Successful imaging requires overcoming three key challenges, notes Stuart Logan, imaging specialist at PerkinElmer, who helped conduct an imaging workshop at the conference.
“The first challenge is dealing with live specimens, where phototoxicity issues can have damaging effects on the organism. Short wavelengths may cause DNA damage. Exposure to fluorescent light may also cause damage through heating. Other concerns are photobleaching (due to the signal rapidly fading under illumination) and the ability to detect low level fluorescence. Finally, because living systems are dynamic, the speed of capture must be fast enough to capture biological changes.”
A second challenge relates to the amount of data generated. “When running long time-lapse experiments, one generates huge volumes of data. It is common to generate greater than 30 gigabytes of data for a single experiment.”
Analyzing the data presents the third major challenge to successful imaging. “Image analysis requires accuracy, reliability, and comparability of statistical information. Software must be able to automatically generate quantitative results for comparison and publication from a large body of image files. Further, because there may be many different users, the software needs to be highly intuitive and easy to use.”
Claire Stewart, associate product manager, described PerkinElmer's approach to solving these issues. “For live-cell imaging, we offer our UltraVIEW® VoX 3D system. It is the only 3-D spinning disk system for acquisition to analysis. Spinning disk microscopy features rotating disks that bear thousands of pinholes on their surfaces. This allows simultaneous collection of multiple data points. One can use a lower dose of laser light because there is faster tracking of the sample. This technology, controlled by software designed for imaging of live specimens, allows capture and storage of images at a very high frame rate minimizing phototoxicity and photobleaching.”
For reliable, statistically unbiased analysis of large numbers of images, particularly from high-content imaging systems, the company suggests the Columbus™ Image Data Management and Analysis System. “Multiple users can easily store, access, and evaluate their data. Further, data can be accessed, evaluated, and measured using a web browser.”
For a more in-depth analysis, PerkinElmer offers Volocity® 3D Image Analysis Software. “To truly understand the biology of your samples, you need to view and evaluate data in 3-D,” Stewart reported. “Only then can you obtain a more complete understanding of the complex interactions that occur among cellular structures.”
Zebrafish live-cell microscopy is gaining in popularity as a model system because it can provide unmatched views of the inner complexity and workings at the resolution of the cell. As further improvements in the instrumentation and ability to analyze complex data occur, researchers will continue to develop more insights into the complex living processes from the single-cell level to functions within the entire organism.