In Vivo Cellular Imaging Using Fluorescent Proteins Methods and Prot
The discovery and genetic engineering of fluorescent proteins has revolutionized cell biology. What was previously invisible in the cell often can be made visible with the use of fluorescent proteins. In Vivo Cellular Imaging Using Fluorescent Prote
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Introduction The first green fluorescent proteins (GFPs) were cloned from hydrozoans—the jelly-fish Aequorea victoria and the sea pansy Renilla reniformis (1, 2). In the last decade, a variety of GFP-type protein homologs have been discovered in hard reef corals belonging to the order Scleractinia and in other anthozoans lacking a hard skeleton (soft corals, sea anemones, zoanthids, corallimorphs), often referred to as “reef FPs” (3–11). They were also discovered in other marine organisms, such as crustaceans and even a basic chordate animal, amphioxus (12, 13). Thus, marine organisms have become a new source of novel and diverse GFP-type proteins.
Robert M. Hoffman (ed.), In Vivo Cellular Imaging Using Fluorescent Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 872, DOI 10.1007/978-1-61779-797-2_15, © Springer Science+Business Media New York 2012
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Reef FPs now provide a multitude of genetically-expressible proteins for use in fluorescence imaging applications. Their colors are significantly more diverse than those of wtGFP, with excitation wavelengths extending from the violet to the orange (400– 590 nm) and emission maxima covering almost the full rainbow color palette, from blue to red (440–660 nm). They frequently have high quantum yields, are typically extremely stable, and resistant to photobleaching (13–15). While highly advantageous for conventional imaging methods, reef-FPs’ resistance to photobleaching limited their applications in dynamic studies using fluorescence recovery after photobleaching (FRAP) techniques, since the light levels needed to bring about bleaching were phototoxic to cells. The recent discovery of the photoactive fluorescent proteins (PAFPs) in anthozoans, which respond to irradiation, by altering their optical properties, now provides an improvement to FRAP-based techniques (8, 15–17). Their fluorescent state can be precisely controlled (dim/bright; or converted from one color to another) by irradiation. PAFPs enable direct color labeling and selective monitoring of labeled proteins, organelles and cells. PAFP-based imaging is less phototoxic to cells, does not require continuous imaging, and is a much more versatile method for studying dynamic processes in living cells. Another novel application of photoactivatable reef FPs is the recent development of super-resolution imaging by photoactivated localization microscopy (PALM) and related methods. Irradiation of PAFPs is used to generate images that precisely localize single fluorophores to within a few tens of nanometers (18). GFP-type proteins contribute a surprisingly high fraction to the overall soluble protein content of anthozoan tissues, ranging from 4.5% to 14.7% (Salih unpublished data; 10). In the majority of cases, several FPs co-occur within the same anthozoan organism whose visual color patterns are determined by the more highly-expressed proteins (7, 9–11, 19). These more abundant FPs frequently mask the rare types when tissues are screened for GFP-type proteins using conventional
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