Practical Considerations for Long-Term Time-Lapse Imaging of Epithelial Morphogenesis in Three-Dimensional Organotypic Cultures

Synapses are continuously formed and eliminated throughout life in the mammalian brain, and emerging evidence suggests that this structural plasticity underlies experience-dependent changes of brain functions such as learning and long-term memory formation. However, it is generally difficult to understand how the rewiring of synaptic circuitry observed in vivo eventually relates to changes in animal's behavior. This is because afferent/efferent connections and local synaptic circuitries are very complicated in most brain regions, hence it is largely unclear how sensorimotor information is conveyed, integrated, and processed through a brain region that is imaged. The cerebellar cortex provides a particularly useful model to challenge this problem because of its simple and well-defined synaptic circuitry. However, owing to the technical difficulty of chronic in vivo imaging in the cerebellum, it remains unclear how cerebellar neurons dynamically change their structures over a long period of time. Here, we showed that the commonly used method for neocortical in vivo imaging was not ideal for long-term imaging of cerebellar neurons, but simple optimization of the procedure significantly improved the success rate and the maximum time window of chronic imaging. The optimized method can be used in both neonatal and adult mice and allows time-lapse imaging of cerebellar neurons for more than 5 mo in ∼80% of animals. This method allows vital observation of dynamic cellular processes such as developmental refinement of synaptic circuitry as well as long-term changes of neuronal structures in adult cerebellum under longitudinal behavioral manipulations.

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Toxicity Evaluation of Two Dental Composites: Three-Dimensional Confocal Laser Scanning Microscopy Time-Lapse Imaging of Cell Behavior

Microscopy and Microanalysis ◽

10.1017/s1431927613000433 ◽

2013 ◽

Vol 19 (3) ◽

pp. 596-607 ◽

Cited By ~ 2

Author(s):

Ghania Nina Attik ◽

Nelly Pradelle-Plasse ◽

Doris Campos ◽

Pierre Colon ◽

Brigitte Grosgogeat

Keyword(s):

Confocal Laser Scanning Microscopy ◽

Laser Scanning ◽

Cell Metabolism ◽

Three Dimensional ◽

Time Lapse ◽

Laser Scanning Microscopy ◽

Confocal Laser Scanning ◽

Dental Composites ◽

Confocal Laser ◽

Time Lapse Imaging

AbstractThe purpose of this study was to investigate thein vitrobiocompatibility of two dental composites (namely A and B) with similar chemical composition used for direct restoration using three-dimensional confocal laser scanning microscopy (CLSM) time-lapse imaging. Time-lapse imaging was performed on cultured human HGF-1 fibroblast-like cells after staining using Live/Dead®. Image analysis showed a higher mortality rate in the presence of composite A than composite B. The viability rate decreased in a time-dependent manner during the 5 h of exposure. Morphological alterations were associated with toxic effects; cells were enlarged and more rounded in the presence of composite A as shown by F-actin and cell nuclei staining. Resazurin assay was used to confirm the active potential of composites in cell metabolism; results showed severe cytotoxic effects in the presence of both no light-curing composites after 24 h of direct contact. However, extracts of polymerized composites induced a moderate decrease in cell metabolism after the same incubation period. Composite B was significantly better tolerated than composite A at all investigated end points and all time points. The finding confirmed that the used CLSM method was sufficiently sensitive to differentiate the biocompatibility behavior of two composites based on similar methacrylate monomers.

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Rapid disruption of the cortical microcirculation after mild traumatic brain injury

10.1101/788455 ◽

2019 ◽

Author(s):

Ellen D. Witkowski ◽

Şefik Evren Erdener ◽

Kıvılcım Kılıç ◽

Sreekanth Kura ◽

Jianbo Tang ◽

...

Keyword(s):

Traumatic Brain Injury ◽

Brain Injury ◽

Vascular Dysfunction ◽

Metabolic Stress ◽

Time Lapse ◽

Post Injury ◽

Injury Model ◽

Time Lapse Imaging

AbstractTraumatic brain injury (TBI) is a major source of cognitive deficits affecting millions annually. The bulk of human injuries are mild, causing little or no macroscopic damage to neural tissue, yet can still lead to long-term neuropathology manifesting months or years later. Although the cellular stressors that ultimately lead to chronic pathology are poorly defined, one notable candidate is metabolic stress due to reduced cerebral blood flow (CBF), which is common to many forms of TBI. Here we used high-resolution in vivo intracranial imaging in a rodent injury model to characterize deficits in the cortical microcirculation during both acute and chronic phases after mild TBI. We found that CBF dropped precipitously during immediate post-injury periods, decreasing to less than half of baseline levels within minutes and remaining suppressed for 1.5-2 hours. Repeated time-lapse imaging of the cortical microvasculature revealed further striking flow deficits in the capillary network, where 18% of vessels were completely occluded for extended periods after injury, and an additional >50% showed substantial stoppages. Decreased CBF was paralleled by extensive vasoconstriction that is likely to contribute to loss of flow. Our data indicate a major role for vascular dysfunction in even mild forms of TBI, and suggest that acute post-injury periods may be key therapeutic windows for interventions that restore flow and mitigate metabolic stress.

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Dendritic space-filling requires a neuronal type-specific extracellular permissive signal inDrosophila

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1707467114 ◽

2017 ◽

Vol 114 (38) ◽

pp. E8062-E8071 ◽

Cited By ~ 20

Author(s):

Amy R. Poe ◽

Lingfeng Tang ◽

Bei Wang ◽

Yun Li ◽

Maria L. Sapar ◽

...

Keyword(s):

Dendritic Growth ◽

Tyrosine Phosphatase ◽

Receptive Fields ◽

Time Lapse ◽

Receptor Protein ◽

Space Filling ◽

Leukocyte Antigen ◽

Somatosensory Neurons ◽

Time Lapse Imaging

Neurons sometimes completely fill available space in their receptive fields with evenly spaced dendrites to uniformly sample sensory or synaptic information. The mechanisms that enable neurons to sense and innervate all space in their target tissues are poorly understood. UsingDrosophilasomatosensory neurons as a model, we show that heparan sulfate proteoglycans (HSPGs) Dally and Syndecan on the surface of epidermal cells act as local permissive signals for the dendritic growth and maintenance of space-filling nociceptive C4da neurons, allowing them to innervate the entire skin. Using long-term time-lapse imaging with intactDrosophilalarvae, we found that dendrites grow into HSPG-deficient areas but fail to stay there. HSPGs are necessary to stabilize microtubules in newly formed high-order dendrites. In contrast to C4da neurons, non–space-filling sensory neurons that develop in the same microenvironment do not rely on HSPGs for their dendritic growth. Furthermore, HSPGs do not act by transporting extracellular diffusible ligands or require leukocyte antigen-related (Lar), a receptor protein tyrosine phosphatase (RPTP) and the only knownDrosophilaHSPG receptor, for promoting dendritic growth of space-filling neurons. Interestingly, another RPTP, Ptp69D, promotes dendritic growth of C4da neurons in parallel to HSPGs. Together, our data reveal an HSPG-dependent pathway that specifically allows dendrites of space-filling neurons to innervate all target tissues inDrosophila.

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Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Journal of Visualized Experiments ◽

10.3791/4426 ◽

2012 ◽

Cited By ~ 4

Author(s):

Kylee M. Peterson ◽

Keiko U. Torii

Keyword(s):

High Resolution ◽

Time Lapse ◽

Time Lapse Imaging

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Passive seismic tomography for three-dimensional time-lapse imaging of mining-induced rock mass changes

The Leading Edge ◽

10.1190/1.3694902 ◽

2012 ◽

Vol 31 (3) ◽

pp. 338-345 ◽

Cited By ~ 15

Author(s):

Erik Westman ◽

K. Luxbacher ◽

S. Schafrik

Keyword(s):

Rock Mass ◽

Seismic Tomography ◽

Three Dimensional ◽

Time Lapse ◽

Passive Seismic ◽

Time Lapse Imaging

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Incubator-independent cell-culture perfusion platform for continuous long-term microelectrode array electrophysiology and time-lapse imaging

Royal Society Open Science ◽

10.1098/rsos.150031 ◽

2015 ◽

Vol 2 (6) ◽

pp. 150031 ◽

Cited By ~ 12

Author(s):

Dirk Saalfrank ◽

Anil Krishna Konduri ◽

Shahrzad Latifi ◽

Rouhollah Habibey ◽

Asiyeh Golabchi ◽

...

Keyword(s):

Cell Culture ◽

Microelectrode Array ◽

Low Cost ◽

Time Lapse ◽

Free Cell ◽

Network Architectures ◽

Hippocampal Networks ◽

Time Lapse Imaging

Most in vitro electrophysiology studies extract information and draw conclusions from representative, temporally limited snapshot experiments. This approach bears the risk of missing decisive moments that may make a difference in our understanding of physiological events. This feasibility study presents a simple benchtop cell-culture perfusion system adapted to commercial microelectrode arrays (MEAs), multichannel electrophysiology equipment and common inverted microscopy stages for simultaneous and uninterrupted extracellular electrophysiology and time-lapse imaging at ambient CO 2 levels. The concept relies on a transparent, replica-casted polydimethylsiloxane perfusion cap, gravity- or syringe-pump-driven perfusion and preconditioning of pH-buffered serum-free cell-culture medium to ambient CO 2 levels at physiological temperatures. The low-cost microfluidic in vitro enabling platform, which allows us to image cultures immediately after cell plating, is easy to reproduce and is adaptable to the geometries of different cell-culture containers. It permits the continuous and simultaneous multimodal long-term acquisition or manipulation of optical and electrophysiological parameter sets, thereby considerably widening the range of experimental possibilities. Two exemplary proof-of-concept long-term MEA studies on hippocampal networks illustrate system performance. Continuous extracellular recordings over a period of up to 70 days revealed details on both sudden and gradual neural activity changes in maturing cell ensembles with large intra-day fluctuations. Correlated time-lapse imaging unveiled rather static macroscopic network architectures with previously unreported local morphological oscillations on the timescale of minutes.

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