STKE Focus Issue Imaging: Lights, Camera, Action!

One of my favorite activities in the lab was sitting at the microscope looking at cells. Advances in microscopy and digital video, combined with the use of fluorescent probes have provided biologists with the ability to literally watch signaling in live cells. One of the pivotal discoveries was green fluorescent protein (GFP) from Aequorea victoria , a protein that folds spontaneously into a light-absorbing and light-emitting chromophore. Since the initial discovery of GFP, this protein has been modified through recombinant DNA techniques to create versions that have enhanced emission or altered spectral properties, that is, absorbing and emitting light at different wavelengths from the original GFP. The beauty of GFP and its variants is that it can be attached to other proteins or portions of proteins to allow it to serve as a probe for those proteins or molecules that interact with the fused domain. These GFP fusion proteins are being used to monitor protein localization, protein interactions with other molecules within cells, or to monitor the changes in second messenger concentrations in response to stimuli or pharmacological manipulation. GFP can also be used as a bioluminescent reporter to monitor gene expression in cells and even living organisms, such as the nematode Caenorhabditis elegans . Microscopic techniques and equipment continue to evolve. Edidin and McCaffrey bring a concise discussion of the highlights and the omissions of Methods in Cellular Imaging , which they describe as "a compendium that is of great value for both beginners and experienced microscopist." Part of this Book Review includes a table of particularly useful Web sites for learning about microscopy or obtaining the needed equipment, supplies, and image analysis software. Lasers and microscopes have applications beyond simply visualizing the cells; but can also be used to activate or inactivate cellular processes. The STKE Archive has two Protocols that describe how highly focused lasers permit localized activation of gene expression (Minden et al .) or localized activation of neurotransmitter receptors (Dodt et al .). Fluorescent microscopes allow scientists to see the glowing proteins labeled either with a GFP tag or by antibodies conjugated to fluorescent molecules. Fluorescent tags exist that have the appropriate characteristics to allow the labeling of multiple proteins, lipids, or DNA simultaneously. Furthermore, several of the fluorescent probes have the proper spectral properties to allow for monitoring of molecular interactions by the transfer of light energy from one fluorophore on one molecule to a different fluorophore on a nearby molecule. When these fluorescent tags are used in combination with laser scanning confocal microscopes, high spatial and temporal image analysis can be performed. This week the STKE features two Protocols that detail methods that rely on GFP and GFP variants to study protein-lipid interactions (Balla and Varnai) or changes in organellar and cytoplasmic calcium concentrations (Shimonozo et al .). The STKE Archive also has a Protocol by Siegel et al . that describes how to use fluorescent resonance energy transfer (FRET) to monitor molecular interactions. The ability to monitor protein interactions and perform live cell imaging allows scientists to justify biochemical experiments with microscopic techniques. Sometimes the biochemical results appear accurate, and sometimes in live cells the associations identified biochemically can be difficult to detect. A Perspective by Edidin from the archive discusses these issues as they relate to lipid rafts and the complexity of reconciling the biochemical data with the in vivo imaging data. This is also a main topic of the Forum on lipid rafts. We invite you to explore the STKE Protocols and join in the Forum on Methodology to share tips or ask for help. If your lab has methods that would be of great interest to the signal transduction community, please let us know by sending us a Feedback message ( ). Featured in This Focus Issue on Imaging Related Resources at STKE