Spelling suggestions: "subject:"light shear microscopy""
1 |
Developing Methods Based on Light Sheet Fluorescence Microscopy for Biophysical Investigations of Larval ZebrafishTaormina, Michael 29 September 2014 (has links)
Adapting the tools of optical microscopy to the large-scale dynamic systems encountered in the development of multicellular organisms provides a path toward understanding the physical processes necessary for complex life to form and function. Obtaining quantitatively meaningful results from such systems has been challenging due to difficulty spanning the spatial and temporal scales representative of the whole, while also observing the many individual members from which complex and collective behavior emerges.
A three-dimensional imaging technique known as light sheet fluorescence microscopy provides a number of significant benefits for surmounting these challenges and studying developmental systems. A thin plane of fluorescence excitation light is produced such that it coincides with the focal plane of an imaging system, providing rapid acquisition of optically sectioned images that can be used to construct a three-dimensional rendition of a sample. I discuss the implementation of this technique for use in larva of the model vertebrate Danio rerio (zebrafish).
The nature of light sheet imaging makes it especially well suited to the study of large systems while maintaining good spatial resolution and minimizing damage to the specimen from excessive exposure to excitation light. I show the results from a comparative study that demonstrates the ability to image certain developmental processes non-destructively, while in contrast confocal microscopy results in abnormal growth due to phototoxicity. I develop the application of light sheet microscopy to the study of a previously inaccessible system: the bacterial colonization of a host organism. Using the technique, we are able to obtain a survey of the intestinal tract of a larval zebrafish and observe the location of microbes as they grow and establish a stable population in an initially germ free fish. Finally, I describe a new technique to measure the fluid viscosity of this intestinal environment in vivo using magnetically driven particles. By imaging such particles as they are oscillated in a frequency chirped field, it is possible to calculate properties such as the viscosity of the material in which they are embedded. Here I provide the first known measurement of intestinal mucus rheology in vivo.
This dissertation includes previously published co-authored material.
|
2 |
Time resolved light sheet microscopyO'Brien, Daniel J. January 2019 (has links)
Understanding and identifying critical protein-protein interactions is just one of the key outcomes in biological research. It can help to confirm key cellular interactions, which in some fields, such as cancer research, can result in a greater understanding of disease pathogenesis, elucidate mechanisms of therapeutic resistance and aid in the development of new specific targets, leading to new methods of prevention and treatment. Time-correlated single photon counting fluorescence lifetime imaging microscopy is just one of the tools used to carry out this line of research. Here we demonstrate a direct interaction between two proteins involved in gene regulation and expression; p21 and FMN2. Furthermore, we also show the capability of this system to measure chromatin compaction in three dimensions. However, fluorescence lifetime imaging has some drawbacks, acquisition times on such a system can range from the tens of seconds to minutes, which is often too long to comprehensively measure many biological events. But microscopy is always developing, aided by new techniques and, perhaps even more so, new technological developments. This thesis also demonstrates two new methods of light sheet microscopy, that use both new equipment made available because of technological developments to allow time resolved imaging and traditional microscopic aspects to form a light sheet system based on polarisation. It outlines the design and how to build these systems and presents their function to show their great promise. Both techniques presented in this thesis utilise aspects of light not conventionally used in light sheet microscopy. Further development of these systems and application of emerging technologies will yield a system capable of outperforming current light sheet fluorescence microscopy-based fluorescence lifetime imaging techniques. The implementation of polarisation control into such a system would enable three-dimensional anisotropy based SPIM-FLIM measurements, an indispensable tool in researching molecular orientation and mobility at a macroscopic level in developing organisms.
|
3 |
All-Optical 4D In Vivo Monitoring And Manipulation Of Zebrafish Cardiac ConductionWeber, Michael 19 May 2015 (has links) (PDF)
The cardiac conduction system is vital for the initiation and maintenance of the heartbeat. Over the recent years, the zebrafish (Danio rerio) has emerged as a promising model organism to study this specialized system. The embryonic zebrafish heart’s unique accessibility for light microscopy has put it in the focus of many cardiac researchers.
However, imaging cardiac conduction in vivo remained a challenge. Typically, hearts had to be removed from the animal to make them accessible for fluorescent dyes and electrophysiology. Furthermore, no technique provided enough spatial and temporal resolution to study the importance of individual cells in the myocardial network.
With the advent of light sheet microscopy, better camera technology, new fluorescent reporters and advanced image analysis tools, all-optical in vivo mapping of cardiac conduction is now within reach. In the course of this thesis, I developed new methods to image and manipulate cardiac conduction in 4D with cellular resolution in the unperturbed zebrafish heart.
Using my newly developed methods, I could detect the first calcium sparks and reveal the onset of cardiac automaticity in the early heart tube. Furthermore, I could visualize the 4D cardiac conduction pattern in the embryonic heart and use it to study component-specific calcium transients. In addition, I could test the robustness of embryonic cardiac conduction under aggravated conditions, and found new evidence for the presence of an early ventricular pacemaker system. My results lay the foundation for novel, non-invasive in vivo studies of cardiac function and performance.
|
4 |
Superresolution Nonlinear Structured Illumination Microscopy By Stimulated Emission DepletionZhang, Han January 2014 (has links)
The understanding of the biological processes at the cellular and subcellular level requires the ability to directly visualize them. Fluorescence microscopy played a key role in biomedical imaging because of its high sensitivity and specificity. However, traditional fluorescence microscopy has a limited resolution due to optical diffraction. In recent years, various approaches have been developed to overcome the diffraction limit. Among these techniques, nonlinear structured illumination microscopy (SIM) has been demonstrated a fast and full field superresolution imaging tool, such as Saturated-SIM and Photoswitching-SIM. In this dissertation, I report a new approach that applies nonlinear structured illumination by combining a uniform excitation field and a patterned stimulated emission depletion (STED) field. The nature of STED effect allows fast switching response, negligible stochastic noise during switching, low shot noise and theoretical unlimited resolution, which predicts STED-SIM to be a better nonlinear SIM. After the algorithm development and the feasibility study by simulation, the STED-SIM microscope was tested on fluorescent beads samples and achieved full field imaging over 1 x 10 micron square at the speed of 2s/frame with 4-fold improved resolution. Our STED-SIM technique has been applied on biological samples and superresolution images with tubulin of U2OS cells and granules of neuron cells have been obtained. In this dissertation, an effort to apply a field enhancement mechanism, surface plasmon resonance (SPR), to nonlinear STED-SIM has been made and around 8 time enhancement on STED quenching effect was achieved. Based on this enhancement on STED, 1D SPR enhanced STED-SIM was built and 50 nm resolution of fluorescence beads sample was achieved. Algorithm improvement is required to achieve full field superresolution imaging with SPR enhanced STED-SIM. The application of nonlinear structured illumination in two photon light-sheet microscopy is also studied in this dissertation. Fluorescent cellular imaging of deep internal organs is highly challenging because of the tissue scattering. By combining two photon Bessel beam light-sheet microscopy and nonlinear SIM, 3D live sample imaging at cellular resolution in depth beyond 200 microns has been achieved on live zebrafish. Two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity.
|
5 |
All-Optical 4D In Vivo Monitoring And Manipulation Of Zebrafish Cardiac ConductionWeber, Michael 05 May 2015 (has links)
The cardiac conduction system is vital for the initiation and maintenance of the heartbeat. Over the recent years, the zebrafish (Danio rerio) has emerged as a promising model organism to study this specialized system. The embryonic zebrafish heart’s unique accessibility for light microscopy has put it in the focus of many cardiac researchers.
However, imaging cardiac conduction in vivo remained a challenge. Typically, hearts had to be removed from the animal to make them accessible for fluorescent dyes and electrophysiology. Furthermore, no technique provided enough spatial and temporal resolution to study the importance of individual cells in the myocardial network.
With the advent of light sheet microscopy, better camera technology, new fluorescent reporters and advanced image analysis tools, all-optical in vivo mapping of cardiac conduction is now within reach. In the course of this thesis, I developed new methods to image and manipulate cardiac conduction in 4D with cellular resolution in the unperturbed zebrafish heart.
Using my newly developed methods, I could detect the first calcium sparks and reveal the onset of cardiac automaticity in the early heart tube. Furthermore, I could visualize the 4D cardiac conduction pattern in the embryonic heart and use it to study component-specific calcium transients. In addition, I could test the robustness of embryonic cardiac conduction under aggravated conditions, and found new evidence for the presence of an early ventricular pacemaker system. My results lay the foundation for novel, non-invasive in vivo studies of cardiac function and performance.
|
6 |
A live imaging paradigm for studying Drosophila development and evolutionSchmied, Christopher 30 March 2016 (has links) (PDF)
Proper metazoan development requires that genes are expressed in a spatiotemporally controlled manner, with tightly regulated levels. Altering the expression of genes that govern development leads mostly to aberrations. However, alterations can also be beneficial, leading to the formation of new phenotypes, which contributes to the astounding diversity of animal forms. In the past the expression of developmental genes has been studied mostly in fixed tissues, which is unable to visualize these highly dynamic processes. We combine genomic fosmid transgenes, expressing genes of interest close to endogenous conditions, with Selective Plane Illumination Microscopy (SPIM) to image the expression of genes live with high temporal resolution and at single cell level in the entire embryo.
In an effort to expand the toolkit for studying Drosophila development we have characterized the global expression patterns of various developmentally important genes in the whole embryo. To process the large datasets generated by SPIM, we have developed an automated workflow for processing on a High Performance Computing (HPC) cluster.
In a parallel project, we wanted to understand how spatiotemporally regulated gene expression patterns and levels lead to different morphologies across Drosophila species. To this end we have compared by SPIM the expression of transcription factors (TFs) encoded by Drosophila melanogaster fosmids to their orthologous Drosophila pseudoobscura counterparts by expressing both fosmids in D. melanogaster. Here, we present an analysis of divergence of expression of orthologous genes compared A) directly by expressing the fosmids, tagged with different fluorophore, in the same D. melanogaster embryo or B) indirectly by expressing the fosmids, tagged with the same fluorophore, in separate D. melanogaster embryos.
Our workflow provides powerful methodology for the study of gene expression patterns and levels during development, such knowledge is a basis for understanding both their evolutionary relevance and developmental function.
|
7 |
A live imaging paradigm for studying Drosophila development and evolutionSchmied, Christopher 27 January 2016 (has links)
Proper metazoan development requires that genes are expressed in a spatiotemporally controlled manner, with tightly regulated levels. Altering the expression of genes that govern development leads mostly to aberrations. However, alterations can also be beneficial, leading to the formation of new phenotypes, which contributes to the astounding diversity of animal forms. In the past the expression of developmental genes has been studied mostly in fixed tissues, which is unable to visualize these highly dynamic processes. We combine genomic fosmid transgenes, expressing genes of interest close to endogenous conditions, with Selective Plane Illumination Microscopy (SPIM) to image the expression of genes live with high temporal resolution and at single cell level in the entire embryo.
In an effort to expand the toolkit for studying Drosophila development we have characterized the global expression patterns of various developmentally important genes in the whole embryo. To process the large datasets generated by SPIM, we have developed an automated workflow for processing on a High Performance Computing (HPC) cluster.
In a parallel project, we wanted to understand how spatiotemporally regulated gene expression patterns and levels lead to different morphologies across Drosophila species. To this end we have compared by SPIM the expression of transcription factors (TFs) encoded by Drosophila melanogaster fosmids to their orthologous Drosophila pseudoobscura counterparts by expressing both fosmids in D. melanogaster. Here, we present an analysis of divergence of expression of orthologous genes compared A) directly by expressing the fosmids, tagged with different fluorophore, in the same D. melanogaster embryo or B) indirectly by expressing the fosmids, tagged with the same fluorophore, in separate D. melanogaster embryos.
Our workflow provides powerful methodology for the study of gene expression patterns and levels during development, such knowledge is a basis for understanding both their evolutionary relevance and developmental function.
|
8 |
Studium maternálně-fetálního mikrochimérismu APC s využitím MHCII/EGFP myšího modelu a clearovacích histologických technik / Study of the materno-fetal microchimerism of the APC using MHCII/EGFP mouse model and clearing histological techniquesKnížková, Karolina January 2020 (has links)
Microchimerism arises from the exchange of cells between genetically distinct individuals. The coexistence of genetically distinct cell populations within a single organism has possible effects on health and functioning of individuals immune systems, but the exact mechanisms of action are often not yet known. With the development of microscopic technologies and software for data analysis, the possibilities of detection and phenotyping of these rare cell populations are expanding. My intention in this work is to find maternal microchimerism in embryonic tissues (E13) and intestines of breastfed pups using MHCII/EGFP knock-in mouse model. Several different technologies potentially suitable for the detection of maternal microchimeric cells in offspring tissues (light sheet fluorescent microscopy - LSFM, virtual slide microscopy and flow cytometry) were selected. Advanced analysis of the obtained samples from the light sheet microscopy using the creation of a neural network was used here. The presence of maternal microchimerism was not demonstrated by flow cytometry. Using LSFM, image data were obtained from intestinal samples of suckling pups, which were processed by the neural network method. Data analysis of embryos (E13) obtained by the same method did not allow data analysis due to high...
|
9 |
CELL TYPE EMERGENCE AND CIRCUIT DISRUPTIONS IN FETAL MODELS OF 15q13.3 MICRODELETION BRAIN DEVELOPMENTKilpatrick, Savannah January 2023 (has links)
The 15q13.3 microdeletion is a common genetic disorder associated with multiple
neurodevelopmental disorders including autism spectrum disorder, epilepsy, and
schizophrenia. Patients have diverse clinical presentations, often prompting genetic
assays that identify the CNV in the clinic. This late-stage screening leaves a considerable
gap in our understanding of the prenatal and prediagnostic developmental impairments in
these individuals, providing a barrier to understanding the disease pathobiology. We
provide the first investigation into embryonic brain development of individuals with the
15q13.3 microdeletion by generating multiple 3D neural organoid models from the
largest clinical cohort in reported literature. We incorporated unguided and guided
forebrain organoid models into our multi-transcriptomic phenotyping pipeline to uncover
changes in cell type emergence and disruptions to circuit development, all of which had
underlying changes to cell adhesion pathways.
Specifically, we identified accelerated growth trajectories in 15q13.3del unguided
neural organoids and used single cell RNA sequencing to identify changes in radial glia
dynamics that affect neurogenesis. We measured changes in the pseudotemporal
trajectory of matured unguided neural organoids, and later identified disruptions in
synaptic signaling modules amongst the primary constituents to neural circuitry,
excitatory and inhibitory neurons.
We leveraged dorsal and ventral forebrain organoid models to better assess circuit
dynamics, as they faithfully produce the excitatory and inhibitory neurons in the pallium
and subpallium, respectively. We then used the entire 15q13.3del cohort and performed
bulk RNA sequencing on each tissue type at two timepoints and discovered convergence on transcriptional dysregulation and disruptions to human-specific zinc finger proteins
localized to chromosome 19. We also identified cell type-specific vulnerabilities to DNA
damage and cell migration amongst the dorsal and ventral organoids, respectively, which
was consistent with the excitatory and inhibitory neural subpopulations amongst the
unguided neural organoids scRNA Seq, respectively.
We then examined neuron migration in a 3D assembloid model by sparsely
labeling dorsal-ventral forebrain organoids from multiple genotype-lineage combinations.
Light sheet microscopy identified deficits in inhibitory neuron migration and
morphology, but not migration distance, suggesting a complex disruption to cortical
circuitry. This novel combination of cell type characterization, pathway identification,
and circuitry phenotyping provides a novel perspective of how the 15q13.3 deletions
impair prenatal development and can be applied to other NDD models to leverage
understanding of early disease pathogenesis. / Dissertation / Doctor of Science (PhD) / The development of the human brain is a highly complex and tightly regulated
process that requires the participation of multiple cell types throughout development.
Disturbances to the emergence, differentiation, or placement of these cell types can cause
disruptions and local miswiring of neural circuits, which is often associated with
neurodevelopmental disorders (NDDs). The 15q13.3 microdeletion syndrome is a highly
complex condition associated with multiple NDDs and has seldom been studied in a
human context. To address this, we used stem cells derived from a 15q13.3 microdeletion
syndrome cohort and their typically developing familial controls to generate unguided
(“whole brain”) and region-specific organoids to investigate early fetal development
across time.
We used the largest 15q13.3 microdeletion cohort in reported literature to identify
shared disruptions in early developmental milestones such as neurogenesis, neural
migration, and neural patterning. We identified expansion of specific cell populations,
including progenitors that later give rise to mature neurons. Abnormalities persisted in
more mature cell populations, including the inhibitory neurons responsible for
establishing critical microcircuitry in the human cortex. By generating guided organoids
that enrich for excitatory and inhibitory neural populations, we were able to merge the
models to form assembloids, where we captured early migratory and morphological
deficits in inhibitory neuron populations, which is supported by the multi-transcriptomics
experiments performed in both organoid models. This study provides a framework for
examining fetal development in a neurodevelopmental disorder context. By using the
15q13.3 microdeletion background, we found novel disruptions in cell type emergence and circuit formation previously unreported in mouse or 2D neuron models, highlighting
the utility of the phenotyping platform for disease modeling.
|
10 |
Ganglion cell translocation across the retina and its importance for retinal laminationIcha, Jaroslav 15 February 2017 (has links) (PDF)
Correct layering (lamination) of neurons in the central nervous system (CNS) is critical for the tissue functionality. Neuronal lamination is established during development, when the majority of neurons have to move from their birthplace to the appropriate layer, where they function. Therefore, to grasp the logic of CNS development, it is essential to understand the kinetics and modes of the variety of neuronal translocation events. Most of our knowledge about neuronal translocation has been gained using fixed tissue or ex vivo imaging, which is not ideal for such a dynamic process heavily dependent on the surrounding environment. To avoid these limitations, I combined translucent zebrafish embryos with light sheet fluorescence microscopy, which together enabled gentle in toto imaging of neuronal translocation.
I studied the translocation of retinal ganglion cells (RGCs) across the developing zebrafish retina. RGCs are the first neurons that differentiate in the vertebrate retina and are born in a proliferative zone at the retinal apical side. From here, they move basally, spanning the complete apico-basal length of the tissue. They are destined to occupy the most basal layer, where their axons form the optic nerve. Although it was described that RGCs move their soma while being attached to both apical and basal sides of the retina, the kinetics and cell biological mechanisms of somal translocation remained unknown.
Extracting single cell behavior of RGCs from high-resolution movies of their translocation allowed for quantitative analysis of RGC movement. I revealed that RGCs cross the retina in less than two hours in a directionally persistent manner. The movement of RGC soma is a cell autonomously generated process, which requires intact microtubules and actin-dependent basal attachment of cells for speed and efficiency. Unexpectedly, interference with somal translocation leads to a shift towards a multipolar migratory mode, previously not observed for RGCs, in which they temporarily lose both apical and basal attachment and apico-basal polarity. The multipolar mode is overall slower and less directionally persistent, but still allows RGCs to reach the basal retina. However, when RGC translocation is inhibited completely, they differentiate ectopically in the center of the retina, which in turn triggers the formation of ectopic layers of later born neurons. These results highlight the importance of establishing the basal layer of ganglion cells for ensuing retinal lamination. Overall, I generated important advances in the understanding of neuronal translocation and lamination, which might be relevant for other parts of the CNS.
|
Page generated in 0.1141 seconds