Recent advances in optical tomography in low scattering media

Low scattering media is the best scenario for optical imaging in thick samples and deep tissue, as it allows to obtain high resolution images without suffering the limitations that the diffusion phenomenon imposes. The high contribution of ballistic light in this regime enabled the development of light sheet microscopy and optical projection tomography, two of the most common techniques nowadays in research laboratories. Their revolutionary approach and wide spectrum of applications and possibilities has lead to a frenetic rhythm of new works and techniques arising every year. The large amount of information available often overwhelms scientists and researchers trying to keep up to date with the last cutting edge advances of the field. This paper aims to give a brief review of the origins and fundamental aspects of these two techniques to focus on the most recent and yet non reviewed works. Apart from novel methods, this document also covers combined multimodal approaches and systems. To conclude, we put a spotlight on the important role that open-source microscopy systems play in the field, as they improve the accessibility to these techniques and promote collaborative networks across the optical imaging community

Development of novel imaging tools for selected biomedical applications

In the quest for better and faster images of cellular and subcellular structures, biology-oriented optical microscopes have advanced significantly in the last few decades. Novel microscopy techniques such as non-linear microscopy (NLM), including two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) microscopy, and light-sheet fluorescence microscopy (LSFM) are emerging as alternatives that overcome some the intrinsic limitations of standard microscopy systems. In this thesis I aimed to advance such techniques even more, and combine them with other photonic technologies to provide novel tools that would help to address complex biological questions. This thesis is organized in two main parts. The first part is dedicated to applications involving femtosecond lasers that are employed for precise microsurgery. For that, damage assessment methodologies based on NLM were developed and tested in relevant biomedical models. In the second part, wavefront engineering methods were employed to enhance the imaging capabilities of light-sheet microscopy systems. These novel methodologies were tested as well in relevant biological applications. This thesis is, therefore, organized as follows: In chapter 1, a brief and comprehensive review of the basic microscopy techniques employed in this thesis is presented, together with the challenges and achievements of this thesis in sequential order. In chapter 2, a multimodal imaging methodology for the assessment of laser induced collateral damage is presented. This was specifically developed for the control of the damage in femtosecond-laser dissection of single axons within a living Caenorhabditis elegans (C. elegans). Here, it is shown that collateral damages at the level of the myosin structure of the muscles adjacent to the axon, can be readily detected. In chapter 3, the optimized multimodal methodology developed in the chapter 2 was employed for minimally invasive dissection of axons of D-type motoneurons in C elegans. Here, a microfluidic chip for C elegans immobilization and a detailed protocol was employed to evaluate the axon regeneration of such neurons. The potential of such platform for testing drugs with regeneration-enhancing capabilities is also presented. In chapter 4, a novel use of TPEF microscopy is presented to characterize and fine tune the laser for photodisruption of excised human crystalline lens samples. In chapter 5, a thorough description of the implementation of a multimodal Digital Scanned Light-Sheet Microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes, is presented. The enhanced capabilities of the developed system is evaluated using in vivo C. elegans samples and multicellular tumor spheroids In chapter 6, the development of a completely new concept in light sheet-based imaging is presented. This is based on the extension of the depth-of-field of the lens in the emission path of the microscope by using wavefront coding (WFC) techniques. Furthermore, I demonstrate the application of the developed methodology for fast volumetric imaging of living biological specimens and 3D particle tracking

Comparative assessment of induced abnormal mitotic events by high-throughput light sheet imaging and image analysis

In recent years, three-dimensional (3D) in vitro cell culture models such as spheroids and organoids have revolutionized life science research by providing a much more reliable context resembling the in vivo microenvironment. These systems yield important cell-to-cell interactions and induce cell differentiation. However, no conventional microscopy setup can provide sufficient imaging throughput as well as spatial and temporal resolution to enable full 3D live imaging and analysis down to subcellular processes. In this project, we established state-of-the-art light sheet microscopy for live, long-term imaging of a short interfering ribonucleic acid (siRNA) treated 3D cell culture model. Due to the high temporal and special resolution of the light sheet microscope, we minimized imaging artifacts and achieved unprecedented visual representations of spheroids throughout development and upon gene knock-down by siRNAs. Furthermore, we deployed a high-throughput image analysis pipeline and machine learning classification to evaluate global, cellular and subcellular features for a precise, quantitative gene knock-down phenotype description. The RNA interference (RNAi) induced gene knock-down phenotypes were replicated and compared by a novel molecular, site-specificepigenome modifying method. Throughout this project, we carefully evaluated every step of the workflow to improved its throughput and increased its reproducibility and usability. We addressed the key challenges in light sheet microscopy, such as sample preparation, data handling, image processing and analysis, thereby establishing quantitative light sheet microscopy screening of 3D cell culture models for many research applications. In total, we believe that our workflow can provide the basis for high-content analysis of 3D cell culture models for future research, enabling much more detailed functional experiments and basic research studies

High-resolution in-depth imaging of optically cleared thick samples using an adaptive SPIM

Today, Light Sheet Fluorescence Microscopy (LSFM) makes it possible to image fluorescent samples through depths of several hundreds of microns. However, LSFM also suffers from scattering, absorption and optical aberrations. Spatial variations in the refractive index inside the samples cause major changes to the light path resulting in loss of signal and contrast in the deepest regions, thus impairing in-depth imaging capability. These effects are particularly marked when inhomogeneous, complex biological samples are under study. Recently, chemical treatments have been developed to render a sample transparent by homogenizing its refractive index (RI), consequently enabling a reduction of scattering phenomena and a simplification of optical aberration patterns. One drawback of these methods is that the resulting RI of cleared samples does not match the working RI medium generally used for LSFM lenses. This RI mismatch leads to the presence of low-order aberrations and therefore to a significant degradation of image quality. In this paper, we introduce an original optical-chemical combined method based on an adaptive SPIM and a water-based clearing protocol enabling compensation for aberrations arising from RI mismatches induced by optical clearing methods and acquisition of high-resolution in-depth images of optically cleared complex thick samples such as Multi-Cellular Tumour Spheroids

Set up of a light sheet fluorescence microscope for cellular studies

Light-sheet fluorescence microscopy (LSFM) has been present in cell biology laboratories for quite some time, mainly as custom-made systems, with imaging applications ranging from single cells (in the μm scale) to small organisms (mm). Such microscopes distinguish themselves for having very low phototoxicity levels and high spatial and temporal resolution, properties that render it ideal for 3D characterization of cell motility in migration and traction force studies. Cellular motion has proven to be essential in biological processes such as tumor metastasis and tissue development. Experimental setups make extensive use of microdevices (bioMEMS) that are providing higher degrees of empirical complexity. The following report details the process of setting-up a functional LSFM device for imaging cell motion in microfluidic devices. It begins with a brief summary of fluorescence imaging and current techniques, important to understand why single-plane illumination microscopy (SPIM) was chosen among other light-sheet methods. Then, the whole SPIM set-up process is described, containing explanations about the physical and material properties of the hardware used, the intricacies of the control system, and important procedures. These procedures include: calibration of the microscope, sample preparation in microdevices, and image acquisition from the software provided. Real fluorescence images acquired serve as evidence of the functionality of the instrument. The current limitations are highlighted, and pointers on how to improve or enhance the device are given. The report contains many diagrams, tables and pictures to aid in the understanding of important concepts. In the Annex, a comprehensive table listing the project costs by category is attached. This table includes links to the manufacturers and providers. The aim of this writing is to serve as an exhaustive guideline and be of reproducible use for researchers aiming to build SPIM systems for similar applications.Ingeniería Biomédic

Development of novel multimodal light-sheet fluorescence microscopes for in-vivo imaging of vertebrate organisms

The observation of biological processes in their native environments is of critical importance for life science. While substantial information can be derived from the examination of in-vitro biological samples, in-vivo studies are necessary to reveal the complexity of the dynamics happening in real-time within a living organism. Between the possible biological model choices, vertebrates represent an important family due to the various characteristics they share with the human organism. The development of an embryo, the effect of a drug, the interaction between the immune system and pathogens, and the cellular machinery activities are all examples of highly-relevant applications requiring in-vivo observations on broadly used vertebrate models such as the zebrafish and the mouse. To perform such observations, appropriate devices have been devised. Fluorescence microscopy is one of the main approaches through which specific sample structures can be detected and registered in high-contrast images. Through micro-injections or transgenic lines, a living specimen can express fluorescence and can be imaged through such microscopes. Various fluorescence microscopy techniques have been developed, such as Widefield Microscopy (WM) and Laser Scanning Confocal Microscopy (LSCM). In WM the entire sample is visualized in a single 2D image, therefore losing the depth information, while LSCM can recover the 3D information of the sample but with inherent limitations, such as phototoxicity and limited imaging speed. In the last two decades, Light-Sheet Fluorescence Microscopy (LSFM) emerged as a technique providing fast and 3D imaging, while minimizing collateral damages to the specimen. However, due to the particular configuration of the microscope’s components, LSFM setups are normally optimized for a single application. Also, sample management is not trivial, as controlling the specimen positioning and keeping it alive for a long time within the microscope needs dedicated environmental conditioning. In this thesis, I aimed at advancing the imaging flexibility of LSFM, with particular attention to sample management. The conjugation of these aspects enabled novel observations and applications on living vertebrate samples. In Chapter 1, a brief review of the concepts employed within this thesis is presented, also pointing to the main challenges that the thesis aims to solve. In Chapter 2, a new design for multimodal LSFM is presented, which enables performing different experiments with the same instrument. Particularly, high-throughput studies would benefit from this imaging paradigm, conjugating the need for fast and reproducible mounting of multiple samples with the opportunity to image them in 3D. Additionally, from this design, a transportable setup has also been implemented. With these systems, I studied the dynamics of the yolk’s microtubule network of zebrafish embryos, describing novel features and underlining the importance of live imaging to have a whole view of the sample’s peculiarities. This is described in Chapter 3. Further applications on challenging live samples have been implemented, monitoring the macrophage recruitment in zebrafish and the development of mouse embryos. For these applications, described in Chapter 4, I devised specific mounting protocols for the samples, keeping them alive during the imaging sessions. In Chapter 5, an additional LSFM system is described, which allows for recording the sub-cellular machinery in a living vertebrate sample, while avoiding its damage thanks to the devised sample mounting. Through this, single-molecule microscopy (SMM) studies, normally performed on cultured cells, can be extended to the nuclei of living zebrafish embryos, which better recapitulate the native environment where biological processes take place. Finally, Chapter 6 recapitulates the conclusions, the impacts, future integrations, and experimental procedures that would be enabled by the work resumed in this thesis.La observación de los procesos biológicos en su entorno es de vital importancia para las ciencias de la vida. Si bien se puede derivar información sustancial desde muestras biológicas in-vitro, los estudios in-vivo son necesarios para revelar la complejidad de la dinámica que ocurre, en tiempo real, dentro de un organismo vivo. Entre las posibles elecciones de modelos biológicos, los vertebrados representan una familia importante debido a las diversas características que comparten con el organismo humano. El desarrollo de un embrión, la interacción entre el sistema inmunitario y los patógenos, el efecto de un fármaco y las actividades celulares son ejemplos de aplicaciones que requieren observaciones in-vivo en modelos de vertebrados, como el pez cebra y el ratón. La microscopía de fluorescencia es uno de los principales métodos mediante los cuales se pueden grabar imágenes, de alto contraste, de estructuras biológicas específicas. Utilizando microinyecciones o líneas transgénicas, es posible inducir una expresión de proteínas fluorescentes en la muestra y entonces puede ser observada a través de dichos microscopios. Existen varias técnicas de microscopía de fluorescencia, entre ellas las más utilizadas son la microscopía ¿widefield¿ (WM) y la microscopía ¿confocal¿ (LSCM). En WM, una sola imagen en 2D representa el volumen entero de la muestra, por lo cual la información de profundidad se pierde. Por otro lado, LSCM puede recuperar la información en 3D con algunas limitaciones como la fototoxicidad y una velocidad de generación de las imágenes limitada. En las últimas dos décadas, la microscopía de fluorescencia de hoja de luz (LSFM) surgió como técnica que ofrece imágenes de manera rápidas y en 3D, y que al mismo tiempo minimiza los daños colaterales de la muestra. Sin embargo, debido a la geometría de los componentes del microscopio, las configuraciones de LSFM normalmente se optimizan para una sola aplicación. Además, la gestión de las muestras no es trivial, ya que controlar su posición y mantenerlas vivas durante largos periodos de tiempo dentro del microscopio requiere una atención especifica. En esta tesis, me propuse mejorar la versatilidad que LSFM puede ofrecer, con especial atención a la gestión de muestras vivas. La conjugación de estos aspectos permitió nuevas observaciones y nuevas aplicaciones en vertebrados vivos. En el Capítulo 1, se presenta un breve resumen de los conceptos empleados dentro de esta tesis, señalando también los principales desafíos que la tesis pretende resolver. En el Capítulo 2, se presenta un nuevo diseño para un LSFM multimodal, que permite realizar diferentes experimentos con el mismo instrumento. Los estudios de High-Throughput se beneficiarían de este diseño, ya que conjuga la necesidad de un montaje rápido y reproducible de varias muestras con las ventajas de LSFM. Además, a partir de este diseño, también se ha desarrollado un otro microscopio LSFM transportable. Con estos sistemas, se estudió la dinámica de la red de microtúbulos en embriones de pez cebra, describiendo características nuevas y acentuando la importancia de los experimentos in-vivo para obtener una visión completa de la muestra. Esto se describe en el Capítulo 3. Para realizar otras aplicaciones, como la observación de la dinámica de macrófagos en el pez cebra y del desarrollo de embriones de ratón, descritas en el Capítulo 4, se establecieron protocolos de montaje específicos para las muestras, manteniéndolas vivas durante las sesiones experimentales. En el Capítulo 5, se describe otro sistema LSFM, que permite extender los estudios de microscopía de moléculas individuales (SMM), normalmente realizados en cultivos de células, a núcleos de embriones de pez cebra vivos, que recrean mejor el entorno natural de los procesos biológicos. Finalmente, el Capítulo 6 recapitula las conclusiones, los impactos, las integraciones futuras y los procedimientos experimentales que serían posibilitados por el trabajo resumido en esta tesis.Postprint (published version

Understanding the Role of the Solid Tumour Microenvironment in Brain Tumour Progression

Glioblastoma (GBM) is the most common malignant brain tumour and has an extremely poor prognosis. The invasion of tumour cells into normal brain tissue makes complete surgical removal impossible; GBM is also resistant to treatment with chemotherapy and radiotherapy. Our aim was to investigate how GBM cell proliferation, survival and invasion is affected by the solid tumour microenvironment. Although GBM is highly vascularised, the abnormal structure and function of tumour blood vessels results in an inadequate supply of oxygen (hypoxia). Hypoxia is known to promote tumour progression; however, the effect of hypoxia on cell proliferation has not been well characterised. We performed a systematic investigation into the effects of different oxygen levels on the cell cycle. In contrast to the prevailing hypothesis, we found that long-term exposure to pathophysiological levels of hypoxia (1–8% O2) does not affect cell proliferation and viability and that even severe hypoxia (0.1% O2) has only minimal effects. We next sought to characterise the effect of hypoxia in multicellular tumour spheroids: 3D cell clusters that replicate important aspects of the tumour microenvironment. We characterised spheroids in terms of proliferation, survival and oxygenation and found that, in this more complex model, hypoxia was associated with reduced proliferation. We then used spheroids to develop a novel method for imaging cellular migration and invasion in 3D using lightsheet fluorescence microscopy (LSFM). We imaged spheroids over 24 h and then quantified the movements of up to 1200 cells per spheroid in terms of speed and straightness of movement. We were able to compare the movement of cells in different regions of spheroids, gaining insight into the behaviour of quiescent cells in the core of large (~500 μm), heterogeneous spheroids that had been exposed to hypoxia. This technique can be used to investigate the effect of the tumour microenvironment on cell motility and to gain insight into the mechanism of drugs that hinder the process of invasion

Microfluidic construction and operation of artificial cell chassis encapsulating living cells and pharmaceutical compounds towards their controlled interaction

Droplet-based microfluidic devices can generate complex, soft-matter emulsion systems towards drug screening applications and artificial cell membrane studies. This thesis investigates a methodology for the eventual ‘programmed’ release of pharmaceuticals to treat breast cancer cells that are encapsulated and cultured within small diameter (<2 mm), artificial cell chassis hydrogel capsules. A pharmaceutical analogue was compartmentalised within smaller, membrane-bound, inner cores, that are arranged inside the overall hydrogel capsule. The membrane was based upon droplet interface bilayers (DIBs), which are widely employed for the study of artificial cell membrane transport properties. The whole capsule and contents were produced using enclosed 3D-printed multi-material, microfluidic devices. Methods to control the (programmed) release of compounds from the inner cores to the hydrogel shell, were investigated. The application-specific study was used as an exemplar for a more generally applicable model system. Monolithic microfluidic devices were fabricated using 3D printing and filaments of cyclic olefin copolymer (COC) and nylon for the production of single, double and triple emulsions. With these devices, monodispersed single-emulsion microgels suitable for cell encapsulation were produced, whilst dual-junction devices generated double-emulsion capsules with a controlled number of oil cores. Multi-junction devices also produced triple emulsion, encapsulated droplet interface bilayers (eDIBs), which were subsequently monitored and characterised. Additionally, to demonstrate the ability of eDIBs to act as programmed pharmaceutical delivery systems, assays were performed to induce core release, using membrane modulation by lysolipids (LPC). Computational simulations and DIB electrophysiology experiments were performed to investigate the effect of LPC on the system. MCF-7 model breast cancer cells were encapsulated in alginate-collagen emulsion capsules and their viability was assessed. Moreover, multicellular tumour spheroids (MCTSs) in oil core microgels showed no response to tested doxorubicin concentrations, while proliferated at certain LPC concentrations. Encapsulated cells in eDIBs formed tumour spheroids, however, the DIB survival was low. The integration of living cells and artificial cell membranes within a single entity presents a hybrid model for studying their interaction, towards applications in synthetic biology and drug delivery/screening