Abstract
Imaging of living cells and tissue is now common in many fields of the life and physical sciences, and is instrumental in revealing a great deal about cellular dynamics and function. It is crucial when performing such experiments that cell viability is at the forefront of any measurement to ensure that the physiological and biological processes that are under investigation are not altered in any way. Many cells and tissues are not normally exposed to light during their life cycle, so it is important for microscopy applications to minimize light exposure, which can cause phototoxicity. To ensure minimal light exposure, it is crucial that microscope systems are optimized to collect as much light as possible. This can be achieved using superior-quality optical components and state-of-the-art detectors. This Commentary discusses how to set up a suitable environment on the microscope stage to maintain living cells. There is also a focus on general and imaging-platform-specific ways to optimize the efficiency of light throughput and detection. With an efficient optical microscope and a good detector, the light exposure can be minimized during live-cell imaging, thus minimizing phototoxicity and maintaining cell viability. Brief suggestions for useful microscope accessories as well as available fluorescence tools are also presented. Finally, a flow chart is provided to assist readers in choosing the appropriate imaging platform for their experimental systems.
Highlights
Live-cell microscopy has been accessible for decades, as is evident from a movie that was taken with 16-mm film over 50 years ago of a neutrophil chasing a bacterium (David Rogers, Vanderbilt University, http://www.biochemweb.org/neutrophil.shtml)
RNAi knockdown of endogenous proteins followed by expression of fluorescent proteins (FPs) constructs by an inducible promoter is recommended to ensure physiological levels of protein expression
We are just scratching the surface with the suggestions provided (Box 2). These probes can be used in combination with photo-activation or fluorescence recovery after photobleaching (FRAP) and other bleaching techniques (Snapp et al, 2003), but care must be taken to ensure that the cells are not exposed to unnecessary amounts of light
Summary
Live-cell microscopy has been accessible for decades, as is evident from a movie that was taken with 16-mm film over 50 years ago of a neutrophil chasing a bacterium (David Rogers, Vanderbilt University, http://www.biochemweb.org/neutrophil.shtml). For live-cell imaging, it is best to reduce the amount of excitation light by optimizing the efficiency of the light path through the microscope, and by using detectors that are optimized to detect most of the fluorescence emission. Cellular contamination Contamination of cells with bacteria, mold or yeast can be visualized when imaging cells (http://www.microscopyu.com/articles/ livecellimaging/livecellmaintenance.html). Image acquisition The key to live-cell fluorescence microscopy is to collect as much fluorescent light as possible so that incident light can be decreased, thereby reducing phototoxic effects. This goal can be achieved by implementing the following three measures. An easy and relatively affordable way to improve the efficiency of light throughput on any microscope is to replace older,
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