Live cell Imaging

A wide variety of cell types, including immune cells, have been observed to frequently interact via transient, long-distance membrane connections [1–17]. However, considerable heterogeneity in their structure, mode of formation and functional properties has emerged, suggesting the existence of distinct subclasses [18–21]. Open-ended tunneling nanotubes allow for the trafficking of cytoplasmic material, e.g. endocytic vesicles, or the transmission of calcium

Advances in regenerative nanomedicine raise a host of ethical, legal, and social questions that healthcare providers and scientists will need to consider. These questions and concerns include definitions, appropriate applications, dual use, potential risks, regulations, and access. In this chapter, we provide an overview of the questions and concerns and recommend proactive consideration and solutions.

One of the ultimate goals of biological research is to understand mechanisms of cell function within living organisms. With this in mind, many sophisticated technologies that allow us to inspect macromolecular structure in exquisite detail have been developed. Although knowledge of structure derived from techniques such as X-ray crystallography and nuclear magnetic resonance is of vital importance, these approaches cannot reveal the remarkable complexity of molecular interactions that exists in vivo. With this in mind, this review focuses on the use of microscopy techniques to analyze cell structure and function. We describe the different basic microscopic methodologies and how the routine techniques are best applied to particular biological problems. We also emphasize the specific capabilities and uses of light and electron microscopy and highlight their individual advantages and disadvantages. For completion, we also comment on the alternative possibilities provided by a variety of advanced imaging technologies. We hope that this brief analysis of the undoubted power of microscopy techniques will be enough to stimulate a wider participation in this rapidly developing area of biological discovery.

Chitosan has been reported to inhibit spore germination and mycelial growth in plant pathogens, but its mode of antifungal action is poorly understood. Following chitosan treatment, we characterized plasma membrane permeabilization, and cell death and lysis in the experimental model, Neurospora crassa. Rhodamine-labeled chitosan was used to show that chitosan is internalized by fungal cells. Cell viability stains and the calcium reporter, aequorin, were used to monitor plasma membrane permeabilization and cell death. Chitosan permeabilization of the fungal plasma membrane and its uptake into fungal cells was found to be energy dependent but not to involve endocytosis. Different cell types (conidia, germ tubes and vegetative hyphae) exhibited differential sensitivity to chitosan with ungerminated conidia being the most sensitive.

Rapid movements of live tissues during the acquisition of 3D image stacks can result in misalignments between successive image slices. The remodeling of the muscles in Drosophila metamorphosis is an example where sporadic motion during image acquisition impede image analysis and volume visualization. Most of the image stack registration algorithms applied in microscopy are aimed at the linear alignment of fixed histological sections. However, live muscles are nonrigid objects and their contractions and relaxations represent nonlinear transformations that cannot be properly rectified by applying purely linear registration methods. We developed a fully automated area-based nonrigid stack registration (NSR) method that minimizes the mean square error of intensities between successive image slices. The mapping function is formulated using the thin plate spline (TPS). A hierarchical linear to nonlinear, coarse to fine matching strategy is applied to ensure stability and fast convergence. Topological structure is preserved by constraining the step size of the nonlinear transformation. To assess the accuracy of 3D reconstruction, we propose a new benchmarking method that measures geometrical features of restored nuclei. We tested our algorithm on image stacks generated by laser scanning confocal microscopy that show live muscles during the prepupal stage of Drosophila metamorphosis. Our registration algorithm is able to restore image stacks that are distorted by periodic contraction of muscles. Quantitative assessment of registration performance agrees well with qualitative visual inspection. Our NSR method is able to restore image stacks for the purpose of visualization and quantitative analysis of Drosophila metamorphosis and, potentially, various other processes in developmental biology studied by 3D live cell microscopy.

cAMP-dependent protein kinase (PKA) mediates key cellular processes via compartmentalized activity, and the ability to track its activity in living cells should help increase our understanding of this precise regulation. Here, through systematic testing of new fluorescent proteins, we developed a new FRET-based A-kinase activity reporter (AKAR), AKAR3, with a dynamic range of 31-41%, twice that of predecessors. Visualization of PKA activity at plasma membrane, cytoplasm, nucleus, and mitochondria was achieved. Targeting AKAR3 to outer mitochondrial membrane revealed that basal PKA activity at mitochondria differs from that in the cytoplasm, indicating differential regulation of PKA activity at different subcellular locations.

Telomeres are specialized nucleoprotein structures, which protect chromosome ends and have been implicated in the ageing process. Telomere shortening has been shown to contribute to a persistent DNA damage response (DDR) during replicative senescence, the irreversible loss of division potential of somatic cells. Similarly, persistent DDR foci can be found in stress-induced senescence, although their nature is not understood. Here we show, using immuno-fluorescent in situ hybridization and ChIP, that up to half of the DNA damage foci in stress-induced senescence are located at telomeres irrespective of telomerase activity. Moreover, live-cell imaging experiments reveal that all persistent foci are associated with telomeres. Finally, we report an age-dependent increase in frequencies of telomere-associated foci in gut and liver of mice, occurring irrespectively of telomere length. We conclude that telomeres are important targets for stress in vitro and in vivo and this has important c...

The identification and characterization of many biological substructures at high resolution requires the use of electron microscopy (EM) technologies. Scanning electron microscopy (SEM) allows the resolution of cellular structures to approximately 3 nm and has facilitated the direct visualization of macromolecular structures, such as nuclear pore complexes (NPCs), which are essential for nucleo-cytoplasmic molecular trafficking. However, SEM generates only static images of fixed samples and therefore cannot give unambiguous information about protein dynamics. The investigation of active processes and analysis of protein dynamics has greatly benefited from the development of molecular biology techniques whereby vectors can be generated and transfected into tissue culture cells for the expression of specific proteins tagged with a fluorescent moiety for real-time light microscopy visualization. As light microscopy is limited in its powers of resolution relative to electron microscopy, it has been important to adapt a protocol for the processing of samples for real-time imaging by conventional light microscopy with protein labels that can also be identified by SEM. This allows correlation of dynamic events with high resolution molecular and structural identification. This method describes the use of GFP for tracking the dynamic distribution of NPC components in real-time throughout the cell cycle and for high resolution immuno-SEM labeling to determine localization at the nanometer level.

Chitosan has been reported to inhibit spore germination and mycelial growth in plant pathogens, but its mode of antifungal action is poorly understood. Following chitosan treatment, we characterized plasma membrane permeabilization, and cell death and lysis in the experimental model, Neurospora crassa. Rhodamine-labeled chitosan was used to show that chitosan is internalized by fungal cells. Cell viability stains and the calcium reporter, aequorin, were used to monitor plasma membrane permeabilization and cell death. Chitosan permeabilization of the fungal plasma membrane and its uptake into fungal cells was found to be energy dependent but not to involve endocytosis. Different cell types (conidia, germ tubes and vegetative hyphae) exhibited differential sensitivity to chitosan with ungerminated conidia being the most sensitive.

The delivery of proteins from the plasma membrane to the lysosome for degradation is essential for normal cellular function. There is now a good understanding of the protein complexes involved in sorting proteins at the plasma membrane and into the intralumenal vesicles of the multi-vesicular body. A combination of cell free content mixing assays and live-cell imaging has dissected out the final step in delivery of macromolecules to the lysosome from the multi-vesicular body and provided insights into the molecular mechanisms by which late endosomes and lysosomes exchange lumenal contents. The endocytic pathway has provided a platform with which to understand the autophagic and phagocytic pathways, but the fine details of how traffic through these pathways is regulated remain to be determined.

Advances in regenerative nanomedicine raise a host of ethical, legal, and social questions that healthcare providers and scientists will need to consider. These questions and concerns include definitions, appropriate applications, dual use, potential risks, regulations, and access. In this chapter, we provide an overview of the questions and concerns and recommend proactive consideration and solutions.