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Live-Cell Imaging at the Next Level

Benefits of Confocal Fluorescence Microscopy for Studying Living Cells

Opterra II logo4 2015

·         Live-Cell Studies

Live-cell studies have become a requirement for biologists researching cellular function to understand basic cell biology, disease causes and cures, and reproductive and developmental processes. Live-cell imaging is now a standard technique used in live-cell studies.

 

Live-cell imaging has its origins in the early 20th century with micro-cinematography, and the extension to video microscopy by pioneers such as Shinye Inoue in the 1980s. The development of fluorescent proteins in the mid-1990s drove the expansion of live-cell microscopy imaging that continues today.

 

The ability to genetically encode expression of fluorescent markers to target specific cellular structures and compartments for the study of cellular function continues to draw increasing numbers of scientists to the methodology. The use of fluorescent markers for live-cell imaging also has been a driver in live-cell confocal instrumentation development. 

 

Multipoint Confocal Microscopy

Imaging with a live-cell microscope and fluorescence probes is subject to the “iron triangle” of resolution, speed and sensitivity. This iron triangle illustrates the immutable balance of speed, intensity, and spatial resolution. To improve performance for one facet requires that some degree of performance in one or more of the other facets be sacrificed.

 

For example, if more spatial resolution is needed, some speed and/or intensity must be forfeited. In most confocal live-cell imaging systems, such as spinning disk, the imaging settings in terms of aperture selection are fixed and these three essential parameters cannot be adjusted. As a result, the imaging cannot be customized to meet specific experimental needs. The newest technology in live-cell microscopy, such as that available with Bruker’s Opterra II system, with its selectable apertures, has the ability to adjust speed, resolution, and intensity as needed to accommodate varying experimental conditions across an array of life science research areas. 

Confocal live-cell imaging is now capable of four dimensional (4D) imaging.  4D microscopy imaging refers to fast three dimensional time-lapse imaging, and it too has become a standard requirement for biological research. 4D time-lapse imaging can record structures labelled by fluorescence-tagged proteins moving in all directions in cells and has been proven capable of revealing previously hidden aspects of cellular processes and protein function.

The Opterra II tightly integrates scanner, CCD camera, illumination, filtering, and motion control devices to provide high-speed 4D imaging capabilities, giving researchers the flexibility they need to adapt acquisition settings to their particular application needs.  It also brings light as a stimulus to live-cell imaging experiments, allowing photomanipulation (bleaching, conversion, ablation) simultaneously with imaging.

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Max projection of C. elegans labeled with GFP.

·         Super-Resolution Microscopy

Other major advances in fluorescent imaging enable single-molecule localization of signals at super-resolution levels (a 10x increase in resolution). Bruker’s Vutara 350 brings video-rate, live-cell 3D imaging to super resolution levels.  Its high acquisition speed, coupled with real-time GPU processing, means that researchers now can study cytoarchitecture dynamics in live-cell imaging paradigms at the super-resolution level.  Vutara’s patented Biplane technology has set a new standard in 3D super resolution localization precision that, coupled with its advanced analysis features, provides a turnkey solution for visualization and measurement of molecular composition and dynamics in sub-cellular structures.

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