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

Definition of a near real time microbiological monitor for space vehicles

Efforts to identify the ideal candidate to serve as the biological monitor on the space station Freedom are discussed. The literature review, the evaluation scheme, descriptions of candidate monitors, experimental studies, test beds, and culture techniques are discussed. Particular attention is given to descriptions of five candidate monitors or monitoring techniques: laser light scattering, primary fluorescence, secondary fluorescence, the volatile product detector, and the surface acoustic wave detector

Theoretical and Experimental Tools for Clinical Translation of Quantitative Tissue Optical Sensing.

Quantitative tissue optical spectroscopy has been considered as a promising method for clinical diagnosis, owing to its ability to non-invasively give an objective assessment of biological tissues at cellular and sub-cellular levels. In spite of recent advances in optics and the computational power, not many quantitative tissue optical sensing technologies have been translated into clinical practice. In order to translate this technology in the clinics, we need to further improve the technology. To name a few, we need accurate and rapid quantification method for a real-time diagnostic feedback. Next, we need computational methods for complex tissue-optics problems. Also, we need a novel approach in probe design for the inaccessible organs. This dissertation focuses on the development, verification and validation of theoretical (mathematical and computational) and experimental (instrumental) tool set to promote the translation of quantitative tissue optical spectroscopy into clinical diagnostic applications. For the mathematical tool, a direct-fit photon tissue interaction (DF-PTI) model that could rapidly and accurately extract the parameters associated biophysical features was developed and validated to characterize the precursor lesions of pancreatic cancer. A rapid scattering model on pancreatic tissue reflectance based on principal components analysis (PCA) results was also developed. The diagnostic capability of scattering properties obtained was demonstrated on an 18-patient data set using a rigorous statistical method, which implied the potential of reflectance spectroscopy for real-time detection of pancreatic cancer. For the computational tool, a ray-traced Monte Carlo (RTMC) simulation for the design of fluorescence spectroscopy or imaging system utilizing complex optics to probe turbid biological tissues was devised. This new method was verified computationally with epithelial tissue models and experimentally using tissue-simulating optical phantoms. For the instrumental tool, the design and development of minimally-invasive diagnostic technologies employing optoelectronic components were discussed. In this dissertation, we focused on detection of pancreatic cancer, which has the worst prognosis among other major cancers. Pancreatic tissues were employed as our model system to validate our developed tools. The developed technology and tools can be applied to a variety of other human tissue sites to help in the translation of quantitative tissue optical sensing in a clinical setting.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111401/1/paulslee_1.pd

Upconverting nanoparticles in ultrasensitive detection of cardiac troponin I

Development of high-sensitivity immunoassays is a continuous interest in medical diagnostics, especially in the case of such diseases where higher sensitivity analyte measurements improve the prognosis of treatment. One such analyte is cardiac troponin I, used for detection of cardiac events. One of the key factors determining immunoassay sensitivity is the reporter, which labels the analytes in the assays and produces the measurable signal. Upconverting nanoparticles (UCNPs) are promising candidates for reporters in sensitive immunoassays. Their unique ability to convert near-infrared light into higher energy visible light by stacking photons, producing emission exhibiting anti-Stokes shift. As no other natural materials are capable of the process, measurement of UCNPs can be designed to completely dismiss any background signal originating from autofluorescence. However, reaching the maximal sensitivity they theoretically enable has been hindered by their tendency to non-specifically bind to solid surfaces in assays and to each other, forming nanoparticle clusters of varying sizes. The extent of these tendencies has been linked to the surface chemistry of the UCNPs. The aim of this thesis was to study the surface chemistry of UCNPs and apply them as reporters in different immunoassay technologies for detection of cTnI. During the research, surface coating of UCNPs with poly(acrylic acid) was studied and successfully improved, leading to reduced non-specific binding and cluster formation tendency. The performance of the UCNPs with the novel surface was compared to other surface chemistry approaches in microtiter plate assays utilizing either analog or digital readout method, and a lateral flow assay. Another aim was to develop the used assay technologies utilizing upconversion to reach extreme sensitivities. Reagents and conditions in analog microtiter plate assay for cTnI were thoroughly investigated to fine-tune the performance, and the limit of detection (LoD) reached an unprecedented low value of 0.13 ng/L for an analog assay. In addition, a mechanical actuator for automation of a lateral flow assay for cTnI was fabricated via 3D-printing, and when combined with the improved UCNPs, an LoD of 1.5 ng/L was reached, bringing high-sensitivity pointof- care detection of cTnI a step closer to reality. KEYWORDS: cardiac biomarker, luminescence, nanoparticle monodispersityHerkkien immunomääritysmenetelmien kehitys on jatkuvan kiinnostuksen aiheena diagnostiikan tutkimuksessa. Herkät biomerkkiainetestit ovat haluttuja etenkin sydänkohtauksen kaltaisissa tiloissa, jossa merkkiaineen pitoisuus verenkierrossa kasvaa samalla, kun hoitoennuste heikkenee. Yksi merkittävimmistä immunomääritysten herkkyyteen vaikuttavista tekijöistä on määritysleima, joka sitoutuu analyyttiin ja tuottaa mitattavan signaalin. Käänteisviritteisten nanopartikkelien (engl. upconverting nanoparticles, UCNP) kyky muuntaa matalaenergistä viritysvaloa korkeaenergiseksi emissioksi tekee niistä lupaavan määritysleiman herkkiin immunomäärityksiin, koska UCNP:iden signaalin mittaus voidaan tehdä niin, ettei autofluoresenssista koituvaa taustasignaalia havaita, mahdollistaen äärimmäisen herkät immunomääritykset. UCNP:iden taipumus sitoutua epäspesifisesti erilaisiin pintoihin ja muodostaa erikokoisia kasaumia on hidastanut niiden käyttöönottoa herkkinä immunomääritysleimoina. Tämän ominaisuuden on osoitettu riippuvan voimakkaasti partikkelien pintakemiasta. Tämän tutkimuksen tavoite oli tutkia UCNP:iden pintakemiaa ja hyödyntää niitä määritysleimoina erilaisissa cTnI:tä havaitsevissa immunomäärityksissä. Tutkimuksessa selvitettiin UCNP:iden poly(akryylihappo) (PAA) -pinnoituksen onnistumiseen vaikuttavia tekijöitä ja onnistuttiin merkittävästi vähentämään PAA-pintaisten UCNPeiden epäspesifistä sitoutumista ja kasautumistaipumusta. Näitä UCNPleimoja verrattiin muulla tavoin pinnoitettuihin UCNP-leimoihin mikrotiitterilevypohjaisessa immunomäärityksessä käyttäen joko analogista tai digitaalista mittausmenetelmää, sekä lateraalivirtausmäärityksessä. Toinen tavoite oli kehittää käytettyjä määritysmenetelmiä äärimmäisten herkkyyksien saavuttamiseksi. Mikrotiitterilevymäärityksen reagenssit ja toteutusmenetelmä tutkittiin tarkoin ja hienosäädettiin. Tällä tekniikalla saavutettiin 0,13 ng/L havaitsemisherkkyys cTnI:lle, mikä on ennätyksellistä analogisissa mikrotiitterilevymäärityksissä. Lisäksi suunniteltiin mekaaninen 3D-tulostettu laite automatisoimaan cTnI:tä havaitseva lateraalivirtausmääritys. Yhdistettynä paranneltuihin PAA-UCNP:ihin, saavutettiin 1,5 ng/L havaitsemisherkkyys, mikä tuo äärimmäisen herkät cTnI:n vieritestausmenetelmät askeleen lähemmäs todellisuutta. ASIASANAT: sydänmerkkiaine, luminesenssi, nanopartikkelien yksittäisyy

Development of a Portable CMOS Time-Domain Fluorescence Lifetime Imager

Modern laboratory equipments to measure the excited-state lifetime of fluorophores usually include an expensive picosecond pulsed-laser excitation source, a fragile photomultiplier tube, and a large instrument body for optics. A portable and robust device to make fluorescence lifetime measurement in nanosecond scale is of great attraction for chemists and biologists. This dissertation reports the development of a portable LED time-domain fluorimeter from an all-solid-state discrete-component prototype to its advanced CMOS integrated circuit implementation. The motivation of the research is to develop a multiplexed fluorimeter for point-of-care diagnosis. Instruments developed by this novel method have higher fill factor, are more portable, and are fabricated at lower cost

Measurement techniques and instruments suitable for life-prediction testing of photovoltaic arrays

Array failure modes, relevant materials property changes, and primary degradation mechanisms are discussed as a prerequisite to identifying suitable measurement techniques and instruments. Candidate techniques and instruments are identified on the basis of extensive reviews of published and unpublished information. These methods are organized in six measurement categories - chemical, electrical, optical, thermal, mechanical, and other physicals. Using specified evaluation criteria, the most promising techniques and instruments for use in life prediction tests of arrays were selected

High-resolution fluorescence endomicroscopy for rapid evaluation of breast cancer margins

Breast cancer is a major public health problem world-wide and the second leading cause of cancer-related female deaths. Breast conserving surgery (BCS), in the form of wide local excision (WLE), allows complete tumour resection while maintaining acceptable cosmesis. It is the recommended treatment for a large number of patients with early stage disease or, in more advanced cases, following neoadjuvant chemotherapy. About 30% of patients undergoing BCS require one or more re-operative interventions, mainly due to the presence of positive margins. The standard of care for surgical margin assessment is post-operative examination of histopathological tissue sections. However, this process is invasive, introduces sampling errors and does not provide real-time assessment of the tumour status of radial margins. The objective of this thesis is to improve intra-operative assessment of margin status by performing optical biopsy in breast tissue. This thesis presents several technical and clinical developments related to confocal fluorescence endomicroscopy systems for real-time characterisation of different breast morphologies. The imaging systems discussed employ flexible fibre-bundle based imaging probes coupled to high-speed line-scan confocal microscope set-up. A preliminary study on 43 unfixed breast specimens describes the development and testing of line-scan confocal laser endomicroscope (LS-CLE) to image and classify different breast pathologies. LS-CLE is also demonstrated to assess the intra-operative tumour status of whole WLE specimens and surgical excisions with high diagnostic accuracy. A third study demonstrates the development and testing of a bespoke LS-CLE system with methylene blue (MB), an US Food and Drug Administration (FDA) approved fluorescent agent, and integration with robotic scanner to enable large-area in vivo imaging of breast cancer. The work also addresses three technical issues which limit existing fibre-bundle based fluorescence endomicroscopy systems: i) Restriction to use single fluorescence agent due to low-speed, single excitation and single fluorescence spectral band imaging systems; ii) Limited Field of view (FOV) of fibre-bundle endomicroscopes due to small size of the fibre tip and iii) Limited spatial resolution of fibre-bundle endomicroscopes due to the spacing between the individual fibres leading to fibre-pixelation effects. Details of design and development of a high-speed dual-wavelength LS-CLE system suitable for high-resolution multiplexed imaging are presented. Dual-wavelength imaging is achieved by sequentially switching between 488 nm and 660 nm laser sources for alternate frames, avoiding spectral bleed-through, and providing an effective frame rate of 60 Hz. A combination of hand-held or robotic scanning with real-time video mosaicking, is demonstrated to enable large-area imaging while still maintaining microscopic resolution. Finally, a miniaturised piezoelectric transducer-based fibre-shifting endomicroscope is developed to enhance the resolution over conventional fibre-bundle based imaging systems. The fibre-shifting endomicroscope provides a two-fold improvement in resolution and coupled to a high-speed LS-CLE scanning system, provides real-time imaging of biological samples at 30 fps. These investigations furthered the utility and applications of the fibre-bundle based fluorescence systems for rapid imaging and diagnosis of cancer margins.Open Acces

Real-Time acoustic emission monitoring of wear-out failure in sic power electronic devices during power cycling tests

C. Choe, C. Chen, S. Nagao and K. Suganuma, "Real-Time Acoustic Emission Monitoring of Wear-Out Failure in SiC Power Electronic Devices During Power Cycling Tests," in IEEE Transactions on Power Electronics, vol. 36, no. 4, pp. 4420-4428, April 2021, doi: 10.1109/TPEL.2020.3024986

High-precision fluorescence photometry for real-time biomarkers detection

Les derniers évènements planétaires et plus particulièrement l'avènement sans précédent du nouveau coronavirus augmente la demande pour des appareils de test à proximité du patient. Ceux-ci fonctionnent avec une batterie et peuvent identifier rapidement des biomarqueurs cibles. Pareils systèmes permettent aux utilisateurs, disposant de connaissances limitées en la matière, de réagir rapidement, par exemple dans la détection d'un cas positif de COVID-19. La mise en œuvre de l'élaboration d'un tel instrument est un projet multidisciplinaire impliquant notamment la conception de circuits intégrés, la programmation, la conception optique et la biologie, demandant tous une maîtrise pointue des détails. De plus, l'établissement des spécifications et des exigences pour mesurer avec précision les interactions lumière-échantillon s'additionnent au besoin d'expérience dans la conception et la fabrication de tels systèmes microélectriques personnalisés et nécessitent en elles-mêmes, une connaissance approfondie de la physique et des mathématiques. Ce projet vise donc à concevoir et à mettre en œuvre un appareil sans fil pour détecter rapidement des biomarqueurs impliqués dans des maladies infectieuses telles que le COVID-19 ou des types de cancers en milieu ambulatoire. Cette détection se fait grâce à des méthodes basées sur la fluorescence. La spectrophotométrie de fluorescence permet aux médecins d'identifier la présence de matériel génétique viral ou bactérien tel que l'ADN ou l'ARN et de les caractériser. Les appareils de paillasse sont énormes et gourmand énergétiquement tandis que les spectrophotomètres à fluorescence miniatuarisés disponibles dans le commerce sont confrontés à de nombreux défis. Ces appareils miniaturisés ont été découverts en tirant parti des diodes électroluminescentes (DEL) à semi-conducteurs peu coûteuses et de la technologie des circuits intégrés. Ces avantages aident les scientifiques à réduire les erreurs possibles, la consommation d'énergie et le coût du produit final utilisé par la population. Cependant, comme leurs homologues de paillasse, ces appareils POC doivent quantifier les concentrations en micro-volume d'analytes sur une large gamme de longueurs d'onde suivant le cadre d'une économie en ressources. Le microsystème envisagé bénéficie d'une approche de haute précision pour fabriquer une puce microélectronique CMOS. Ce procédé se fait de concert avec un boîtier personnalisé imprimé en 3D pour réaliser le spectrophotomètre à la fluorescence nécessaire à la détection quantitative d'analytes en microvolume. En ce qui a trait à la conception de circuits, une nouvelle technique de mise à auto-zeroing est appliquée à l'amplificateur central, celui-ci étant linéarisé avec des techniques de recyclage et de polarisation adaptative. Cet amplificateur central est entièrement différentiel et est utilisé dans un amplificateur à verrouillage pour récupérer le signal d'intérêt éclipsé par le bruit. De plus, l'augmentation de la sensibilité de l'appareil permet des mesures quantitatives avec des concentrations en micro-volume d'analytes ayant moins d'erreurs de prédiction de concentration. Cet avantage cumulé à une faible consommation d'énergie, un faible coût, de petites dimensions et un poids léger font de notre appareil une solution POC prometteuse dans le domaine de la spectrophotométrie de fluorescence. La validation de ce projet s'est fait en concevant, fabriquant et testant un prototype discret et sans fil. Son article de référence a été publié dans IEEE LSC 2018. Quant à la caractérisation et l'interprétation du prototype d'expériences in vitro à l'aide d'une interface MATLAB personnalisée, cet article a été publié dans IEEE Sensors journal (2021). Les circuits intégrés et les photodétecteurs ont été fabriqués ont été conçus et fabriqués par Cadence en 2019. Relativement aux solutions de circuit proposées, elles ont été fabriquées avec la technologie CMOS 180 nm et publiées lors de la conférence IEEE MWSCAS 2020. Tout comme cette dernière contribution, les expériences in vitro avec le dispositif proposé incluant la puce personnalisée et le boîtier imprimé en 3D ont été réalisés et les résultats électriques et optiques ont été soumis au IEEE Journal of Solid-State Circuits (JSSC 2022).The most recent and unprecedented experience of the novel coronavirus increases the demand for battery-operated near-patient testing devices that can rapidly identify the target biomarkers. Such systems enable end-users with limited resources to quickly get feedback on various medical tests, such as detecting positive COVID-19 cases. Implementing such a device is a multidisciplinary project dealing with multiple areas of expertise, including integrated circuit design, programming, optical design, and biology, each of which needs a firm grasp of details. Alongside the need for experience in designing and manufacturing custom microelectronic systems, establishing the specifications and requirements to precisely measure the light-sample interactions requires an in-depth knowledge of physics and mathematics. This project aims to design and implement a wireless point-of-care (POC) device to rapidly detect biomarkers involved in infectious diseases such as COVID-19 or different types of cancers in an ambulatory setting using fluorescence-based methods. Fluorescence spectrophotometry allows physicians to identify and characterize viral or bacterial genetic materials such as DNAs or RNAs. The benchtop devices that are currently available are bulky and power-hungry, whereas the commercially available miniaturized fluorescence spectrophotometers are facing many challenges. Many of these difficulties have been resolved in literature thanks to inexpensive semiconductor light-emitting diodes (LEDs) and integrated circuits technology. Such advantages aid scientists in decreasing the size, power consumption, and cost of the final product for end-users. However, like the benchtop counterparts, such POC devices must quantify micro-volume concentrations of analytes across a wide wave length range under an economy of resources. The envisioned microsystem benefits from a high-precision approach to fabricating a CMOS microelectronic chip combined with a custom 3D-printed housing. This implementation results in a fluorescence spectrophotometer for qualitative and quantitative detection of micro-volume analytes. In terms of circuit design, a novel switched-biasing ping-pong auto-zeroed technique is applied to the core amplifier, linearized with recycling and adaptive biasing techniques. The fully differential core amplifier is utilized within a lock-in amplifier to retrieve the signal of interest overshadowed by noise. Increasing the device's sensitivity allows quantitative measurements down to micro-volume concentrations of analytes with less concentration prediction error. Such an advantage, along with low-power consumption, low cost, low weight, and small dimensions, make our device a promising POC solution in the fluorescence spectrophotometry area. The approach of this project was validated by designing, fabricating, and testing a discrete and wireless prototype. Its conference paper was published in IEEE LSC 2018, and the prototype characterization and interpretation of in vitro experiments using a custom MATLAB interface were published in IEEE Sensors Journal (2021). The integrated circuits and photodetectors were designed and fabricated by the Cadence circuit design toolbox (2019). The proposed circuit solutions were fabricated with 180-nm CMOS technology and published at IEEE MWSCAS 2020 conference. As the last contribution, the in vitro experiments with the proposed device, including the custom chip and 3D-printed housing, were performed, and the electrical and optical results were submitted to the IEEE Journal of Solid-State Circuits (JSSC 2022)