaging systems. Hyperspectral imaging (HSI) facilitates the detection of both spatial and spectral information in a single data acquisition, however, instrumentation for HSI is typically complex, bulky and expensive. We sought to overcome these limitations using a novel robust and low cost HSI camera based on a spectrally resolved detector array (SRDA). We integrated this HSI camera into a wide-field reflectance-based imaging system operating in the near-infrared range to assess the suitability for $\textit{in vivo}$ imaging of exogenous fluorescent contrast agents. Using this fluorescence HSI (fHSI) system, we were able to accurately resolve the presence and concentration of at least 7 fluorescent dyes in solution. We also demonstrate high spectral unmixing precision, signal linearity with dye concentration and at depth in tissue mimicking phantoms, and delineate 4 fluorescent dyes $\textit{in vivo}$. Our approach, including statistical background removal, could be directly generalised to broader spectral ranges, for example, to resolve tissue reflectance or autofluorescence and in future be tailored to video rate applications requiring snapshot HSI data acquisition.This work was funded by CRUK (C47594/A16267, C14303/A17197), the EPSRC-CRUK Cancer Imaging Centre in Cambridge and Manchester (grant no. C197/A16465) and the EU FP7 agreement FP7-PEOPLE-2013-CIG-630729. Additional funds were provided by the University of Cambridge MRC Confidence in Concept Award

Advancing fluorescent contrast agent recovery methods for surgical guidance applications

Fluorescence-guided surgery (FGS) utilizes fluorescent contrast agents and specialized optical instruments to assist surgeons in intraoperatively identifying tissue-specific characteristics, such as perfusion, malignancy, and molecular function. In doing so, FGS represents a powerful surgical navigation tool for solving clinical challenges not easily addressed by other conventional imaging methods. With growing translational efforts, major hurdles within the FGS field include: insufficient tools for understanding contrast agent uptake behaviors, the inability to image tissue beyond a couple millimeters, and lastly, performance limitations of currently-approved contrast agents in accurately and rapidly labeling disease. The developments presented within this thesis aim to address such shortcomings. Current preclinical fluorescence imaging tools often sacrifice either 3D scale or spatial resolution. To address this gap in high-resolution, whole-body preclinical imaging tools available, the crux of this work lays on the development of a hyperspectral cryo-imaging system and image-processing techniques to accurately recapitulate high-resolution, 3D biodistributions in whole-animal experiments. Specifically, the goal is to correct each cryo-imaging dataset such that it becomes a useful reporter for whole-body biodistributions in relevant disease models. To investigate potential benefits of seeing deeper during FGS, we investigated short-wave infrared imaging (SWIR) for recovering fluorescence beyond the conventional top few millimeters. Through phantom, preclinical, and clinical SWIR imaging, we were able to 1) validate the capability of SWIR imaging with conventional NIR-I fluorophores, 2) demonstrate the translational benefits of SWIR-ICG angiography in a large animal model, and 3) detect micro-dose levels of an EGFR-targeted NIR-I probe during a Phase 0 clinical trial. Lastly, we evaluated contrast agent performances for FGS glioma resection and breast cancer margin assessment. To evaluate glioma-labeling performance of untargeted contrast agents, 3D agent biodistributions were compared voxel-by-voxel to gold-standard Gd-MRI and pathology slides. Finally, building on expertise in dual-probe ratiometric imaging at Dartmouth, a 10-pt clinical pilot study was carried out to assess the technique’s efficacy for rapid margin assessment. In summary, this thesis serves to advance FGS by introducing novel fluorescence imaging devices, techniques, and agents which overcome challenges in understanding whole-body agent biodistributions, recovering agent distributions at greater depths, and verifying agents’ performance for specific FGS applications

Snapshot Hyperspectral Imaging for Complete Fundus Oximetry

In this work, a snapshot hyperspectral imager capable of tuning its average spectral resolution from 22.7 nm to 13.9 nm in a single integrated form is presented. The principle of this system will enable future snapshot systems to dynamically adapt to a wide range of imaging situations. Additionally, the system overcomes datacube size limitations imposed by detector array size limits. The work done in this thesis also advances oximetry of the retina using data collected by the Image Mapping spectrometer (IMS), a snapshot spectrometer. Hyperspectral images of the retina are acquired, and oximetry of individual vessels in four diseased eyes is presented. Further, oximetry of the entire fundus is performed using a novel algorithm with data collected with the IMS. We present oxyhemoglobin concentration maps of the eye and demonstrate oxygen sensitivity of the maps by comparing normal and diseased eyes. The aim of this work is to advance the general capabilities of snapshot hyperspectral imagers and to advance the integration of retinal oximetry into the standard ophthalmology instrument repertoire

Multi-Modal Imaging Techniques for Early Cancer Diagnostics

Cancer kills more Americans under the age of 75 than any other disease. Although most cancers occur in epithelial surfaces that can be directly visualized, the majority of cases are detected at an advanced stage. Optical imaging and spectroscopy may provide a solution to the need for non-invasive and effective early detection tools. These technologies are capable of examining tissue over a wide range of spatial scales, with widefield macroscopic imaging typically spanning several square-centimeters, and high resolution in vivo microscopy techniques enabling cellular and subcellular features to be visualized. This work presents novel technologies in two important areas of optical imaging: high resolution imaging and widefield imaging. For subcellular imaging applications, new high resolution endomicroscope techniques are presented with improved lateral resolution, larger field-of-view, increased contrast, decreased background signal, and reduced cost compared to existing devices. A new widefield optical technology called multi-modal spectral imaging is also developed. This technique provides real-time in vivo spectral data over a large field-of-view, which is useful for detecting biochemical alterations associated with neoplasia. The described devices are compared to existing technologies, tested using ex vivo tissue specimens, and evaluated for diagnostic potential in a multi-patient oral cancer clinical trial

2D and 3D high-speed multispectral optical imaging systems for in-vivo biomedical research

Functional optical imaging encompasses the use of optical imaging techniques to study living biological systems in their native environments. Optical imaging techniques are well-suited for functional imaging because they are minimally-invasive, use non ionizing radiation, and derive contrast from a wide range of biological molecules. Modern transgenic labeling techniques, active and inactive exogenous agents, and intrinsic sources of contrast provide specific and dynamic markers of in-vivo processes at subcellular resolution. A central challenge in building functional optical imaging systems is to acquire data at high enough spatial and temporal resolutions to be able to resolve the in-vivo process(es) under study. This challenge is particularly highlighted within neuroscience where considerable effort in the field has focused on studying the structural and functional relationships within complete neurovascular units in the living brain. Many existing functional optical techniques are limited in meeting this challenge by their imaging geometries, light source(s), and/or hardware implementations. In this thesis we describe the design, construction, and application of novel 2D and 3D optical imaging systems to address this central challenge with a specific focus on functional neuroimaging applications. The 2D system is an ultra-fast, multispectral, wide-field imaging system capable of imaging 7.5 times faster than existing technologies. Its camera-first design allows for the fastest possible image acquisition rates because it is not limited by synchronization challenges that have hindered previous multispectral systems. We present the development of this system from a bench top instrument to a portable, low-cost, modular, open source, laptop based instrument. The constructed systems can acquire multispectral images at >75 frames per second with image resolutions up to 512 x 512 pixels. This increased speed means that spectral analysis more accurately reflects the instantaneous state of tissues and allows for significantly improved tracking of moving objects. We describe 3 quantitative applications of these systems to in-vivo research and clinical studies of cortical imaging and calcium signaling in stem cells. The design and source code of the portable system was released to the greater scientific community to help make high-speed, multispectral imaging more accessible to a larger number of dynamic imaging applications, and to foster further development of the software package. The second system we developed is an entirely new, high-speed, 3D fluorescence microscopy platform called Laser-Scanning Intersecting Plane Tomography (L-SIPT). L-SIPT uses a novel combination of light-sheet illumination and off-axis detection to provide en-face 3D imaging of samples. L-SIPT allows samples to move freely in their native environments, enabling a range of experiments not possible with previous 3D optical imaging techniques. The constructed system is capable of acquiring 3D images at rates >20 volumes per second (VPS) with volume resolutions of 1400 x 50 x 150 pixels, over a 200 fold increase over conventional laser scanning microscopes. Spatial resolution is set by choice of telescope design. We developed custom opto-mechanical components, computer raytracing models to guide system design and to characterize the technique's fundamental resolution limits, and phantoms and biological samples to refine the system's performance capabilities. We describe initial applications development of the system to image freely moving, transgenic Drosophila Melanogaster larvae, 3D calcium signaling and hemodynamics in transgenic and exogenously labeled rodent cortex in-vivo, and 3D calcium signaling in acute transgenic rodent cortical brain slices in-vitro

Hyperspectral Imaging for Landmine Detection

This PhD thesis aims at investigating the possibility to detect landmines using hyperspectral imaging. Using this technology, we are able to acquire at each pixel of the image spectral data in hundreds of wavelengths. So, at each pixel we obtain a reflectance spectrum that is used as fingerprint to identify the materials in each pixel, and mainly in our project help us to detect the presence of landmines. The proposed process works as follows: a preconfigured drone (hexarotor or octorotor) will carry the hyperspectral camera. This programmed drone is responsible of flying over the contaminated area in order to take images from a safe distance. Various image processing techniques will be used to treat the image in order to isolate the landmine from the surrounding. Once the presence of a mine or explosives is suspected, an alarm signal is sent to the base station giving information about the type of the mine, its location and the clear path that could be taken by the mine removal team in order to disarm the mine. This technology has advantages over the actually used techniques: • It is safer because it limits the need of humans in the searching process and gives the opportunity to the demining team to detect the mines while they are in a safe region. • It is faster. A larger area could be cleared in a single day by comparison with demining techniques • This technique can be used to detect at the same time objects other than mines such oil or minerals. First, a presentation of the problem of landmines that is expanding worldwide referring to some statistics from the UN organizations is provided. In addition, a brief presentation of different types of landmines is shown. Unfortunately, new landmines are well camouflaged and are mainly made of plastic in order to make their detection using metal detectors harder. A summary of all landmine detection techniques is shown to give an idea about the advantages and disadvantages of each technique. In this work, we give an overview of different projects that worked on the detection of landmines using hyperspectral imaging. We will show the main results achieved in this field and future work to be done in order to make this technology effective. Moreover, we worked on different target detection algorithms in order to achieve high probability of detection with low false alarm rate. We tested different statistical and linear unmixing based methods. In addition, we introduced the use of radial basis function neural networks in order to detect landmines at subpixel level. A comparative study between different detection methods will be shown in the thesis. A study of the effect of dimensionality reduction using principal component analysis prior to classification is also provided. The study shows the dependency between the two steps (feature extraction and target detection). The selection of target detection algorithm will define if feature extraction in previous phase is necessary. A field experiment has been done in order to study how the spectral signature of landmine will change depending on the environment in which the mine is planted. For this, we acquired the spectral signature of 6 types of landmines in different conditions: in Lab where specific source of light is used; in field where mines are covered by grass; and when mines are buried in soil. The results of this experiment are very interesting. The signature of two types of landmines are used in the simulations. They are a database necessary for supervised detection of landmines. Also we extracted some spectral characteristics of landmines that would help us to distinguish mines from background

Transient absorption imaging of hemeprotein in fresh muscle fibers

2022 Summer.Includes bibliographical references.Mitochondrial diseases affect 1 in 4000 individuals in the U.S. among adults and children of all races and genders. Nevertheless, these diseases are hard to diagnose because they affect each person differently. Meanwhile the gold standard diagnosis methods are usually invasive and time- consuming. Therefore, a non-invasive and in-vivo diagnosis method is highly demanded in this area. Our goal is to develop a non-invasive diagnosis method based on the endogenous nonlinear optical effect of the live tissues. Mitochondrial disease is frequently the result of a defective electron transport chain (ETC). Our goal is to develop a non-invasive way to measure redox within the ETC, specifically, of cytochromes. Cytochromes are iron porphyrins that are essential to the ETC. Their redox states can indicate cellular oxygen consumption and mitochondrial ATP production. So being able to differentiate the redox states of cytochromes will offer us a method to characterize mitochondrial function. Meanwhile, Chergui's group found out that the two redox states of cytochrome c have different pump-probe spectroscopic responses, meaning that the transient absorption (TA) decay lifetime can be a potential molecular contrast for cytochrome redox state discrimination. Their research leads us to utilize the pump-probe spectroscopic idea to develop a time-resolved optical microscopic method to differentiate not only cytochromes from other chemical compounds but also reduced cytochromes from oxidized ones. This dissertation describes groundbreaking experiments where transient absorption is used to reveal excited-state lifetime differences between healthy controls and an animal model of mitochondrial disease, in addition to differences between reduced and oxidized ETC in isolated mitochondria and fresh preparations of muscle fibers. For our initial experiments, we built a pump-probe microscopic system with a fiber laser source, producing 530nm pump and 490nm probe using a 3.5kHz laser scanning rate. The pulse durations of pump and probe are both 800fs. For the preliminary results, we have successfully achieved TA decay contrast between reduced and oxidized cytochromes in solution form. Then we have achieved SNR enhanced pump-probe image of BGO crystal particles with the help of the software- based adaptive filter noise canceling method. We also have installed a FPGA-based adaptive filter to enhance the pump-probe signals of the electrophoresis gels that contain different mitochondrial respiratory chain supercomplexes. However, because the noise floor was still 30 dB higher than shot noise limit, cytochrome imaging in live tissues was still problematic. We then built another pump-probe microscope with a solid- state ultrafast laser source. In that way, we do not need to worry about laser relative intensity noise (RIN) anymore, since the noise floor of the solid-state laser source can reach the shot noise limit at MHz region. One other advantage of the new laser source is that it can provide one tunable laser output that can be directly converted to the probe pulse with tunable center wavelength. Its tunability can cover the entire visible spectrum. We realized a pump-probe microscopy with a 520nm pump pulse and a tunable probe pulse. The tunability on the probe arm allows us to explore better pump-probe contrast between two redox states. What's more, I will introduce my preliminary results of utilizing supercontinuum generation in a photonic crystal fiber (PCF) to realize tunability on pump wavelength. In that way, more possibilities will be unlocked. And the hyperspectral pump-probe microscope will be able to distinguish more molecules