The Fluorescent Toolbox for Assessing Protein Location and Function
Ben N. G. Giepmans1,2,
Stephen R. Adams2,
Mark H. Ellisman1 and
Roger Y. Tsien2,3*
1 National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA.
2 Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA.
3 Department of Chemistry and Biochemistry and Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093, USA.
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Fig. 1. Characteristics and applications of types of fluorophores in protein detection. Applications of different targeting methods and types of fluorophores are illustrated on connexin43 (green) and  -tubulin (red) in fibroblasts (A and B) and connexin43 in HeLa cells (D and E) as indicated. The structures of different types of targeting proteins and fluorophores (C) are shown to scale. [Scale bar is 2 nm; reproduced from (58).] Endogenous proteins are labeled using primary antibodies followed by secondary antibodies conjugated to small organic dyes (A) or Fab fragments attached to QDs (B), which are also detected at the EM level; (B) (right), QD 565 at connexin43–based gap junction. Genetically encoded intrinsic FPs (D) or tetracysteine tags labeled with biarsenicals (E) rely on ectopic expression. Tetracysteines on connexin43 were pulse-labeled with FlAsH (green) and subsequently ReAsH (red), thus distinguishing old from new connexins, respectively. ReAsH is also visualized in EM using photooxidation (E) (right). [Reproduced from (37).] Scale bars in (A), (B), and (D), 20 µm (LM); (B), 50 nm (EM); (C), 2 nm; (E), 2 µm.
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Fig. 2. Advanced fluorescent applications with genetic tags. (A to I) Principles of advanced techniques are depicted and explained. Barrels represent cyan, green, yellow, and red fluorescent proteins. X, Y, and Z represent target proteins. Light intensity is indicated by the thickness of the waves. See text for further details.
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Fig. 3. Parallel application of targeting methods and fluorophores. HeLa cells transfected with GFP–-tubulin and tetracysteine–ß-actin were stained with ReAsH. After fixation, cells were immunolabeled for the Golgi matrix protein giantin with QDs and for the mitochondrial enzyme cytochrome c with Cy5 as indicated. DNA was stained with Hoechst 33342. Images were acquired from Z planes that best represent each structure using excitation and emission wavelengths as indicated. Individual channels are false-colored (middle) and merged (bottom). Scale bars, 20 µm.
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