Utilizing the Organizational Power of DNA Scaffolds for New Nanophotonic Applications

AbstractRapid development of DNA technology has provided a feasible route to creating nanoscale materials. DNA acts as a self‐assembled nanoscaffold capable of assuming any three‐dimensional shape. The ability to integrate dyes and new optical materials such as quantum dots and plasmonic nanoparticles precisely onto these architectures provides new ways to exploit their near‐ and far‐field interactions. A fundamental understanding of these optical processes will help drive development of next‐generation photonic nanomaterials. This review is focused on latest progress in DNA‐based photonic materials and highlights DNA scaffolds for rapidly assembling and prototyping nanoscale optical devices. Three areas are discussed including intrinsically active DNA structures displaying chiral properties, DNA scaffolds hosting plasmonic nanomaterials, and fluorophore‐labeled DNAs that engage in Förster resonance energy transfer and give rise to complex molecular photonic wires. An explanation of what is desired from these optical processes when harnessed sets the tone for what DNA scaffolds are providing toward each focus. Examples from the literature illustrate current progress along with a discussion of challenges to overcome for further improvements. Opportunities to integrate diverse classes of optically active molecules including light‐generating enzymes, fluorescent proteins, nanoclusters, and metal–chelates in new structural combinations on DNA scaffolds are also highlighted

Pursuing Excitonic Energy Transfer with Programmable DNA-Based Optical Breadboards

DNA nanotechnology has now enabled the self-assembly of almost any prescribed 3-dimensional nanoscale structure in large numbers and with high fidelity. These structures are also amenable to site-specific modification with a variety of small molecules ranging from drugs to reporter dyes. Beyond obvious application in biotechnology, such DNA structures are being pursued as programmable nanoscale optical breadboards where multiple different/identical fluorophores can be positioned with sub-nanometer resolution in a manner designed to allow them to engage in multistep excitonic energy-transfer (ET) via Förster resonance energy transfer (FRET) or other related processes. Not only is the ability to create such complex optical structures unique, more importantly, the ability to rapidly redesign and prototype almost all structural and optical analogues in a massively parallel format allows for deep insight into the underlying photophysical processes. Dynamic DNA structures further provide the unparalleled capability to reconfigure a DNA scaffold on the fly in situ and thus switch between ET pathways within a given assembly, actively change its properties, and even repeatedly toggle between two states such as on/off. Here, we review progress in developing these composite materials for potential applications that include artificial light harvesting, smart sensors, nanoactuators, optical barcoding, bioprobes, cryptography, computing, charge conversion, and theranostics to even new forms of optical data storage. Along with an introduction into the DNA scaffolding itself, the diverse fluorophores utilized in these structures, their incorporation chemistry, and the photophysical processes they are designed to exploit, we highlight the evolution of DNA architectures implemented in the pursuit of increased transfer efficiency and the key lessons about ET learned from each iteration. We also focus on recent and growing efforts to exploit DNA as a scaffold for assembling molecular dye aggregates that host delocalized excitons as a test bed for creating excitonic circuits and accessing other quantum-like optical phenomena. We conclude with an outlook on what is still required to transition these materials from a research pursuit to application specific prototypes and beyond

Multidimensional Fluorescence Imaging and Super-resolution Exploiting Ultrafast Laser and Supercontinuum Technology

This thesis centres on the development of multidimensional fluorescence imaging tools, with a particular emphasis on fluorescence lifetime imaging (FLIM) microscopy for application to biological research. The key aspects of this thesis are the development and application of tunable supercontinuum excitation sources based on supercontinuum generation in microstructured optical fibres and the development of stimulated emission depletion (STED) microscope capable of fluorescence lifetime imaging beyond the diffraction limit. The utility of FLIM for biological research is illustrated by examples of experimental studies of the molecular structure of sarcomeres in muscle fibres and of signalling at the immune synapse. The application of microstructured optical fibre to provide tunable supercontinuum excitation source for a range of FLIM microscopes is presented, including wide-field, Nipkow disk confocal and hyper-spectral line scanning FLIM microscopes. For the latter, a detailed description is provided of the supercontinuum source and semi-confocal line-scanning microscope configuration that realised multidimensional fluorescence imaging, resolving fluorescence images with respect to excitation and emission wavelength, fluorescence lifetime and three spatial dimensions. This included the first biological application of a fibre laser-pumped supercontinuum exploiting a tapered microstructured optical fibre that was able to generate a spectrally broad output extending to ~ 350 nm in the ultraviolet. The application of supercontinuum generation to the first super-resolved FLIM microscope is then described. This novel microscope exploited the concept of STED with a femtosecond mode-locked Ti:Sapphire laser providing a tunable excitation beam by pumping microstructured optical fibre for supercontinuum generation and directly providing the (longer wavelength) STED beam. This STED microscope was implemented in a commercial scanning confocal microscope to provide compatibility with standard biological imaging, and exploited digital holography using a spatial light modulator (SLM) to provide the appropriate phase manipulation for shaping the STED beam profile and to compensate for aberrations. The STED microscope was shown to be capable of recording super resolution in both the lateral and axial planes, according to the settings of the SLM

Fast fluorescence lifetime imaging and sensing via deep learning

Error on title page – year of award is 2023.Fluorescence lifetime imaging microscopy (FLIM) has become a valuable tool in diverse disciplines. This thesis presents deep learning (DL) approaches to addressing two major challenges in FLIM: slow and complex data analysis and the high photon budget for precisely quantifying the fluorescence lifetimes. DL's ability to extract high-dimensional features from data has revolutionized optical and biomedical imaging analysis. This thesis contributes several novel DL FLIM algorithms that significantly expand FLIM's scope. Firstly, a hardware-friendly pixel-wise DL algorithm is proposed for fast FLIM data analysis. The algorithm has a simple architecture yet can effectively resolve multi-exponential decay models. The calculation speed and accuracy outperform conventional methods significantly. Secondly, a DL algorithm is proposed to improve FLIM image spatial resolution, obtaining high-resolution (HR) fluorescence lifetime images from low-resolution (LR) images. A computational framework is developed to generate large-scale semi-synthetic FLIM datasets to address the challenge of the lack of sufficient high-quality FLIM datasets. This algorithm offers a practical approach to obtaining HR FLIM images quickly for FLIM systems. Thirdly, a DL algorithm is developed to analyze FLIM images with only a few photons per pixel, named Few-Photon Fluorescence Lifetime Imaging (FPFLI) algorithm. FPFLI uses spatial correlation and intensity information to robustly estimate the fluorescence lifetime images, pushing this photon budget to a record-low level of only a few photons per pixel. Finally, a time-resolved flow cytometry (TRFC) system is developed by integrating an advanced CMOS single-photon avalanche diode (SPAD) array and a DL processor. The SPAD array, using a parallel light detection scheme, shows an excellent photon-counting throughput. A quantized convolutional neural network (QCNN) algorithm is designed and implemented on a field-programmable gate array as an embedded processor. The processor resolves fluorescence lifetimes against disturbing noise, showing unparalleled high accuracy, fast analysis speed, and low power consumption.Fluorescence lifetime imaging microscopy (FLIM) has become a valuable tool in diverse disciplines. This thesis presents deep learning (DL) approaches to addressing two major challenges in FLIM: slow and complex data analysis and the high photon budget for precisely quantifying the fluorescence lifetimes. DL's ability to extract high-dimensional features from data has revolutionized optical and biomedical imaging analysis. This thesis contributes several novel DL FLIM algorithms that significantly expand FLIM's scope. Firstly, a hardware-friendly pixel-wise DL algorithm is proposed for fast FLIM data analysis. The algorithm has a simple architecture yet can effectively resolve multi-exponential decay models. The calculation speed and accuracy outperform conventional methods significantly. Secondly, a DL algorithm is proposed to improve FLIM image spatial resolution, obtaining high-resolution (HR) fluorescence lifetime images from low-resolution (LR) images. A computational framework is developed to generate large-scale semi-synthetic FLIM datasets to address the challenge of the lack of sufficient high-quality FLIM datasets. This algorithm offers a practical approach to obtaining HR FLIM images quickly for FLIM systems. Thirdly, a DL algorithm is developed to analyze FLIM images with only a few photons per pixel, named Few-Photon Fluorescence Lifetime Imaging (FPFLI) algorithm. FPFLI uses spatial correlation and intensity information to robustly estimate the fluorescence lifetime images, pushing this photon budget to a record-low level of only a few photons per pixel. Finally, a time-resolved flow cytometry (TRFC) system is developed by integrating an advanced CMOS single-photon avalanche diode (SPAD) array and a DL processor. The SPAD array, using a parallel light detection scheme, shows an excellent photon-counting throughput. A quantized convolutional neural network (QCNN) algorithm is designed and implemented on a field-programmable gate array as an embedded processor. The processor resolves fluorescence lifetimes against disturbing noise, showing unparalleled high accuracy, fast analysis speed, and low power consumption

The BrightEyes-TTM: an open-source time-tagging module for fluorescence lifetime imaging microscopy applications

The aim of this Ph.D. work is to reason and show how an open-source multi-channel and standalone time-tagging device was developed, validated and used in combination with a new generation of single-photon array detectors to pursue super-resolved time-resolved fluorescence lifetime imaging measurements. Within the compound of time-resolved fluorescence laser scanning microscopy (LSM) techniques, fluorescence lifetime imaging microscopy (FLIM) plays a relevant role in the life-sciences field, thanks to its ability of detecting functional changes within the cellular micro-environment. The recent advancements in photon detection technologies, such as the introduction of asynchronous read-out single-photon avalanche diode (SPAD) array detectors, allow to image a fluorescent sample with spatial resolution below the diffraction limit, at the same time, yield the possibility of accessing the single-photon information content allowing for time-resolved FLIM measurements. Thus, super-resolved FLIM experiments can be accomplished using SPAD array detectors in combination with pulsed laser sources and special data acquisition systems (DAQs), capable of handling a multiplicity of inputs and dealing with the single-photons readouts generated by SPAD array detectors. Nowadays, the commercial market lacks a true standalone, multi-channel, single-board, time-tagging and affordable DAQ device specifically designed for super-resolved FLIM experiments. Moreover, in the scientific community, no-efforts have been placed yet in building a device that can compensate such absence. That is why, within this Ph.D. project, an open-source and low-cost device, the so-called BrightEyes-TTM (time tagging module), was developed and validated both for fluorescence lifetime and time-resolved measurements in general. The BrightEyes-TTM belongs to a niche of DAQ devices called time-to-digital converters (TDCs). The field-gate programmable array (FPGA) technology was chosen for implementing the BrightEyes-TTM thanks to its reprogrammability and low cost features. The literature reports several different FPGA-based TDC architectures. Particularly, the differential delay-line TDC architecture turned out to be the most suitable for this Ph.D. project as it offers an optimal trade-off between temporal precision, temporal range, temporal resolution, dead-time, linearity, and FPGA resources, which are all crucial characteristics for a TDC device. The goal of the project of pursuing a cost-effective and further-upgradable open-source time-tagging device was achieved as the BrigthEyes-TTM was developed and assembled using low-cost commercially available electronic development kits, thus allowing for the architecture to be easily reproduced. BrightEyes-TTM was deployed on a FPGA development board which was equipped with a USB 3.0 chip for communicating with a host-processing unit and a multi-input/output custom-built interface card for interconnecting the TTM with the outside world. Licence-free softwares were used for acquiring, reconstructing and analyzing the BrightEyes-TTM time-resolved data. In order to characterize the BrightEyes-TTM performances and, at the same time, validate the developed multi-channel TDC architecture, the TTM was firstly tested on a bench and then integrated into a fluorescent LSM system. Yielding a 30 ps single-shot precision and linearity performances that allows to be employed for actual FLIM measurements, the BrightEyes-TTM, which also proved to acquire data from many channels in parallel, was ultimately used with a SPAD array detector to perform fluorescence imaging and spectroscopy on biological systems. As output of the Ph.D. work, the BrightEyes-TTM was released on GitHub as a fully open-source project with two aims. The principal aim is to give to any microscopy and life science laboratory the possibility to implement and further develop single-photon-based time-resolved microscopy techniques. The second aim is to trigger the interest of the microscopy community, and establish the BrigthEyes-TTM as a new standard for single-photon FLSM and FLIM experiments

Surface plasmon-coupled emission for applications in biomedical diagnostics

Surface plasmon-coupled emission (SPCE) is a phenomenon whereby the light emitted from a fluorescent molecule can couple into the surface plasmon of an adjacent metal layer resulting in highly directional emission in the region of the surface plasmon resonance (SPR) angle. As well as the high directionality of emission, SPCE has the added advantage of surface selectivity in that the coupling depends on the distance from the surface. This effect can be exploited in bioassays whereby a fluorescing background from the sample can be suppressed. This thesis reports, both theoretically and experimentally, the SPCE effect for a fluorophorespacersilver layer system. Both the angular dependence and the dependence of SPCE emission intensity on fluorophore-metal separation were investigated. It is demonstrated that SPCE leads to lower total fluorescence signal than that obtained in the absence of a metal layer (e.g. when a supercritical angle fluorescence approach is adopted). The experimental results are in good agreement with the theoretical model and with recently published work. Despite the lower overall intensities achievable with SPCE, the advantages of highly directional emission and surface selectivity make it a useful tool for development of high performance fluorescence-based biosensors. The SPCE principles were exploited to achieve an enhanced optical bioassay using a novel, disposable parabolic biochip. This biochip is designed to capture the light generated near the interface with high efficiency. The plasmonic structure is integrated into the chip by depositing a thin metal film on top of the recognition area and by carrying out appropriate surface modification. An optical reader was designed and validated by raytracing simulations and aspects of illumination and polarisation were broadly discussed. The use of various materials was assessed in terms of both their chemical stability and compatibility with the biochip design. The proof-of-concept has been demonstrated by performing a model human Immunoglobulin G sandwich immunoassay and a limit of detection below 5ng/ml of the analyte was achieved. Real time antibody-antigen binding was also demonstrated. This works shows the potential of SPCE to become a useful analytical technique which has a high degree of surface sensitivity and the inherent capability to reject background luminescence. Among the additional advantages are compatibility with the electrochemiluminescence (ECL) technique and multiwavelength discrimination

Fluorescent-based nanosensors for selective detection of a wide range of biological macromolecules: A comprehensive review

Thanks to their unique attributes, such as good sensitivity, selectivity, high surface-to-volume ratio, and versatile optical and electronic properties, fluorescent-based bioprobes have been used to create highly sensitive nano -biosensors to detect various biological and chemical agents. These sensors are superior to other analytical instrumentation techniques like gas chromatography, high-performance liquid chromatography, and capillary electrophoresis for being biodegradable, eco-friendly, and more economical, operational, and cost-effective. Moreover, several reports have also highlighted their application in the early detection of biomarkers associ-ated with drug-induced organ damage such as liver, kidney, or lungs. In the present work, we comprehensively overviewed the electrochemical sensors that employ nanomaterials (nanoparticles/colloids or quantum dots, carbon dots, or nanoscaled metal-organic frameworks, etc.) to detect a variety of biological macromolecules based on fluorescent emission spectra. In addition, the most important mechanisms and methods to sense amino acids, protein, peptides, enzymes, carbohydrates, neurotransmitters, nucleic acids, vitamins, ions, metals, and electrolytes, blood gases, drugs (i.e., anti-inflammatory agents and antibiotics), toxins, alkaloids, antioxidants, cancer biomarkers, urinary metabolites (i.e., urea, uric acid, and creatinine), and pathogenic microorganisms were outlined and compared in terms of their selectivity and sensitivity. Altogether, the small dimensions and capability of these nanosensors for sensitive, label-free, real-time sensing of chemical, biological, and pharma-ceutical agents could be used in array-based screening and in-vitro or in-vivo diagnostics. Although fluorescent nanoprobes are widely applied in determining biological macromolecules, unfortunately, they present many challenges and limitations. Efforts must be made to minimize such limitations in utilizing such nanobiosensors with an emphasis on their commercial developments. We believe that the current review can foster the wider incorporation of nanomedicine and will be of particular interest to researchers working on fluorescence tech-nology, material chemistry, coordination polymers, and related research areas

DNA-mediated assembly of optical components for light manipulation at the nanoscale

Die Selbstorganisation von DNA ist eine leistungsstarke Bottom-Up-Herstellungstechnik, die es ermöglicht, Billionen von Nanostrukturen parallel zu produzieren. Dank der molekularen Erkennungseigenschaften und der intrinsisch hohen räumlichen Auflösung des DNA-Moleküls sind alle diese Strukturen hochgradig adressierbar und erlauben die präzise Steuerung der Position von Nanokomponenten und deren Distanzen zueinander. Diese Eigenschaften ermöglichen die Manipulation der Licht-Materie-Wechselwirkung auf der Nanoskala, wo die physikalischen Phänomene und die Eigenschaften von Strukturen von der gegenseitigen Anordnung und dem Abstand ihrer Komponenten abhängen. In dieser Arbeit werden verschiedene optisch aktive Nanokomponenten wie Fluorophore, Halbleiter-Quantenpunkte und metallische Nanopartikel in maßgeschneiderten Konfigurationen angeordnet, um verschiedene physikalische Phänomene zu untersuchen. Im ersten Teil dieser Arbeit werden DNA-Nanostrukturen verwendet, um zwei spezifische Prozesse der Lichtsammlung nachzuahmen: Farbstoff-Farbstoff-Kopplung und Fern-Energietransport. Die Kopplung von zwei Cyanin-3-Farbstoffen wird durch kovalente Verknüpfung der Moleküle mit einem DNA-Gerüst gesteuert. Durch Variation ihrer Entfernung im Sub-Nanometer-Maßstab wird eine H-Dimerisierung beobachtet. Um den Langstrecken-Energietransport zu untersuchen, wird eine 16 nm lange photonische Leitung auf einer DNA-Origami-Plattform montiert. Die Energiekaskade von einem primären Donor zu einem letzten Akzeptor wird durch Fluorophore der gleichen Art vermittelt, die in der Lage sind, einen Energietransfer durchzuführen. Dank der Modularität des DNA-Origami ist es möglich zu bestimmen, dass das Phänomen der Homo-Energieübertragung zu einer insgesamt verbesserten Ende-zu-Ende-Übertragungseffizienz führen kann. Im zweiten Teil dieser Arbeit wird die DNA-Selbstorganisation verwendet, um die Exziton-Plasmon-Kopplung zu untersuchen, indem ein kolloidaler Quantenpunkt in der Lücke zwischen zwei Goldnanopartikeln positioniert wird. Zu Beginn wird eine neue Methode zur Funktionalisierung kolloidaler Quantenpunkte mit DNA entwickelt. Diese neue Technik beruht auf der Affinität zwischen DNA-Basen und der Oberfläche der Nanopartikel. Im Gegensatz zu bestehenden Verfahren erfordert es keine chemisch modifizierte DNA oder spezielle Ausrüstung und kann bei Raumtemperatur in nur 15 Minuten durchgeführt werden. Anschließend werden einzelne Quantenpunkte in den Hot Spot von Plasmonenantennen aus 40 nm Goldnanopartikeln platziert. Die Anordnung basiert auf DNA-Komplementarität, Stöchiometrie und sterische Hinderung und kann auf verschiedene Materialien erweitert werden. Da nur kurze DNA-Stränge benötigt werden, um die Komponenten zu verbinden, besitzen diese Antennen eine sehr kleine Lücke (~ 6 nm), die wichtig ist, um hohe Purcell-Faktoren und plasmonische Verstärkung zu erreichen. Mit diesen Strukturen wird eine bis zu 30-fache Fluoreszenzzunahme im Vergleich zu Quantenpunkten ohne Antenne erreicht.DNA self-assembly is a powerful bottom-up fabrication technique that enables the realization of trillions of nanodevices in a parallel manner. Thanks to the molecular recognition properties and the intrinsically high spatial resolution of the DNA molecule, all of these devices are highly addressable and allow sub nanometer precise control over the distance and the positioning of nano-components. These features are highly desirable for the manipulation of light-matter interaction at the nanoscale, where the physical phenomena and the properties of devices depend on the reciprocal arrangement and distance of their components. In the work presented here, DNA self-assembly is used to arrange different optically active nano-components, such as fluorophores, semiconductor quantum dots and metallic nanoparticles, in custom-tailored configurations, in order to explore specific physical phenomena. In the first part of this work, DNA nanostructures are used to mimic two specific processes of light harvesting: dye-dye coupling and long-range energy transport. The coupling of two cyanine 3 dyes is controlled by covalently linking the molecules to a DNA stand. By varying their distance at sub nanometer scale, an H-type dimerization is observed. To study long-range energy transport, a 16 nm photonic wire is assembled on a DNA origami platform. The energy cascade from a primary donor to a final acceptor is mediated by fluorophores of the same kind able in order to perform homo-energy transfer. Thanks to the modularity of DNA origami, it is possible to demonstrate that the homo-energy transfer phenomenon can indeed lead to an overall enhancement in end-to-end transfer efficiency. In the second part of this work, DNA self-assembly is used to study exciton-plasmon coupling by positioning a colloidal quantum dot in the gap between two gold nanoparticles. To start, a new method for functionalizing colloidal quantum dots with DNA is developed. This new technique relies on the affinity between DNA bases and the capping shell of the nanoparticles. Opposed to existing methods, it is fast, does not require chemically modified DNA or specialized equipment, and it can be carried out at room temperature in as short as 15 minutes. Subsequently, single quantum dots are placed inside the hot-spot of plasmonic antennas consisting of pairs of 40 nm gold nanoparticles. The assembly is based on DNA complementarity, stoichiometry, and steric-hindrance principles, and can be extended to different materials. Since only short DNA strands are required to link the components, these antennas possess a very small gap (~6 nm), which is important to achieve high Purcell factors and plasmonic enhancement. With these devices, an increase in fluorescence of up to 30-fold is obtained in comparison to quantum dots that are not placed within the antenna