Strategies for pushing nonlinear microscopy towards its performance limits

The requirement for imaging living structures with higher contrast and resolution has been covered by the inherent advantages offered by nonlinear microscopy (NLM). However, to achieve its full potential there are still several issues that must be addressed. To do so, it is very important to identify and adapt the key elements in a NLM for achieving an optimized interaction among them. These are 1) the laser source 2) the optics and 3) the sample properties for contrast generation. In this thesis, three strategies have been developed for pushing NLM towards its limits based on the light sample interaction optimization. In the first strategy it is experimentally demonstrated how to take advantage of the sample optical properties to generate label-free contrast, eliminating the requirement of modifying the sample either chemically or genetically. This is carried out by implementing third harmonic generation (THG) microscopy. Here, it is shown how the selection of the ultra-short pulsed laser (USPL) operating wavelength (1550 nm) is crucial for generating a signal that matches the peak sensitivity of most commercial detectors. This enables reducing up to seven times the light dose applied to a sample while generating an efficient signal without the requirement of amplification schemes and specialized optics (such as the need of ultraviolet grade). To show the applicability of the technique, a full developmental study of in vivo Caenorhabditis elegans embryos is presented together with the observation of wavelength induced effects. The obtained results demonstrate the potential of the technique at the employed particular wavelength to be used to follow morphogenesis processes in vivo. In the second strategy the limits of NLM are pushed by using a compact, affordable and maintenance free USPL sources. Such device was designed especially for two-photon excited fluorescence (TPEF) imaging of one the most widely used fluorescent markers in bio-imaging research: the green fluorescent protein. The system operating parameters and its emission wavelength enables to demonstrate how matching the employed fluorescent marker two-photon action cross-section is crucial for efficient TPEF signal production at very low powers. This enables relaxing the peak power conditions (40 W) to excite the sample. The enhanced versatility of this strategy is demonstrated by imaging both fixed and in vivo samples containing different dyes. More over the use of this laser is employed to produce second harmonic generation images of different samples. Several applications that can benefit by using such device are outlined. Then a comparison of the employed USPL source is performed versus the Titanium sapphire laser (the most used excitation source in research laboratories). The final goal of this strategy is to continue introducing novel laser devices for future portable NLM applications. In this case, the use of chip-sized semiconductor USPL sources for TPEF imaging is demonstrated. This will allow taking NLM technology towards the sample and make it available for any user. In the last strategy, the light interaction with the optical elements of a NLM workstation and the sample were optimized. The first enhancement was carried out in the laser-microscope optical path using an adaptive element to spatially shape the properties of the incoming beam wavefront. For an efficient light-sample interaction, aberrations caused by the index mismatch between the objective, immersion fluid, cover-glass and the sample were measured. To do so the nonlinear guide-star concept, developed in this thesis, was employed for such task. The correction of optical aberrations in all the NLM workstation enable in some cases to have an improvement of more than one order of magnitude in the total collected signal intensity. The obtained results demonstrate how adapting the interaction among the key elements of a NLM workstation enables pushing it towards its performance limits.La creciente necesidad de observar estructuras complicadas cada vez con mayor contraste y resolución han sido cubiertas por las ventajas inherentes que ofrece la microscopia nolineal. Sin embargo, aun hay ciertos aspectos que deben ser ajustados para obtener su máximo desempeño. Para ello es importante identificar y adaptar los elementos clave que forman un microscopio optimizar la interacción entre estos. Dichos elementos son: 1) el laser, 2) la óptica y 3) las propiedades de la muestra. En esta tesis, se realizan tres estrategias para llevar la eficiencia de la microscopia nolineal hacia sus límites. En la primera estrategia se demuestra de forma experimental como obtener ventaja de las propiedades ópticas de la muestra para generar contraste sin el uso de marcadores mediante la generación de tercer harmónico. Aquí se muestra como la selección de la longitud de onda del láser de pulsos ultracortos es crucial para que la señal obtenida concuerde con la máxima sensibilidad del detector utilizado. Esto permite una reducción de la dosis de luz con la que se expone la muestra, elimina intrínsecamente el requerimiento de esquemas de amplificación de señal y de óptica de tipo ultravioleta (generalmente empleada en este tipo de microscopios). Mediante un estudio comparativo con un sistema convencional se demuestra que los niveles de potencia óptica pueden ser reducidos hasta siete veces. Para demostrar las ventajas de dicha técnica se realiza un estudio completo sobre el desarrollo embrionario de Caenorhabditis elegans y los efectos causados por la exposición de la muestra a dicha longitud de onda. Los resultados demuestran el potencial de la técnica para dar seguimiento a procesos morfogénicos en muestras vivas a la longitud de onda utilizada. En la segunda estrategia se diseñó una fuente de pulsos ultracortos que es compacta, de costo reducido y libre de mantenimiento para excitar mediante la absorción de dos fotones uno de los marcadores más utilizados en el entorno biológico, la proteína verde fluorescente. Los parámetros de operación en conjunto con la longitud de onda emitida por el sistema proporcionan la máxima eficiencia permitiendo el uso de potencias pico muy bajas (40 W), ideales para relajar la exposición de la muestra. La versatilidad de esta estrategia se demuestra empleando muestras fijas y vivas con diferentes marcadores fluorescentes. Este láser también es empleado para la obtención de señal de segundo harmónico en diferentes muestras. Adicionalmente, se llevó a cabo un estudio comparativo entre la fuente desarrollada y un sistema Titanio zafiro (uno de los láseres más utilizados en laboratorios de investigación). El objetivo final de esta estrategia es introducir fuentes novedosas para aplicaciones portátiles basadas en procesos nolineales. En base a esto se demuestra el uso de dispositivos construidos sobre un microchip para generar imágenes de fluorescencia de dos fotones. Esto permitirá llevar la tecnología hacia la muestra biológica y hacerla disponible para cualquier usuario. En la última estrategia se optimiza de la interacción de la luz con los elementos ópticos del microscopio y la muestra. La primera optimización se lleva a cabo en la trayectoria óptica que lleva el láser hacia el microscopio empleando un elemento adaptable que modifica las propiedades espaciales de la luz. Para mejorar la interacción de la luz y la muestra se miden las aberraciones causadas por la diferencia de índices refractivos entre el objetivo, el medio de inmersión y la muestra. Esto se realizo empleando el concepto de la “estrella guía nolineal” desarrollado en esta tesis. Mediante la corrección de las aberraciones en el sistema de microscopia nolineal se obtiene una mejora, en algunos casos de un orden de magnitud, en la intensidad total medida. Los resultados obtenidos en esta tesis demuestran como el adaptar la interacción entre los elementos clave en un microscopio nolineal permiten llevar su desempeño hacia los límites.Postprint (published version

High-resolution remote thermography using luminescent low-dimensional tin-halide perovskites

While metal-halide perovskites have recently revolutionized research in optoelectronics through a unique combination of performance and synthetic simplicity, their low-dimensional counterparts can further expand the field with hitherto unknown and practically useful optical functionalities. In this context, we present the strong temperature dependence of the photoluminescence (PL) lifetime of low-dimensional, perovskite-like tin-halides, and apply this property to thermal imaging with a high precision of 0.05 {\deg}C. The PL lifetimes are governed by the heat-assisted de-trapping of self-trapped excitons, and their values can be varied over several orders of magnitude by adjusting the temperature (up to 20 ns {\deg}C-1). Typically, this sensitive range spans up to one hundred centigrade, and it is both compound-specific and shown to be compositionally and structurally tunable from -100 to 110 {\deg} C going from [C(NH2)3]2SnBr4 to Cs4SnBr6 and (C4N2H14I)4SnI6. Finally, through the innovative implementation of cost-effective hardware for fluorescence lifetime imaging (FLI), based on time-of-flight (ToF) technology, these novel thermoluminophores have been used to record thermographic videos with high spatial and thermal resolution.Comment: 25 pages, 4 figure

hybrid materials for integrated photonics

In this review materials and technologies of the hybrid approach to integrated photonics (IP) are addressed. IP is nowadays a mature technology and is the most promising candidate to overcome the main limitations that electronics is facing due to the extreme level of integration it has achieved. IP will be based on silicon photonics in order to exploit the CMOS compatibility and the large infrastructures already available for the fabrication of devices. But silicon has severe limits especially concerning the development of active photonics: its low efficiency in photons emission and the limited capability to be used as modulator require finding suitable materials able to fulfill these fundamental tasks. Furthermore there is the need to define standardized processes to render these materials compatible with the CMOS process and to fully exploit their capabilities. This review describes the most promising materials and technological approaches that are either currently implemented or may be used in the coming future to develop next generations of hybrid IP devices

Investigation of Interfacial Charge Transfer in Solution Processed Cs2SnI6 Thin Films

Cesium tin halide based perovskite Cs2SnI6 has been subjected to in-depth investigations owing to its potentiality toward the realization of environment benign Pb free and stable solar cells. In spite of the fact that Cs2SnI6 has been successfully utilized as an efficient hole transport material owing to its p-type semiconducting nature, however, the nature of the majority carrier is still under debate. Therefore, intrinsic properties of Cs2SnI6 have been investigated in detail to explore its potentiality as light absorber along with facile electron and hole transport. A high absorption coefficient (5 × 104 cm–1) at 700 nm indicates the penetration depth of 700 nm light to be 0.2 μm, which is comparable to conventional Pb based solar cells. Preparation of pure and CsI impurity free dense thin films with controllable thicknesses of Cs2SnI6 by the solution processable method has been reported to be difficult owing to its poor solubility. An amicable solution to circumvent such problems of Cs2SnI6 has been provided utilizing spray-coating in combination with spin-coating. The presence of two emission peaks at 710 and 885 nm in the prepared Cs2SnI6 thin films indicated coexistence of quantum dot and bulk parts which were further supported by transmission electron microscopy (TEM) investigations. Time-resolved photoluminescence (PL) and transient absorption spectroscopy (TAS) were employed to investigate the excitation carrier lifetime, which revealed fast decay kinetics in the picoseconds (ps) to nanoseconds (ns) time domains. Time-resolved microwave photoconductivity decay (MPCD) measurement provided the mobile charge carrier lifetime exceeding 300 ns, which was also in agreement with the nanosecond transient absorption spectroscopy (ns-TAS) indicating slow charge decay lasting up to 20 μs. TA assisted interfacial charge transfer investigations utilizing Cs2SnI6 in combination with n-type PCBM and p-type P3HT exhibited both intrinsic electron and hole transport

Femtosecond Cr⁴⁺: forsterite laser for applications in telecommunications and biophotonics

In this thesis, the development of a femtosecond Cr⁴⁺:forsterite solid-state laser is described where the mode-locking procedure was initiated using two novel saturable absorbers. One was a GaInNAs quantum-well device and the other a quantum-dot-based saturable absorber. These devices had not previously been exploited for the generation of femtosecond pulses from a solid-state laser but in the course of this project, successful mode-locked laser operation in the femtosecond domain was demonstrated for both devices. When the GaInNAs device was incorporated in the Cr⁴⁺:forsterite laser, transform-limited pulses with durations as short as 62fs were obtained. The performance of this femtosecond laser was significantly superior to that for previous quantum-well based saturable absorbers in the 1300nm spectral region. The dynamics of the device were investigated with the aim of refining subsequent devices and to explore the potential to grow future devices for use at longer wavelengths. At the outset of my research work quantum-dot based saturable absorbers had not be used for the mode locking of solid-state lasers in the femtosecond regime. The work presented in this thesis showed that quantum-dot structures could be exploited very effectively for this purpose. This was initially achieved with the quantum-dot element being inclined at an off-normal incidence within the cavity but experimental assessment together with further development of the device allowed for implementation at normal incidence. Reliable operation of the femtosecond laser was demonstrated very convincingly where transform-limited pulses of 160fs duration were generated. Having developed practical femtosecond Cr⁴⁺:forsterite lasers, the final part of the project research was directed towards exemplar applications for a laser operating in the 1300nm spectral region. These were biophotonics experiments in which assessments of both deep tissue penetration and two-photon chromosome cutting were undertaken. This work confirmed the suitability of the 1300nm laser radiation for propagation through substantial thicknesses of biological tissue (~15cm). The demonstration of highly localised two-photon cutting of Muntjac deer chromosomes also represented a novel result because single-photon absorption could be avoided effectively and the temporal broadening of the femtosecond pulses in the delivery optics arising from group velocity dispersion around 1300nm was minimal

Nanoantennas and Nanoradars: The Future of Integrated Sensing and Communication at the Nanoscale

Nanoantennas, operating at optical frequencies, are a transformative technology with broad applications in 6G wireless communication, IoT, smart cities, healthcare, and medical imaging. This paper explores their fundamental aspects, applications, and advancements, aiming for a comprehensive understanding of their potential in various applications. It begins by investigating macroscopic and microscopic Maxwell's equations governing electromagnetic wave propagation at different scales. The study emphasizes the critical role of Surface Plasmon Polariton (SPP) wave propagation in enhancing light-matter interactions, contributing to high data rates, and enabling miniaturization. Additionally, it explores using two-dimensional materials like graphene for enhanced control in terahertz communication and sensing. The paper also introduces the employment of nanoantennas as the main building blocks of Nano-scale Radar (NR) systems for the first time in the literature. NRs, integrated with communication signals, promise accurate radar sensing for nanoparticles inside a nano-channel, making them a potential future application in integrated sensing and communication (ISAC) systems. These nano-scale radar systems detect and extract physical or electrical properties of nanoparticles through transmitting, receiving, and processing electromagnetic waves at ultra-high frequencies in the optical range. This task requires nanoantennas as transmitters/receivers/transceivers, sharing the same frequency band and hardware for high-performance sensing and resolution

Two-Dimensional Halide Perovskites for Emerging New- Generation Photodetectors

Compared to their conventional three-dimensional (3D) counterparts, two-dimensional (2D) halide perovskites have attracted more interests recently in a variety of areas related to optoelectronics because of their unique structural characteristics and enhanced performances. In general, there are two distinct types of 2D halide perovskites. One represents those perovskites with an intrinsic layered crystal structure (i.e. MX6 layers, M = metal and X = Cl, Br, I), the other defines the perovskites with a 2D nanostructured morphology such as nanoplatelets and nanosheets. Recent studies have shown that 2D halide perovskites hold promising potential for the development of new-generation photodetectors, mainly arising from their highly efficient photoluminescence and absorbance, color tunability in the visible-light range and relatively high stability. In this chapter, we present the summary and highlights of latest researches on these two types of 2D halide perovskites for developing photodetectors, with an emphasis on synthesis methods, structural characterization, optoelectronic properties, and theoretical analysis and simulations. We also discuss the current challenging issues and future perspective. We hope this chapter would add new elements for understanding halide perovskite-based 2D materials and for developing their more efficient optoelectronic devices

Single photon kilohertz frame rate imaging of neural activity

Establishing the biological basis of cognition and its disorders will require high precision spatiotemporal measurements of neural activity. Recently developed genetically encoded voltage indicators (GEVIs) report both spiking and subthreshold activity of identified neurons. However, maximally capitalizing on the potential of GEVIs will require imaging at millisecond time scales, which remains challenging with standard camera systems. Here, application of single photon avalanche diode (SPAD) sensors is reported to image neural activity at kilohertz frame rates. SPADs are electronic devices that when activated by a single photon cause an avalanche of electrons and a large electric current. An array of SPAD sensors is used to image individual neurons expressing the GEVI Voltron‐JF525‐HTL. It is shown that subthreshold and spiking activity can be resolved with shot noise limited signals at frame rates of up to 10 kHz. SPAD imaging is able to reveal millisecond scale synchronization of neural activity in an ex vivo seizure model. SPAD sensors may have widespread applications for investigation of millisecond timescale neural dynamics