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

Functional characterization of developing heart in embryos using Electric Potential Sensors

The characterization of the electrocardiographic activity of the living zebrafish heart during early developmental stages is a challenging task. Most of the available techniques are limited to heartbeat rate quantification being this inaccurate. Other invasive methodologies require the insertion of electrodes noise isolated environments and advanced amplification stages making these techniques very expensive. In this paper, we present a novel and non-invasive sensor development to characterize the functional activity of the developing heart of in vivo zebrafish embryos. The design is based on the Electric Potential Sensing technology patented at Sussex which has been developed to achieve reproducibility and continuous detection. We present preliminary functional characterization data of the developing zebrafish heart starting at 3 days-post-fertilization. Results show that using the proposed system for mapping the electrocardiographic activity of the zebrafish heart at early developmental stages is successfully accomplished. This is the first time that such a sensitive sensor has been developed for measuring the electrical changes occurring on micron sized (< 100 µm) living samples such as the zebrafish heart

An experimental method for bio-signal denoising using unconventional sensors

In bio-signal denoising, current methods reported in literature consider purely simulated envi-ronments, requiring high computational powers and signal processing algorithms that may in-troduce signal distortion. To achieve an efficient noise reduction, such methods require previous knowledge of the noise signals or to have certain periodicity and stability, making the noise es-timation difficult to predict. In this paper, we solve these challenges through the development of an experimental method applied for bio-signal denoising using a combined approach. This is based on the implementation of unconventional electric field sensors used for creating a noise replica required to obtain the ideal Wiener filter transfer function and achieve further noise reduction. This work aims to investigate the suitability of the proposed approach for the real-time noise reduction affecting bio-signal recordings. The experimental evaluation presented considers two scenarios: a) human bio-signals trials including electrocardiogram, electromyogram and elec-trooculogram; and b) bio-signal recordings from the MIT-MIH arrhythmia database. The per-formance of the proposed method is evaluated using qualitative (i.e. power spectral density) and quantitative criteria (i.e. signal-to-noise ratio and mean square error) followed by a comparison between the proposed methodology and state of the art denoising methods. The results indicate that the combined approach proposed in this paper can be used for noise reduction in electro-cardiogram, electromyogram and electrooculogram signals achieving noise attenuation levels of 26.4 dB, 21.2 dB and 40.8 dB, respectively

Two-photon fluorescence imaging with 30 fs laser system tunable around 1 micron

We developed a low-cost, low-noise, tunable, high-peak-power, ultrafast laser system based on a SESAM-modelocked, solid-state Yb tungstate laser plus spectral broadening via a microstructured fiber followed by pulse compression. The spectral selection, tuning, and pulse compression are performed with a simple prism compressor. The output pulses are tunable from 800 to 1250 nm, with the pulse duration down to 25 fs, and average output power up to 150 mW, at 80 MHz pulse repetition rate. We introduce the figure of merit (FOM) for the two-photon and multi-photon imaging (or other nonlinear processes), which is a useful guideline in discussions and for designing the lasers for an improved microscopy signal. Using a 40 MHz pulse repetition rate laser system, with twice lower FOM, we obtained high signal-to-noise ratio two-photon fluorescence images with or without averaging, of mouse intestine section and zebra fish embryo. The obtained images demonstrate that the developed system is capable of nonlinear (TPE, SHG) imaging in a multimodal operation. The system could be potentially used in a variety of other techniques including, THG, CARS and applications such as nanosurgery

Neo-SENSE: a non-invasive smart sensing mattress for cardiac monitoring of babies

Within the first minute of life a newborn must take its first breath to make the transition from life inside the womb to the outside world. If a baby does not start breathing, its heart rate will drop and the circulation of blood carrying oxygen to the organs will be seriously affected. The damage done to a newborn who is deprived of oxygen happens so quickly that rapid response is imperative. During birth, the attending neonatal staff manually listen to the baby´s heart and count the heart rate; however, this has proven inaccurate and inefficient. Nowadays, there is not a reliable method to monitor newborn heart rate promptly throughout birth. In this paper, we report the design and development of a novel smart mattress device to measure the babies’ electrocardiogram and respiration non-invasively. The device is based on electrometer-based amplifier sensors combined with novel screen-printing techniques. Proof of concept tests are carried out to demonstrate the suitability of the smart-mattress for new born ECG monitoring. We perform tests with a young infant and demonstrate the potential of this sensing technology to provide a quick and reliable application as ECG readings were displayed within a time < 30 seconds. This will aid the neonatal staff to assess the success of the resuscitation technology aiming to lower the incidence of long-term consequences of poor adaptation to life outside the womb

Novel 3D printed device with integrated macroscale magnetic field triggerable anti-cancer drug delivery system

With the growing demand for personalized medicine and medical devices, the impact of on-demand triggerable (e.g., via magnetic fields) drug delivery systems increased significantly in recent years. The three-dimensional (3D) printing technology has already been applied in the development of personalized dosage forms because of its high-precision and accurate manufacturing ability. In this study, a novel magnetically triggerable drug delivery device composed of a magnetic polydimethylsiloxane (PDMS) sponge cylinder and a 3D printed reservoir was designed, fabricated and characterized. This system can realize a switch between “on” and “off” state easily through the application of different magnetic fields and from different directions. Active and repeatable control of the localized drug release could be achieved by the utilization of magnetic fields to this device due to the shrinking extent of the macro-porous magnetic sponge inside. The switching “on” state of drug-releasing could be realized by the magnetic bar contacted with the side part of the device because the times at which 50%, 80% and 90% (w/w) of the drug were dissolved are observed to be 20, 55 and 140 min, respectively. In contrast, the switching “off” state of drug-releasing could be realized by the magnetic bar placed at the bottom of the device as only 10% (w/w) of the drug could be released within 12 h. An anti-cancer substance, 5-fluorouracil (FLU), was used as the model drug to illustrate the drug release behaviour of the device under different strengths of magnetic fields applied. In vitro cell culture studies also demonstrated that the stronger the magnetic field applied, the higher the drug release from the deformed PDMS sponge cylinder and thus more obvious inhibition effects on Trex cell growth. All results confirmed that the device can provide a safe, long-term, triggerable and reutilizable way for localized disease treatment such as cancer

Characterisation of textile embedded electrodes for use in a neonatal smart mattress electrocardiography system

Heart rate monitoring is the predominant quantitative health indicator of a newborn in the delivery room. A rapid and accurate heart rate measurement is vital during the first minutes after birth. Clinical recommendations suggest that electrocardiogram (ECG) monitoring should be widely adopted in the neonatal intensive care unit to reduce infant mortality and improve long term health outcomes in births that require intervention. Novel non-contact electrocardiogram sensors can reduce the time from birth to heart rate reading as well as providing unobtrusive and continuous monitoring during intervention. In this work we report the design and development of a solution to provide high resolution, real time electrocardiogram data to the clinicians within the delivery room using non-contact electric potential sensors embedded in a neonatal intensive care unit mattress. A real-time high-resolution electrocardiogram acquisition solution based on a low power embedded system was developed and textile embedded electrodes were fabricated and characterised. Proof of concept tests were carried out on simulated and human cardiac signals, producing electrocardiograms suitable for the calculation of heart rate having an accuracy within ±1 beat per minute using a test ECG signal, ECG recordings from a human volunteer with a correlation coefficient of ~ 87% proved accurate beat to beat morphology reproduction of the waveform without morphological alterations and a time from application to heart rate display below 6 s. This provides evidence that flexible non-contact textile-based electrodes can be embedded in wearable devices for assisting births through heart rate monitoring and serves as a proof of concept for a complete neonate electrocardiogram monitoring system

Evaluation of screen-printing techniques for embedding ECG sensors in medical devices

Heart rate monitoring is the most important indicator to evaluate the clinical status of a newborn during birth. Approximately 90% of newborn infants make the transition from the intrauterine to extra uterine environment without major complications; however, the remaining 10% of newborn infants require assistance during this transition. Heart rate monitoring is required for guiding further interventions in the event of complications such as the need for resuscitation. In this work we evaluate the suitability of embedding electrometer-based-amplifier sensors employing novel screen-printing techniques into medical devices. We compare our results with traditional copper based wired electrodes. Our implementation was able to acquire electrocardiogram with enough signal to noise ratio, suitable for heart rate detection with a 1% loss of heart rate accuracy, compared with the copper-based electrodes. Our device has the potential to be embedded in devices for assisting births though heart rate monitoring

Non-invasive sensor methods used in monitoring newborn babies after birth, a clinical perspective

Background Reducing the global new-born mortality is a paramount challenge for humanity. There are approximately 786,323 live births in the UK each year according to the office for National Statistics; around 10% of these newborn infants require assistance during this transition after birth. Each year around, globally around 2.5 million newborns die within their first month. The main causes are complications due to prematurity and during delivery. To act in a timely manner and prevent further damage, health professionals should rely on accurate monitoring of the main vital signs heart rate and respiratory rate. Aims To present a clinical perspective on innovative, non-invasive methods to monitor heart rate and respiratory rate in babies highlighting their advantages and limitations in comparison with well-established methods. Methods Using the data collected in our recently published systematic review we highlight the barriers and facilitators for the novel sensor devices in obtaining reliable heart rate measurements. Details about difficulties related to the application of sensors and interfaces, time to display, and user feedback are explored. We also provide a unique overview of using a non-invasive respiratory rate monitoring method by extracting RR from the pulse oximetry trace of newborn babies. Results Novel sensors to monitor heart rate offer the advantages of minimally obtrusive technologies but have limitations due to movement artefact, bad sensor coupling, intermittent measurement, and poor-quality recordings compared to gold standard well established methods. Respiratory rate can be derived accurately from pleth recordings in infants. Conclusion Some limitations have been identified in current methods to monitor heart rate and respiratory rate in newborn babies. Novel minimally invasive sensors have advantages that may help clinical practice. Further research studies are needed to assess whether they are sufficiently accurate, practical, and reliable to be suitable for clinical use