Description
Cell biologists are increasingly using live-cell imaging techniques to provide clues into the fundamental nature of cellular and tissue structure and function. These investigations are being aided by the explosive rate of developments in fluorescent protein, quantum dot, and synthetic fluorophore technology. A number of technical challenges must be overcome in order to perform successful live-cell imaging experiments, including the ability to maintain cells in a healthy state on the microscope stage for extended periods of time. New developments in instrumentation (microscope systems, cameras, filter technology, and illumination devices) have enabled imaging of a variety of dynamic events with high spatial and temporal resolution over a wide range of time scales.
How this lab uses this technology
The Leica STELLARIS platform can be used for live-cell imaging and time-lapse fluorescence microscopy, allowing biological processes to be monitored in living cells over time. When combined with an appropriate environmental control system, temperature, humidity and CO₂ concentration can be maintained during image acquisition to preserve cell viability and physiological conditions.
The system enables repeated acquisition of single optical sections, z-stacks or multiple positions at defined time intervals. This makes it possible to analyse dynamic processes such as cell migration, cell division, intracellular trafficking, organelle dynamics, cytoskeletal reorganization, protein translocation, signal transduction and interactions between cells and microorganisms.
Confocal acquisition parameters can be optimized to reduce photobleaching, phototoxicity and cellular stress during prolonged experiments. This includes adjusting laser intensity, scanning speed, acquisition frequency, spatial resolution and the number of optical sections collected. Sensitive fluorescence detection also facilitates imaging with reduced excitation power, helping to preserve weak fluorescent signals and sample viability.
Depending on the experimental configuration, live-cell imaging can be combined with multichannel fluorescence acquisition, three-dimensional imaging, spectral detection, fluorescence intensity measurements and quantitative analysis of changes occurring over time. The resulting datasets can be used to generate time-lapse sequences and four-dimensional reconstructions, in which three-dimensional spatial information is monitored throughout the experiment.
Live-cell imaging applications include:
Monitoring cell migration, morphology and proliferation.
Analysis of mitosis, cell division and cell death.
Visualization of intracellular transport and vesicle trafficking.
Study of organelle movement and structural changes.
Monitoring protein localization and translocation.
Analysis of calcium, pH, redox or other fluorescent biosensors.
Investigation of cell–cell and host–microorganism interactions.
Time-lapse acquisition of multiple fields or sample positions.
Three-dimensional and four-dimensional imaging of dynamic processes.
The availability of controlled temperature, CO₂, humidity and other environmental conditions depends on the incubation and live-cell modules installed on the microscope.
Technical Resources
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