Development of high-speed imaging techniques for C. elegans nervous system studies

We report high-speed imaging techniques for C. elegans nervous systems studies. We introduce C. elegans, the main model organism in this dissertation, and neuroscientific and biomedical studies using C. elegans involving calcium imaging, nerve regeneration, and drug screening. We review technologies including confocal microscopy and microfluidic devices used in the neuroscientific and biomedical studies We discuss development of a high-speed laser scanning confocal microscope capable of flexible control of imaging conditions, fast imaging speed, and large field-of-view. We provides the design principles used in the development of the confocal microscope including the optical, electrical, and software implementation, and the details of the confocal microscope we built based on the design principles. We present the performance characterization of the confocal microscope, then a few sample images obtained with the confocal microscope. We present development of time-lapse volumetric confocal imaging of whole animal C. elegans Ca²⁺ dynamics. We provide the design of the time-lapse volumetric confocal imaging system including a microfluidic device to accommodate the whole animal within the field-of-view of the imaging system. We examine the feasibility of the volumetric confocal imaging of a whole animal, and demonstrate imaging of the whole animal C. elegans neurons’ response to NaCl within a 630 × 150 × 25 μm³ volume at 2 Hz rate. We report a high-throughput automated imaging platform for C. elegans nerve regeneration study. We describe the design of the automated imaging platform and the automation flow, and characterizes the performance of the platform. The imaging platform can obtain high-resolution 3D confocal images of 20 animals in 10 minutes. We show sample images of C. elegans anterior lateral microtubule nerve regeneration examples acquired via the automated imaging platform. We demonstrate a planar laser activated neuronal scanning platform (PLANS), a high-throughput animal examination system for drug screening. We explain the construction of PLANS involving the optics, the microfluidic device, and the electronics. The PLANS system can scan an animal in less than 5 ms with a spatial sampling resolution of 3 μm FWHM. We show sample scanning results of a Huntington’s disease model of C. elegans. We summarize the studies discussed in this dissertation, and suggest relevant future research to follow up on the studies.Electrical and Computer Engineerin

High-Throughput Nonlinear Optical Microscopy

High-resolution microscopy methods based on different nonlinear optical (NLO) contrast mechanisms are finding numerous applications in biology and medicine. While the basic implementations of these microscopy methods are relatively mature, an important direction of continuing technological innovation lies in improving the throughput of these systems. Throughput improvement is expected to be important for studying fast kinetic processes, for enabling clinical diagnosis and treatment, and for extending the field of image informatics. This review will provide an overview of the fundamental limitations on NLO microscopy throughput. We will further cover several important classes of high-throughput NLO microscope designs with discussions on their strengths and weaknesses and their key biomedical applications. Finally, this review will close with a perspective of potential future technological improvements in this field.National Institutes of Health (U.S.) (9P41EB015871-26A1)National Institutes of Health (U.S.) (R01-EX017656)National Institutes of Health (U.S.) (5 R01 NS051320)National Institutes of Health (U.S.) (4R44EB012415-02)National Science Foundation (U.S.) (CBET-0939511)Singapore-MIT AllianceSkolkovo Institute of Science and TechnologySingapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology)Wellcome Trust (London, England) (Massachusetts Institute of Technology. Postdoctoral Fellowship 093831/Z/10/Z

Design and implementation of a control system for use of galvanometric scanners in laser micromachining applications

In the recent years, laser machining technology has been used widely in industrial applications usually with the aim of increasing the production capability of mass production lines - especially for fast marking, engraving type of applications where speed is an important concern - or manufacturing quality of a certain facility by increasing the level of accuracy in material processing applications such as drilling, cutting; or any scientific research oriented work where high precision machining of parts in sub millimeter scale might be required. A galvanometric scanner is a high precision device that is able to steer a laser beam with a mirror attached to a motor, whose rotor angular range is usually limited with tens of degrees in both directions of rotation; and position is controlled either by voltage or current. Due to their lightness, the rotor and the mirror can move very fast, allowing fast marking (burning out) operation with the laser beam. This can be evaluated as a great advantage compared to slower mechanical appliances used for cutting/machining of different materials. This study concentrates on the analysis of galvanometric scanner system components; and the design and implementation of a hardware and software based control system for a dual-axis galvo setup; and their adaptation for use in laser micromachining applications either as a standalone system or a modular subsystem. Analysis part of the thesis work contains: evaluation of dominant laser micromachining techniques, an overview of the galvanometric scanner system based approach and related components (e.g. electromechanical, electrical, optical), understanding of working principles and related simulation work, compatibility issues with the target micromachining applications. Design part of the thesis work includes: the design and implementation of electronic controller board, intermediate drive electronics stage, microcontroller programming for machining control algorithm, interfacing with graphical user interface based control software and production of necessary mechanical parts. The study has been finalized with experimental work and evaluation of obtained results. The results of these studies are promising and motivate the use of laser galvanometric scanner systems in laser micromachining applications

Optogenetic Interrogation and Manipulation of Vascular Blood Flow in Cortex

Understanding blood flow regulatory mechanisms that correlate the regional blood flow with the level of local neuronal activity in brain is an ongoing research. Discerning different aspects of this coupling is of substantial importance in interpretation of functional imaging results, such as functional magnetic resonance imaging (fMRI), that rely on hemodynamic recordings to detect and image brain neuronal activity. Moreover, this understanding can provide insight into blood flow disorders under different pathophysiological conditions and possible treatments for such disorders. The blood regulatory mechanisms can be studied at two different; however, complementary levels: at the cellular level or at the vascular level. To fully understand the regulatory mechanisms in brain, it is essential to discern details of the coupling mechanism in each level. While, the cellular pathways of the coupling mechanism has been studied extensively in the past few decades, our understanding of the vascular response to brain activity is fairly basic. The main objective of this dissertation is to develop proper methods and instrumentation to interrogate regional cortical vasodynamics in response to local brain stimulation. For this purpose we offer the design of a custom-made OCT scanner and the necessary lens mechanisms to integrate the OCT system, fluorescence imaging, and optogenetic stimulation technologies in a single system. The design uses off-the-shelf components for a cost-effective design. The modular design of the device allows scientists to modify it in accordance with their research needs. With this multi-modal system we are able to monitor blood flow, blood velocity, and lumen diameter of pial vessels, simultaneously. Additionally, the system design provides the possibility of generating arbitrary spatial stimulation light pattern on brain. These abilities enables researchers to capture more diverse datasets and, eventually, obtain a more comprehensive picture of the vasodynamics in the brain. Along with the device we also proposed new biological experiments that are tailored to investigate the spatio-temporal properties of the vascular response to optical neurostimulation of the excitatory neurons. We demonstrate the ability of the proposed methods to investigate the effect of length and amplitude of stimulation on the temporal pattern of response in the blood flow, blood velocity, and diameter of the pial vessels. Moreover, we offer systemic approaches to investigate the spatial characteristics of the response in a vascular network. In these methods we apply arbitrary spatial patterns of optical stimulation to the cortex of transgenic mice and monitor the attributes of surrounding vessels. With this flexibility we were able to image the brain region that is influenced by a pial artery. After characterizing the spatio-temporal properties of the vascular blood flow response to optical neuro-modulation, we demonstrate the design and application of an optogenetic-based closed-loop controller mechanism in the brain. This controller, uses a proportional–integral–derivative (PID) compensator to engineer temporal optogenetic stimulation light pulses and maintain the flow of blood at various user defined levels in a set of selected arteries. Upon tuning the gain values of the PID controller we obtained a near to critically-damped response in the blood flow of selected arterial vessels

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

Modular multimodal platform for classical and high throughput light sheet microscopy

Light-sheet fluorescence microscopy (LSFM) has become an important tool for biological and biomedical research. Although several illumination and detection strategies have been developed, the sample mounting still represents a cumbersome procedure as this is highly dependent on the type of sample and often this might be time consuming. This prevents the use of LSFM in other promising applications in which a fast and straightforward sample-mounting procedure and imaging are essential. These include the high-throughput research fields, e.g. in drug screenings and toxicology studies. Here we present a new imaging paradigm for LSFM, which exploits modularity to offer multimodal imaging and straightforward sample mounting strategy, enhancing the flexibility and throughput of the system. We describe its implementation in which the sample can be imaged either as in any classical configuration, as it flows through the light-sheet using a fluidic approach, or a combination of both. We also evaluate its ability to image a variety of samples, from zebrafish embryos and larvae to 3D complex cell cultures.The authors acknowledge financial support from the Spanish Ministerio de Economía y Competitividad (MINECO) through the “Severo Ochoa” program for Centres of Excellence in R&D (CEX2019-000910-S [MCIN/ AEI/10.13039/501100011033]), Fundació Privada Cellex, Fundació Mir-Puig, and Generalitat de Catalunya through CERCA program; MINECO/FEDER Ramón y Cajal program (RYC-2015-17935); Laserlab- Europe EU-H2020 GA no. 871124; European Union’s Horizon 2020 Framework Programme (H2020 Marie Skłodowska-Curie Innovative Training Networks ImageInLife N. 721537). We thank Verena Ruprecht (CRG- Center of Genomic Regulation, Barcelona), Paz Herráez (Universidad de León), Ester Antón-Galindo and Noelia Fernández-Castillo (Universitat de Barcelona), Marymar Becerra (Universidad Nacional Autónoma de México), Georges Lutfalla, Mai Nguyen Chi and Tamara Sipka (Université de Montpellier), Catarina Brito (ITQB/IBEQ, Lisbon), Antonia Weberling and Magdalena Zernicka-Goetz (University of Cambridge), and Corinne Lorenzo (ITAV – CNRS, Toulouse) for the samples provided. We also thank Maria Marsal and Jordi Andilla for many fruitful discussions.Postprint (published version

A platform for brain-wide imaging and reconstruction of individual neurons

The structure of axonal arbors controls how signals from individual neurons are routed within the mammalian brain. However, the arbors of very few long-range projection neurons have been reconstructed in their entirety, as axons with diameters as small as 100 nm arborize in target regions dispersed over many millimeters of tissue. We introduce a platform for high-resolution, three-dimensional fluorescence imaging of complete tissue volumes that enables the visualization and reconstruction of long-range axonal arbors. This platform relies on a high-speed two-photon microscope integrated with a tissue vibratome and a suite of computational tools for large-scale image data. We demonstrate the power of this approach by reconstructing the axonal arbors of multiple neurons in the motor cortex across a single mouse brain.Howard Hughes Medical InstitutePublished versio

Rapid Assembly of Small Materials Building Blocks (Voxels) into Large Functional 3D Metamaterials

Herein, various 3D additive manufacturing approaches are reviewed in terms of two important figures of merit: maximum voxel printing rate and minimum voxel size. Voxel sizes from several 100 µm down to the 100 nm scale are covered. Original results on multifocus two‐photon printing at around voxel printing rates of 107 voxels s−1 are presented in this context, which significantly surpass previous best values. These advances are illustrated by and applied to the making of microstructured 3D (chiral) mechanical metamaterials that are composed of more than one‐hundred‐thousand unit cells in three dimensions. Previous best values for unit cells of similar complexity are smaller by two orders of magnitude

Desktop microfactory

Micro technology is continuously progressing towards smaller, smarter and reliable forms. Consequently, demand for such miniature and complex systems is arising rapidly in various fields such as industry, medicine, aerospace and automotive. Such fast development of micro technology is achieved thanks to improvements in micromanufacturing tools and techniques. Miniaturization of the machinery and manufacturing equipment is emerging to be an attractive idea that would eventually solve many of the issues existing in conventional micro-manufacturing. This work presents a modular and reconfigurable desktop microfactory for high precision assembly and machining of micro mechanical parts as proof of concept inspired by the downsizing trend of the production tools. The system is constructed based on primary functional and performance requirements such as miniature size, operation with sub-millimeter precision, modular and reconfigurable structure, parallel processing capability, ease of transportation and integration. Proposed miniature factory consists of downsized functional modules such as two parallel kinematic robots for manipulation and assembly, galvanometric laser beam scanning system for micromachining, high precision piezoelectric positioning stage, camera system for detection and inspection, and a rotational conveyor system. Each of the listed modules is designed and tested for fine precision tasks separately and results are presented. Design comprises development of mechanics, electronics and controller for the modules individually. Once stand-alone operation of each unit is achieved further assembly to a single microfactory system is considered. The overall mechanical structure of the proposed microfactory facilitates parallel processing, flexible rearrangement of the layout, and ease of assembling and disassembling capabilities. These important steps are taken to investigate possibilities of a microfactory concept for production of microsystems in near future