Fluorescence microscopy: Established and emerging methods, experimental strategies, and applications in immunology

Cutting-edge biophysical technologies including total internal reflection fluorescence microscopy, single molecule fluorescence, single channel opening events, fluorescence resonance energy transfer, high-speed exposures, two-photon imaging, fluorescence lifetime imaging, and other tools are becoming increasingly important in immunology as they link molecular events to cellular physiology, a key goal of modern immunology. The primary concern in all forms of microscopy is the generation of contrast; for fluorescence microscopy contrast can be thought of as the difference in intensity between the cell and background, the signal-to-noise ratio. High information-content images can be formed by enhancing the signal, suppressing the noise, or both. As improved tools, such as ICCD and EMCCD cameras, become available for fluorescence imaging in molecular and cellular immunology, it is important to optimize other aspects of the imaging system. Numerous practical strategies to enhance fluorescence microscopy experiments are reviewed. The use of instrumentation such as light traps, cameras, objectives, improved fluorescent labels, and image filtration routines applicable to low light level experiments are discussed. New methodologies providing resolution well beyond that given by the Rayleigh criterion are outlined. Ongoing and future developments in fluorescence microscopy instrumentation and technique are reviewed. This review is intended to address situations where the signal is weak, which is important for emerging techniques stressing super-resolution or live cell dynamics, but is less important for conventional applications such as indirect immunofluorescence. This review provides a broad integrative discussion of fluorescence microscopy with selected applications in immunology. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56150/1/20455_ftp.pd

Spatial Dynamics and the Mechanoresponse in CD4+ T Cell Activation

The activation of naïve CD4+ T cells by antigen presenting cells is a critical step in the response of the immune system to foreign pathogens and in its acclimation to host tissues. Activation of naïve T cells proceeds through TCR engagement and is further augmented by CD28 costimulation: ensuring T cell survival and conferring numerous functional capabilities. The work in this dissertation highlights the spatial and temporal dynamics that regulate the initial coupling of CD28 with TCR signaling and also dissects the mechanical properties conferred by downstream effectors that are required to relay CD28 costimulation. A reaction-diffusion model that describes the spatial regulation of costimulation in activating human T cells is developed. The Src kinase Lck, though predominantly cytosolic, is an ideal candidate for the coupling of the TCR and CD28 pathways. Membrane associations bring Lck in contact with these receptors, where mediation of its active state by kinase activity and regulation of its spatial dynamics dictate its capacity to integrate early TCR and CD28 signaling. This developed reaction-diffusion model focusing on Lck is then extrapolated to mouse cells that do not share similar sensitivity to segregation of TCR and CD28 triggering: indicating that while Lck is essential for costimulation, it does not confer spatial sensitivity in activating mouse T cells. A comparison of human and mouse cells demonstrate underlying differences in the diffusivity of Lck across the membrane and the enrichment of the cytoskeleton at the interface. The role of the cytoskeleton in generating TCR-driven contractile forces is then investigated through use of micropillar arrays. This approach also enables the quantification of forces generated by T cells during cellular activation. The impact of CD28 costimulation on TCR-driven force generation is assessed and noted to increase cellular forces by 80% beyond what is induced through TCR triggering. By manipulating the presentation of CD28 activation, CD28 is determined to be a mechanoresponsive receptor that is not directly responsible for mechanosensitivty. Rather, CD28 mediates a change in cellular forces through PI3 kinase, whose inhibition normalizes force generation in T cells activated by TCR and those costimulated with TCR and CD28. Downstream of PI3 kinase, PDK1 is identified as being essential in both TCR and CD28 costimulatory force generation; inhibition of PDK1 fully abrogates cellular forces. Lastly, we qualitatively characterize T cell activation on micropillar arrays, where their complex topology reveals a multiphasic behavior during activation. Whereas T cells activated on planar surfaces are relatively stationary, T cells activated on micropillars slowly migrate towards the base of the array. Forces exerted during this migration are substantially greater than those previously measured, and the slow migration leads to the characterization of multiple phases and the relocalization of key cellular proteins

Image processing and analysis methods in quantitative endothelial cell biology

This thesis details the development of computerised image processing and analysis pipelines for quantitative evaluation of microscope image data acquired in endothelial vascular biology experimentation. The overarching objective of this work was to advance our understanding of the cell biology of cardiovascular processes; principally involving haemostasis, thrombosis, and inflammation. Bioinformatics techniques are increasingly necessary to extract and evaluate information from biological experimentation. In cell biology advances in microscopy and the increased acquisition of large scale digital image data sets have created a need for automated image processing and data analysis. The development, testing, and evaluation of three computerised workflows for analysis of microscopy images investigating cardiovascular cell biology are described here. The first image analysis pipeline extracts morphometric features from high-throughput experiments imaging endothelial cells and organelles. Segmentation of endothelial cells and their organelles followed by extraction of morphometric features provides a rich quantitative data set to investigate haemostatic mechanisms. A second image processing workflow was applied to platelet images obtained from super-resolution microscopy, and used in a proof-of-principle study of a new platelet dense-granule deficiency diagnostic method. The method was able to efficiently differentiate between healthy volunteers and three patients with Hermansky-Pudlak syndrome. This was achieved by segmenting and counting the number of CD63-positive structures per platelet, allowing for the differentiation of patients from control volunteers with 99\% confidence. The final workflow described is a video analysis method that quantifies interactions of leukocytes with an endothelial monolayer. Phase contrast microscopy videos were analysed with a Haar-like features object detection and custom tracking method to quantify the dynamic interaction of rolling leukocytes. This technique provides much more information than a manual evaluation and was found to give a tracking accuracy of 92\%. These three methodologies provide a toolkit to further biological understanding of multiple facets of cardiovascular behaviour

Swim Like Your Lifecycle Depends On It. Investigating Motility of Leishmania mexicana; its Impact on Parasite Lifecycle Progression and Infectivity.

The motility of Leishmania promastigote parasites is important for survival during host transitions and lifecycle progression. An oscillating flagellum at the anterior end of the promastigotes pulls it through environmental conditions which change significantly during the lifecycle. The parasite morphologically transforms to optimise infection potential. This study adapts a unique method of high-speed, three-dimensional imaging called digital inline holographic microscopy (DIHM) allowing us to examine the movements of Leishmania mexicana promastigotes. We have tracked distinct stages of promastigote parasites over multiple frames to gain information on the swimming patterns of these cells. Quantification of the 3D trajectories reveals stage-specific differences in swimming behaviour. Using this technique we reveal that mammalian-infective metacyclic promastigotes are more capable of swimming in highly viscous solutions, a result which has interesting implications in the ability of this specific stage to transmit through promastigote secretory gel. Additionally, the DIHM technique has allowed us to investigate whether these different stages of Leishmania promastigote are capable of sensing chemicals in their environment. We reveal how distinct chemotaxic capabilities could play a role in the uptake of parasites by host cells during early infection. Mathematically quantifying the cell movements of L. mexicana within contrasting, biologically relevant environments has revealed swimming mechanisms that are essential for the parasite to remain unencumbered by environmental pressures and adapt their motility to reach preferred conditions

Review on Photomicrography based Full Blood Count (FBC) Testing and Recent Advancements

With advancements in related sub-fields, research on photomicrography in life science is emerging and this is a review on its application towards human full blood count testing which is a primary test in medical practices. For a prolonged period of time, analysis of blood samples is the basis for bio medical observations of living creatures. Cell size, shape, constituents, count, ratios are few of the features identified using DIP based analysis and these features provide an overview of the state of human body which is important in identifying present medical conditions and indicating possible future complications. In addition, functionality of the immune system is observed using results of blood tests. In FBC tests, identification of different blood cell types and counting the number of cells of each type is required to obtain results. Literature discuss various techniques and methods and this article presents an insightful review on human blood cell morphology, photomicrography, digital image processing of photomicrographs, feature extraction and classification, and recent advances. Integration of emerging technologies such as microfluidics, micro-electromechanical systems, and artificial intelligence based image processing algorithms and classifiers with cell sensing have enabled exploration of novel research directions in blood testing applications.

F-actin bundles direct the initiation and orientation of lamellipodia through adhesion-based signaling

During cell migration, F-actin bundles/filopodia serve as templates for formation and orientation of lamellipodia and prime their stabilization by adhesion-based PI3K signaling.Mesenchymal cells such as fibroblasts are weakly polarized and reorient directionality by a lamellipodial branching mechanism that is stabilized by phosphoinositide 3-kinase (PI3K) signaling. However, the mechanisms by which new lamellipodia are initiated and directed are unknown. Using total internal reflection fluorescence microscopy to monitor cytoskeletal and signaling dynamics in migrating cells, we show that peripheral F-actin bundles/filopodia containing fascin-1 serve as templates for formation and orientation of lamellipodia. Accordingly, modulation of fascin-1 expression tunes cell shape, quantified as the number of morphological extensions. Ratiometric imaging reveals that F-actin bundles/filopodia play both structural and signaling roles, as they prime the activation of PI3K signaling mediated by integrins and focal adhesion kinase. Depletion of fascin-1 ablated fibroblast haptotaxis on fibronectin but not platelet-derived growth factor chemotaxis. Based on these findings, we conceptualize haptotactic sensing as an exploration, with F-actin bundles directing and lamellipodia propagating the process and with signaling mediated by adhesions playing the role of integrator

Pre-Clinical Development of Best-in-Class Zn0.4Fe2.6O4 Magnetic Nanoparticles for Thermal Treatment of Brain Glioblastoma

Nanomaterials are intensely researched and developed for a wide range of applications. The focus of this work is on developing novel nanomaterials with augmented physicochemical properties for biomedical applications. Specifically, developing magnetic nanoparticles for thermal treatment of neoplasms as this method offers a potentially drug-free approach to cancer treatment currently approved for clinical use for a limited number of malignancies and undergoing further trials for assessing its effect on others. To date, several procedures have been established to produce nanoparticles with variable shapes, sizes and compositions and their effect on various technologies are intensely investigated. Among both anisotropic and isotropic magnetic nanoparticles synthesised as part of this work, superparamagnetic zinc doped ferrite nanoparticles were the most suitable for further development owing to their high magnetisation, superparamagnetic nature, low anisotropy and biocompatibility as characterised by their chemical and physical attributes and compared with iron oxide nanoparticles of same size and morphology. These nanoparticles have been developed using liquid chemistry routes under high temperature and pressure. Their extensive characterisation renders them as the best-in-class nanoparticles in terms of their magnetic properties and size exceeding the magnetic properties of the next most magnetic zinc ferrite synthesised to date whilst having ten times smaller magnetic volume. Their ability to convert magnetic energy to heat (magnetothermal) and light to heat (photothermal) has been assessed with photothermia being far more efficient than magnetothermia both in suspension and in cellular confinement. Magnetothermal conversion was similar to other superparamagnetic materials and of limited clinical use while photothermal conversion showed enhanced performance achieving complete cell death in 10 minutes using clinically relevant settings. The nanoparticles showed extensive cellular uptake in vitro on brain glioblastoma cells as indicated by imaging and magnetometry. The biocompatibility of the nanoparticles has been assessed with several techniques to assess mitochondrial function, cell membrane integrity and clonogenicity indicating a well-tolerated material of similar toxicity to iron oxide which itself is cleared for medical use by the Food and Drug Administration and the European medicines Agency. A practical treatment time has been determined to induce preferentially irreversible apoptosis than necrosis in in vitro experiments as a result of apoptosis-related proteins expression or inhibition and reactive oxygen species formation before, during and after thermal treatment. Biodistribution studies made use of nuclear medicine tomographic imaging techniques to monitor the biodistribution profile of the nanomaterial in real-time including positron emission tomography and single photon emission computed tomography integrated with computed tomography on physiological and immunocompromised mice via intravenous and intranasal administration. The nanomaterial mainly accumulates in organs involved in the clearance pathway; the liver and the kidneys with a small amount of material reaching the brain

Scaffold dimensionality and confinement determine single cell morphology and migration

This thesis describes a highly interdisciplinary approach to discern the differing impact of scaffold dimensionality and physical space restrictions on the behavior of single cells. Rolled-up nanotechnology is employed to fabricate three-dimensional (3D) SiO/SiO2 microtube geometries of varied diameter, that after a biofunctionalization step are shown to support the growth of U2OS and six different types of stem cells. Cell confinement quantifiable through the given microtube diameter is tolerated by U2OS cells through a remarkable elongation of the cell body and nucleus down to a certain threshold, while the integrity of the DNA is maintained. This confinement for NSPCs also leads to the approaching of the in vivo morphology, underlining the space-restrictive property of live tissue. The dimensionality of the cell culture scaffold however is identified as the major determiner of NSPC migration characteristics and leads to a morphologically distinct mesenchymal to amoeboid migration mode transition. The 3D microtube migration is characterized by exclusively filopodia protrusion formation, a higher dependence on actin polymerization and adopts aspects of in vivo-reported saltatory movement. The reported findings contribute to the determination of biomaterial scaffold design principles and advance our current understanding of how physical properties of the extracellular environment affect cell migration characteristics