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

Beam halo and bunch purity monitoring

Beam halo measurements imply measurements of beam profiles with a very high dynamic range; in transverse and also longitudinal planes. This lesson gives an overview of high dynamic range instruments for beam halo measurements. In addition halo definitions and quantifications in view of beam instrumentation are discussed.Comment: 29 pages, contribution to the CAS - CERN Accelerator School: Beam Instrumentation, 2-15 June 2018, Tuusula, Finland. arXiv admin note: text overlap with arXiv:1303.676

A Point-of-Care Device for Molecular Diagnosis Based on CMOS SPAD Detectors with Integrated Microfluidics

We describe the integration of techniques and technologies to develop a Point-of-Care for molecular diagnosis PoC-MD, based on a fluorescence lifetime measurement. Our PoC-MD is a low-cost, simple, fast, and easy-to-use general-purpose platform, aimed at carrying out fast diagnostics test through label detection of a variety of biomarkers. It is based on a 1-D array of 10 ultra-sensitive Single-Photon Avalanche Diode (SPAD) detectors made in a 0.18 μm High-Voltage Complementary Metal Oxide Semiconductor (HV-CMOS) technology. A custom microfluidic polydimethylsiloxane cartridge to insert the sample is straightforwardly positioned on top of the SPAD array without any alignment procedure with the SPAD array. Moreover, the proximity between the sample and the gate-operated SPAD sensor makes unnecessary any lens or optical filters to detect the fluorescence for long lifetime fluorescent dyes, such as quantum dots. Additionally, the use of a low-cost laser diode as pulsed excitation source and a Field-Programmable Gate Array (FPGA) to implement the control and processing electronics, makes the device flexible and easy to adapt to the target label molecule by only changing the laser diode. Using this device, reliable and sensitive real-time proof-of-concept fluorescence lifetime measurement of quantum dot QdotTM 605 streptavidin conjugate is demonstrated

Instrumentation for fluorescence lifetime measurement using photon counting

We describe the evolution of HORIBA Jobin Yvon IBH Ltd, and its time-correlated single-photon counting (TCSPC) products, from university research beginnings through to its present place as a market leader in fluorescence lifetime spectroscopy. The company philosophy is to ensure leading-edge research capabilities continue to be incorporated into instruments in order to meet the needs of the diverse range of customer applications, which span a multitude of scientific and engineering disciplines. We illustrate some of the range of activities of a scientific instrument company in meeting this goal and highlight by way of an exemplar the performance of the versatile DeltaFlex instrument in measuring fluorescence lifetimes. This includes resolving fluorescence lifetimes down to 5 ps, as frequently observed in energy transfer, nanoparticle metrology with sub-nanometre resolution and measuring a fluorescence lifetime in as little as 60 μs for the study of transient species and kinetics

Capillary and microchip gel electrophoresis using multiplexed fluorescence detection with both time-resolved and spectral-discrimination capabilities: applications in DNA sequencing using near-infrared fluorescence

Increasing the information content obtainable from a single assay and system miniaturization has continued to be important research areas in analytical chemistry. The research presented in this dissertation involves the development of a two-color, time-resolved fluorescence microscope for the acquisition of both steady-state and time-resolved data during capillary and microchip electrophoresis. The utility of this hybrid fluorescence detector has been demonstrated by applying it to DNA sequencing applications. Coupling color discrimination with time-resolved fluorescence offers increased multiplexing capabilities because the lifetime data adds another layer of information. An optical fiber-based fluorescence microscope was constructed, which utilized fluorescence in near-IR region, greatly simplifying the hardware and allowing superior system sensitivity. Time-resolved data was processed using electronics configured in a time-correlated single photon counting format. Cross-talk between color channels was successfully eliminated by utilizing the intrinsic time-resolved capability associated with the detector. The two-color, time-resolved microscope was first coupled to a single capillary and carried out two-color, two-lifetime sequencing of an M13 template, achieving a read length of 650 bps at a calling accuracy of 95.1%. The feasibility of using this microscope with microchips (glass-based chips) for sequencing was then demonstrated. Results from capillaries and microchips were compared, with the microchips providing faster analysis and adequate electrophoretic performance. Lifetimes of a set of fluorescent dyes were determined with favorable precision, in spite of the low loading levels associated with the microchips. The sequencing products were required to be purified and concentrated prior to electrophoretic sorting to improve data quality. PMMA-based microchips for DNA sequencing application were evaluated. The microchips were produced from thermo plastics, which allowed rapid and inexpensive production of microstructures with high aspect ratios. It was concluded that surface coating was needed on the polymer chips in order to achieve single-base resolution required for DNA sequencing. The capability of the two-color time-resolved microscope operated in a scanning mode was further explored. The successful construction of the scanner allows scanning of multi-channel microchips for high throughput processing

Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research

Single Photon Counting Detectors for Low Light Level Imaging Applications

This dissertation presents the current state-of-the-art of semiconductor-based photon counting detector technologies. HgCdTe linear-mode avalanche photodiodes (LM-APDs), silicon Geiger-mode avalanche photodiodes (GM-APDs), and electron-multiplying CCDs (EMCCDs) are compared via their present and future performance in various astronomy applications. LM-APDs are studied in theory, based on work done at the University of Hawaii. EMCCDs are studied in theory and experimentally, with a device at NASA\u27s Jet Propulsion Lab. The emphasis of the research is on GM-APD imaging arrays, developed at MIT Lincoln Laboratory and tested at the RIT Center for Detectors. The GM-APD research includes a theoretical analysis of SNR and various performance metrics, including dark count rate, afterpulsing, photon detection efficiency, and intrapixel sensitivity. The effects of radiation damage on the GM-APD were also characterized by introducing a cumulative dose of 50 krad(Si) via 60 MeV protons. Extensive development of Monte Carlo simulations and practical observation simulations was completed, including simulated astronomical imaging and adaptive optics wavefront sensing. Based on theoretical models and experimental testing, both the current state-of-the-art performance and projected future performance of each detector are compared for various applications. LM-APD performance is currently not competitive with other photon counting technologies, and are left out of the application-based comparisons. In the current state-of-the-art, EMCCDs in photon counting mode out-perform GM-APDs for long exposure scenarios, though GM-APDs are better for short exposure scenarios (fast readout) due to clock-induced-charge (CIC) in EMCCDs. In the long term, small improvements in GM-APD dark current will make them superior in both long and short exposure scenarios for extremely low flux. The efficiency of GM-APDs will likely always be less than EMCCDs, however, which is particularly disadvantageous for moderate to high flux rates where dark noise and CIC are insignificant noise sources. Research into decreasing the dark count rate of GM-APDs will lead to development of imaging arrays that are competitive for low light level imaging and spectroscopy applications in the near future

Probing Molecular Stoichiometry by Photon Antibunching and Nanofluidics Assisted Imaging in Solution

A mechanistic understanding of biological function requires a quantitative determination of macromolecular subunit architecture and interaction. Optical microscopy and spectroscopy provide a noninvasive method to characterize the stoichiometric ratios of molecular complexes. Though target-bound fluorescence labeling techniques can help to detect single molecules, counting molecules in a molecular complex remains challenging. In solution, diffusion limits the observation times of single molecules and, thus reduces the number of detectable photons. Current methods have limited resolving power or are constrained by a complex experimental configuration. Therefore, they are not able to precisely quantify the number of labeled fluorophores. In this dissertation, I first explore the ability of photon antibunching to probe molecular stoichiometry in solution. The underlying theoretical model is elucidated and subsequently applied to samples of different labeling stoichiometry. It enables determining the average number of emitters per molecular complex. In the second part of my thesis, to obtain the full distribution of species with a particular number of fluorescent labels, another method is developed. It is based on molecular brightness analysis using imaging-based photon counting histograms. This is assisted by a nanofluidic device that enables direct imaging of diffusing molecules with extended observation time. I performed a systematic study of the experimental conditions which guarantee an optimal performance of this method. The capability of correctly determining distributions of stoichiometries of molecular mixtures is verified by both simulation and measurements of small molecules. The nanofluidics system allows both single-molecule detection and manipulation under microscopic imaging, which is simple and implementation-friendly

Nonlinear optical signals and spectroscopy with quantum light

Conventional nonlinear spectroscopy uses classical light to detect matter properties through the variation of its response with frequencies or time delays. Quantum light opens up new avenues for spectroscopy by utilizing parameters of the quantum state of light as novel control knobs and through the variation of photon statistics by coupling to matter. We present an intuitive diagrammatic approach for calculating ultrafast spectroscopy signals induced by quantum light, focusing on applications involving entangled photons with nonclassical bandwidth properties - known as "time-energy entanglement". Nonlinear optical signals induced by quantized light fields are expressed using time ordered multipoint correlation functions of superoperators. These are different from Glauber's g- functions for photon counting which use normally ordered products of ordinary operators. Entangled photon pairs are not subjected to the classical Fourier limitations on the joint temporal and spectral resolution. After a brief survey of properties of entangled photon pairs relevant to their spectroscopic applications, different optical signals, and photon counting setups are discussed and illustrated for simple multi-level model systems