Emerging Optical Techniques for Detection of Oral, Cervical and Anal Cancer in Low-Resource Settings

Cancers of the oral and anogenital regions are a growing global health problem that disproportionately impact women and men living in developing countries. The high death rate in developing countries is largely due to the fact that these countries do not have the appropriate medical infrastructure and resources to support the organized screening and diagnostic programs that are available in the developed world. Emerging optical diagnostics techniques, such as optical spectroscopy, reflectance imaging, and fluorescence imaging, are noninvasive techniques that are sensitive to multiple cancer biomarkers and have shown the potential as a cost–effective and fast tool for diagnosis of early precancerous changes in the cervix, oral cavity and anus. This paper provides a review of current strategies for prevention, screening and diagnostic tests of oral, cervical and anal cancers and development in optical diagnostic techniques that could potentially be used to improve current practice in resource–limited settings

A portable device for time-resolved fluorescence based on an array of CMOS SPADs with integrated microfluidics

[eng] Traditionally, molecular analysis is performed in laboratories equipped with desktop instruments operated by specialized technicians. This paradigm has been changing in recent decades, as biosensor technology has become as accurate as desktop instruments, providing results in much shorter periods and miniaturizing the instrumentation, moving the diagnostic tests gradually out of the central laboratory. However, despite the inherent advantages of time-resolved fluorescence spectroscopy applied to molecular diagnosis, it is only in the last decade that POC (Point Of Care) devices have begun to be developed based on the detection of fluorescence, due to the challenge of developing high-performance, portable and low-cost spectroscopic sensors. This thesis presents the development of a compact, robust and low-cost system for molecular diagnosis based on time-resolved fluorescence spectroscopy, which serves as a general-purpose platform for the optical detection of a variety of biomarkers, bridging the gap between the laboratory and the POC of the fluorescence lifetime based bioassays. In particular, two systems with different levels of integration have been developed that combine a one-dimensional array of SPAD (Single-Photon Avalanch Diode) pixels capable of detecting a single photon, with an interchangeable microfluidic cartridge used to insert the sample and a laser diode Pulsed low-cost UV as a source of excitation. The contact-oriented design of the binomial formed by the sensor and the microfluidic, together with the timed operation of the sensors, makes it possible to dispense with the use of lenses and filters. In turn, custom packaging of the sensor chip allows the microfluidic cartridge to be positioned directly on the sensor array without any alignment procedure. Both systems have been validated, determining the decomposition time of quantum dots in 20 nl of solution for different concentrations, emulating a molecular test in a POC device.[cat] Tradicionalment, l'anàlisi molecular es realitza en laboratoris equipats amb instruments de sobretaula operats per tècnics especialitzats. Aquest paradigma ha anat canviant en les últimes dècades, a mesura que la tecnologia de biosensor s'ha tornat tan precisa com els instruments de sobretaula, proporcionant resultats en períodes molt més curts de temps i miniaturitzant la instrumentació, permetent així, traslladar gradualment les proves de diagnòstic fora de laboratori central. No obstant això i malgrat els avantatges inherents de l'espectroscòpia de fluorescència resolta en el temps aplicada a la diagnosi molecular, no ha estat fins a l'última dècada que s'han començat a desenvolupar dispositius POC (Point Of Care) basats en la detecció de la fluorescència, degut al desafiament que suposa el desenvolupament de sensors espectroscòpics d'alt rendiment, portàtils i de baix cost. Aquesta tesi presenta el desenvolupament d'un sistema compacte, robust i de baix cost per al diagnòstic molecular basat en l'espectroscòpia de fluorescència resolta en el temps, que serveixi com a plataforma d'ús general per a la detecció òptica d'una varietat de biomarcadors, tancant la bretxa entre el laboratori i el POC dels bioassaigs basats en l'anàlisi de la pèrdua de la fluorescència. En particular, s'han desenvolupat dos sistemes amb diferents nivells d'integració que combinen una matriu unidimensional de píxels SPAD (Single-Photon Avalanch Diode) capaços de detectar un sol fotó, amb un cartutx microfluídic intercanviable emprat per inserir la mostra, així com un díode làser UV premut de baix cost com a font d'excitació. El disseny orientat a la detecció per contacte de l'binomi format pel sensor i la microfluídica, juntament amb l'operació temporitzada dels sensors, permet prescindir de l'ús de lents i filtres. Al seu torn, l'empaquetat a mida de l'xip sensor permet posicionar el cartutx microfluídic directament sobre la matriu de sensors sense cap procediment d'alineament. Tots dos sistemes han estat validats determinant el temps de descomposició de "quantum dots" en 20 nl de solució per a diferents concentracions, emulant així un assaig molecular en un dispositiu POC

Diagnostic Biosensors for Detection of Blood-Derived Biomarkers

Standard diagnostic tools used from patient samples, specifically from blood draws, require specialized equipment, personnel, and facilities. Conventional techniques can often be very laborious and time consuming due to required sample preparation. The evident delay from sample collection to a patient’s result immensely impacts their outcome. The aims of this research are to design diagnostic biosensors that decrease time-to-results, minimize reagent and sample handling, and incorporate automated simple optical transduction and user interfaces for the detection of blood-derived biomarkers. Specifically, four biosensing detection mechanisms performed on 3 different point-of-care platforms will be discussed. First is a static loop-mediated isothermal amplification (LAMP) of nucleic acid aqueous droplet on a silicone chip platform immersed in mineral oil. The target-of-interest is a nucleic acid sequence as a biomarker for antibiotic resistant bacteria. The biosensing technique used related changes in interfacial tension (IFT) at the water-oil interface by measuring the change in contact angle (geometrical-effects) over time. Initially the system was characterized as a linear response in relation to concentration of bacteria in a buffer system down to the limit of detection (LOD) of 100 CFU per uL. Subsequently, with the addition of bacterial infected blood sample models, the system became a binary assay (i.e. yes or no) as low as 10 CFU per uL within 10 min of reaction. Secondly, a two-layered, paper microfluidic chip was utilized to quantify cancer cells from a buffy coat sample matrix by two detection mechanisms: 1) on-chip particle enumeration via smartphone microscope and 2) capillary flow dynamics via smartphone video processing. The assay resulted in a LOD as low as 1 cell per uL for the on-chip imaging aspect of platform and 0.1 cell per uL for the capillary flow analysis within 13 to 22s post application of blood sample. Lastly, the same concepts previously described in the first platform utilizes changes in IFT due to amplicon presence in an aqueous solution immersed in mineral oil. An emulsion LAMP platform was investigated to determine the relation between angle-dependent light scatter intensity (based off Mie scatter theory) and nucleic acid amplification progression. The phenomenon attributing to changes in light scatter intensities is due to the interfacial changes occurring in the emulsion droplets, where amplicon amount increases the IFT decreases, resulting in smaller diameter emulsions. Changes in light scatter intensity within 3 min of the reaction shows statistical difference in comparison to no target control (NTC) for 10^3 CFU per uL of bacteria dosed into aqueous sample. These four detection mechanisms and three platforms offer but a few alternatives as biosensing methods for blood-derived diagnostic biosensors

Beyond solid-state lighting: Miniaturization, hybrid integration, and applications og GaN nano- and micro-LEDs

Gallium Nitride (GaN) light-emitting-diode (LED) technology has been the revolution in modern lighting. In the last decade, a huge global market of efficient, long-lasting and ubiquitous white light sources has developed around the inception of the Nobel-price-winning blue GaN LEDs. Today GaN optoelectronics is developing beyond lighting, leading to new and innovative devices, e.g. for micro-displays, being the core technology for future augmented reality and visualization, as well as point light sources for optical excitation in communications, imaging, and sensing. This explosion of applications is driven by two main directions: the ability to produce very small GaN LEDs (microLEDs and nanoLEDs) with high efficiency and across large areas, in combination with the possibility to merge optoelectronic-grade GaN microLEDs with silicon microelectronics in a fully hybrid approach. GaN LED technology today is even spreading into the realm of display technology, which has been occupied by organic LED (OLED) and liquid crystal display (LCD) for decades. In this review, the technological transition towards GaN micro- and nanodevices beyond lighting is discussed including an up-to-date overview on the state of the art

Microchip imaging cytometer: making healthcare available, accessible, and affordable

The Microchip Imaging Cytometer (MIC) is a class of integrated point-of-care detection systems based on the combination of optical microscopy and flow cytometry. MIC devices have the attributes of portability, cost-effectiveness, and adaptability while providing quantitative measurements to meet the needs of laboratory testing in a variety of healthcare settings. Based on the use of microfluidic chips, MIC requires less sample and can complete sample preparation automatically. Therefore, they can provide quantitative testing results simply using a finger prick specimen. The decreased reagent consumption and reduced form factor also help improve the accessibility and affordability of healthcare services in remote and resource-limited settings. In this article, we review recent developments of the Microchip Imaging Cytometer from the following aspects: clinical applications, microfluidic chip integration, imaging optics, and image acquisition. Following, we provide an outlook of the field and remark on promising technologies that may enable significant progress in the near future

Pulsed Laser Manipulation of Cells Decorated by Plasmonic Nanoparticles

Au cours des dernières décennies, la thérapie cellulaire a suscité un intérêt considérable dans les domaines de la recherche biomédicale et théranostique. Une large gamme d’applications biomédicales a obtenu des avantages fructueux par une manipulation directe de cellules (cellules seuls ou groupes de cellules individuelles) en contrôlant la concentration de nanoparticules plasmoniques fonctionnelles et de lasers à impulsions. Cependant, tous ces protocoles de transfection, accompagnés d’une efficacité et d’une toxicité de transfection cellulaire variées, sont applicables pour une condition spécifique. Ainsi, l’optoporation doit être explorée, optimisée et généralisée pour un large éventail de thérapies cellulaires délicates et complexes par le laser. Un suivi en temps réel des cellules optoporées révèle les mécanismes impliqués dans une transfection réussie sans induire de cytotoxicité. Dans cette thèse, le laser pulsé nanoseconde (532 nm) utilisé pour optoporer une seule cellule cancéreuse humaine du sein (MDA-MB-231) montre clairement les effets des fluences du laser pulsé. L’optoporation intériorise les molécules exogènes (Iodure de Propidium) dans l’intervalle fonctionnel compris entre 0.3 et 0.7 J/cm2 sans provoquer d’effet secondaire (confirmé par Calcein AcetoxyMethyl). La position du faisceau détermine clairement dans quels compartiments subcellulaires les molécules exogènes à intérioriser avec précision. Le faisceau laser focalisé près du noyau dirige intensément l’Iodure de Propidium (PI) pour qu’il réagisse avec les nucléotides cellulaires, alors que pour le faisceau focalisé loin du noyau, le PI se déverse à peine dans le site d’action. Ce protocole d’optoporation, indiquant le rôle critique des nanoparticules plasmoniques liées, nécessite un faisceau laser dirigé vers la position de la nanoparticule plasmonique sur la membrane cellulaire. L’éclairage latéral par LED développé ici visualise simplement la nanoparticule plasmonique liée sur la membrane cellulaire dans une grande zone. Par la suite, un laser femtoseconde dans le proche infrarouge (800 nm) est également utilisé pour optoporer des cellules de lymphocyte T humaines supersensibles (Jurkat) avec un taux de survie négligeable après une exposition de courte durée à une fluence relativement élevée (252 mJ/cm2). En explorant plusieurs fluences laser et durées d’irradiation, nous obtenons donc une gamme applicable de fluences laser (63 à 71 mJ/cm2) et de temps d’irradiation (10 ms) pour l’optoporation de cellules de Jurkat liées à des nanoparticules plasmoniques sans réduire leur viabilité cellulaire. Ces résultats fondamentaux indiquent comment effectuer une optoporation réussie en ajustant les paramètres du laser (fluence, durée d’irradiation, position, etc.) sur différentes lignées cellulaires afin d’atteindre une haute transfection pour une large gamme d’applications biomédicales.----------ABSTRACT In the last decades, cell therapy has attracted tremendous interests in biomedical and theranostic research fields. A wide range of biomedical applications has gained fruitful benefits by a direct manipulation of cells (whether single cell or bunch of individual cells) by controlling the concentration of functional plasmonic nanoparticles and pulsed lasers. However, all these transfection protocols, accompanied by a varied cellular transfection efficiency and toxicity, are applicable for a specific condition. Thus, the optoporation needs to be explored, optimized, and generalized for a broad range of delicate and complex laser mediated cell therapies. A real-time monitoring of the optoporated cells reveals mechanisms involved in a successful transfection without inducing cytotoxicity. In this thesis, the nanosecond pulsed laser (532 nm) used to optoporate a single adherent human breast cancer cell (MDA-MB-231) clearly shows effects of the pulsed laser fluences. The optoporation internalizes the exogenous molecules (Propidium Iodide) at the functional range between 0.3 to 0.7 J/cm2 without causing a side effect (confirmed by Calcein AcetoxyMethyl). The beam pointing location clearly determines in which subcellular compartments the exogenous molecules to be internalized precisely. The laser beam localized close to the nucleus intensively directs the Propidium Iodide there to react with cellular nucleotides, whereas the beam far away from the nucleus barely fluxes into the site of action. This optoporation protocol, indicates the critical role of the bound plasmonic nanoparticles, is required a laser beam to be directed to the position of the plasmonic nanoparticle on the cellular membrane. The lateral LED illumination developed here simply visualizes the bound plasmonic nanoparticle on the cell membrane in a large area. Afterward, near-infrared femtosecond laser (800 nm) is also employed to optoporate supersensitive human T lymphocyte cells (Jurkat) with a negligible survival rate after a short time exposure to a relatively high fluence (252 mJ/cm2). Exploring several laser fluences and irradiation durations, we therefore obtain an applicable range of laser fluences (63 to 71 mJ/cm2) and irradiation time (10 ms) for the optoporation of plasmonic nanoparticles bound Jurkat cells without reducing their cell viability. These fundamental results indicate how to perform a successful optoporation by adjusting laser parameters (i.e., fluence, irradiation duration, position, etc.) on different cell lines in order to achieve high transfection for a wide range of biomedical applications

Intraoperative Fluorescence Imaging for Personalized Brain Tumor Resection: Current State and Future Directions

abstract: Introduction: Fluorescence-guided surgery is one of the rapidly emerging methods of surgical “theranostics.” In this review, we summarize current fluorescence techniques used in neurosurgical practice for brain tumor patients as well as future applications of recent laboratory and translational studies. Methods: Review of the literature. Results: A wide spectrum of fluorophores that have been tested for brain surgery is reviewed. Beginning with a fluorescein sodium application in 1948 by Moore, fluorescence-guided brain tumor surgery is either routinely applied in some centers or is under active study in clinical trials. Besides the trinity of commonly used drugs (fluorescein sodium, 5-aminolevulinic acid, and indocyanine green), less studied fluorescent stains, such as tetracyclines, cancer-selective alkylphosphocholine analogs, cresyl violet, acridine orange, and acriflavine, can be used for rapid tumor detection and pathological tissue examination. Other emerging agents, such as activity-based probes and targeted molecular probes that can provide biomolecular specificity for surgical visualization and treatment, are reviewed. Furthermore, we review available engineering and optical solutions for fluorescent surgical visualization. Instruments for fluorescent-guided surgery are divided into wide-field imaging systems and hand-held probes. Recent advancements in quantitative fluorescence-guided surgery are discussed. Conclusion: We are standing on the threshold of the era of marker-assisted tumor management. Innovations in the fields of surgical optics, computer image analysis, and molecular bioengineering are advancing fluorescence-guided tumor resection paradigms, leading to cell-level approaches to visualization and resection of brain tumors.View the article as published at http://journal.frontiersin.org/article/10.3389/fsurg.2016.00055/ful

Ultracompact fluorescence smartphone attachment using built-in optics for protoporphyrin-IX quantification in skin

Smartphone-based fluorescence imaging systems have the potential to provide convenient quantitative image guidance at the point of care. However, common approaches have required the addition of complex optical attachments, which reduce translation potential. In this study, a simple clip-on attachment appropriate for fluorescence imaging of protoporphyrin-IX (PpIX) in skin was designed using the built-in light source and ultrawide camera sensor of a smartphone. Software control for image acquisition and quantitative analysis was developed using the 10-bit video capability of the phone. Optical performance was characterized using PpIX in liquid tissue phantoms and endogenously produced PpIX in mice and human skin. The proposed system achieves a very compact form factor (\u3c30 cm3) and can be readily fabricated using widely available low-cost materials. The limit of detection of PpIX in optical phantoms was \u3c10 nM, with good signal linearity from 10 to 1000 nM (R2 \u3e0.99). Both murine and human skin imaging verified that in vivo PpIX fluorescence was detected within 1 hour of applying aminolevulinic acid (ALA) gel. This ultracompact handheld system for quantification of PpIX in skin is well-suited for dermatology clinical workflows. Due to its simplicity and form factor, the proposed system can be readily adapted for use with other smartphone devices and fluorescence imaging applications. Hardware design and software for the system is made freely available on GitHub (https://github.com/optmed/CompactFluorescenceCam)

Development of instrumentation for autofluorescence spectroscopy and its application to tissue autofluorescence studies and biomedical research

Autofluorescence spectroscopy is a promising non-invasive label-free approach to characterise biological samples and has shown potential to report structural and biochemical changes occurring in tissue owing to pathological transformations. This thesis discusses the development of compact and portable single point fibre-optic probe-based instrumentation for time-resolved spectrofluorometry, utilising spectrally resolved time-correlated single photon counting (TCSPC) detection and white light reflectometry. Following characterisation and validation, two of these instruments were deployed in clinical settings and their potential to report structural and metabolic alterations in tissue associated with osteoarthritis and heart disease was investigated. Osteoarthritis is a chronic and progressive disease of the joint characterised by irreversible destruction of articular cartilage for which there is no effective treatment. Working with the Kennedy Institute of Rheumatology, we investigated the potential of time-resolved autofluorescence spectroscopy as a diagnostic tool for early detection and monitoring of the progression of osteoarthritis. Our studies in enzymatically degenerated porcine and murine cartilage, which serve as models for osteoarthritis, suggest that autofluorescence lifetime is sensitive to disruption of the two major extracellular matrix components, aggrecan and collagen. Preliminary autofluorescence lifetime data were also obtained from ex vivo human tissue presenting naturally occurring osteoarthritis. Overall, our studies indicate that autofluorescence lifetime may offer a non-invasive readout to monitor cartilage matrix integrity that could contribute to future diagnosis of early cartilage defects as well as monitoring the efficacy of therapeutic agents. This thesis also explored the potential of time-resolved autofluorescence spectroscopy and steady-state white-light reflectometry of tissue to report structural and metabolic changes associated with cardiac disease, both ex vivo and in vivo, in collaboration with clinical colleagues from the National Heart and Lung Institute. Using a Langendorff rat model, the autofluorescence signature of cardiac tissue was investigated following different insults to the heart. We were able to correlate and translate results obtained from ex vivo Langendorff data to an in vivo myocardial infarction model in rats, where we report structural and functional alterations in the infarcted and remote myocardium at different stages following infarction. This investigation stimulated the development of a clinically viable instrument to be used in open-chest surgical procedures in humans, of which progress to date is described. 4 The impact of time-resolved autofluorescence spectroscopy for label-free diagnosis of diseased would be significantly enhanced if the cost of the instrumentation could be reduced below what is achievable with commercial TCSPC-based technology. The last part of this thesis concerns the development of compact and portable instrumentation utilising low-cost FPGA-based circuitry that can be used with laser diodes and photon-counting photomultipliers. A comprehensive description of this instrument is presented together with data from its application to both fluorescence lifetime standards and biological tissue. The lower potential cost of this instrument could enhance the potential of autofluorescence lifetime metrology for commercial development and clinical deployment.Open Acces