The time interval between the images should be selected depending on the speed of the target protein movement. Based on the proposed rate of protein redistribution, or on the preliminary data concerning this rate, the frequency of image acquisition within a time series should be reduced as much possible. Indeed, for long time series, too often image acquisition will require many images to be captured, leading to undesirable photobleaching of the photoactivated protein. Generally, 10–30 consecutive images are enough to measure protein mobility and at the same time to avoid undesirable photobleaching/photoconversion effects during visualization.
Examples of Dendra2 Tracking
Here we provide examples of photoactivation and tracking of Dendra2 using different laser scanning confocal microscopes.
Example 1: tracking of Dendra2 fast redistribution within HeLa nucleus; photoactivation by 405-nm laser. A Zeiss LSM 5 LIVE Duoscan confocal microscope with Plan Neofluar 40×/1.3 oil objective (Carl Zeiss, Jena, Germany) was used. HeLa cells were transiently transfected with pDendra2-C vector (Evrogen). Photoactivation was performed at the edge of the HeLa cell nucleus by a short pulse of 405-nm laser light. Further time series was obtained, 1 frame/0.6 s, in two channels: channel 1, excitation 532 nm 8%, emission collected BP 560–675; channel 2, excitation 488 nm 0.1%, emission collected BP 495–555. Diffusion of the photoactivated red fluorescent protein within the nucleus could be accurately monitored, along with its replacement with the nonactivated green fluorescent form (Figure 2).
Example 2: tracking of Dendra2 redistribution from nucleus to cytosol of HeLa cell; photoactivation by 405-nm laser.An Olympus FluoView™ FV1000 with objective UPLSAPO 60 × O NA:1.35 (Olympus, Tokyo, Japan) was used. HeLa cells were transiently transfected with pDendra2-C vector. The protein was photoactivated in the center of nucleus by a 15% 405-nm laser line, SIM Tornado (Olympus) for 200 ms. Further redistribution of both protein forms was tracked. We used the following settings to obtain a time series (1 frame/20 s): zoom 2.4 (88.064 × 88.064 µm image size); 1024 × 1024 pixels; sampling speed 2 µs per pixel; line sequential mode: channel 1, excitation with 488-nm laser line, emission 500–541 nm; channel 2, excitation with 561-nm laser line, emission 576–676 nm; DM405/488/561/633; integration count 3. Redistribution of both the photoactivated red fluorescent protein and the nonactivated green fluorescent form between the nucleus and cytosol was observed (Figure 3).
Example 3: activation and tracking Dendra2 fused with nucleolus protein fibrillarin; photoactivation by 488-nm laser. This experiment was performed using a Leica confocal inverted microscope DMIRE2 TCS SP2 equipped with HCX PL APO Ibd.BL 63× 1.4 NA oil objective (Leica, Wetzlar, Germany) and 125 mW Ar and 1 mW HeNe lasers. Figure 4 shows Dendra2-fibrillarin tracking in nucleus of a HeLa cell transiently transfected with pDendra2-fibrillarin vector (Evrogen). We used the following settings: mode, xyt; format, 512 × 512 pixels; zoom, 13 (18 × 18 µm field of view); scan speed, 400 Hz; beam expander, 3; pinhole, 140 µm; laser, 488 nm 1% power, PMT1, 500–535 nm, gain 725 V (for green fluorescence detection); or laser, 543 nm 20% power, PMT2, 560–680 nm; gain 700 V (for red fluorescence detection). Activation was done at a point within smaller nucleolus using point bleach mode by 25% 488-nm laser for 200 ms. After that, 20 images in red channel were taken with 3-s time interval. As a result, we were able to observe a drastic increase of red signal in the activated nucleolus [region of interest (ROI)1] and further migration of red signal in the nucleoplasm and adjacent nucleolus. A transient wave of Dendra2-fibrillarin migration was clearly detected in a nucleoplasm region near the activation point (ROI 2); only in about 45 s red signal became equalized across the nucleoplasm (compare ROI 2 and 3). Analysis of red signal within the nonactivated nucleolus showed that Dendra2-fibrillarin easily migrates through the nucleolus with a rate comparable to that in the nucleoplasm. So, Dendra2-fibrillarin accumulation occurred first at the side closest to the activated nucleolus (ROI 4), then in the central part (ROI 5), then at the opposite side (ROI 6), but not first at the periphery, then in the central part (as it could be expected). Apparently, such detailed information about migration of fibrillarin in nucleus cannot be obtained in a single experiment using classical approaches based on photobleaching.
We are grateful to T. Zimmermann (Centre de Regulació Genòmica, Barcelona, Spain), D. Ossipov and W. Hempell (Olympus), R. Wolleschensky and M. Kempe (Zeiss), and J. Schroeder (Leica-Microsystems). This work was supported by Russian Academy of Sciences for the program Molecular and Cell Biology, EC FP-6 Integrated Project LSHG-CT-2003-503259, National Institutes of Health (GM070358). D.M.C. and K.A.L. are supported by Grants of the President of Russian Federation MK-8236.2006.4 and Russian Science Support Foundation.
1.) Chudakov, D.M., S. Lukyanov, and K.A. Lukyanov. 2005. Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 23:605-613.
2.) Lippincott-Schwartz, J., N. Altan-Bonnet, and G.H. Patterson. 2003. Photobleaching and photoactivation: following protein dynamics in living cells. Nat. Cell Biol. Suppl:S7-S14.
3.) Lukyanov, K.A., D.M. Chudakov, S. Lukyanov, and V.V. Verkhusha. 2005. Innovation: photoactivatable fluorescent proteins. Nat. Rev. Mol. Cell Biol. 6:885-891.
4.) Zhang, L., N.G. Gurskaya, E.M. Merzlyak, D.B. Stanoverov, N.N. Mudrik, O.N. Samarkina, L.M. Vinokurov, S. Lukyanov, and K.A. Lukyanov. 2007. Method for real-time monitoring of protein degradation at the single cell level. BioTechniques 42:446-450.
5.) Demarco, I.A., A. Periasamy, C.F. Booker, and R.N. Day. 2006. Monitoring dynamic protein interactions with photoquenching FRET. Nat. Methods 3:519-524.
6.) Chudakov, D.M., T.V. Chepurnykh, V.V. Belousov, S. Lukyanov, and K.A. Lukyanov. 2006. Fast and precise protein tracking using repeated reversible photoactivation. Traffic 7:1304-1310.
7.) Hofmann, M., C. Eggeling, S. Jakobs, and S.W. Hell. 2005. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc. Natl. Acad. Sci. USA 102:17565-17569.
8.) Betzig, E., G.H. Patterson, R. Sougrat, O.W. Lindwasser, S. Olenych, J.S. Bonifacino, M.W. Davidson, J. Lippincott-Schwartz, and H.F. Hess. 2006. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642-1645.
9.) Patterson, G.H., and J. Lippincott-Schwartz. 2002. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873-1877.
10.) Chudakov, D.M., V.V. Verkhusha, D.B. Staroverov, E.A. Souslova, S. Lukyanov, and K.A. Lukyanov. 2004. Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol. 22:1435-1439.
11.) van Thor, J.J., T. Gensch, K.J. Hellingwerf, and L.N. Johnson. 2002. Phototransformation of green fluorescent protein with UV and visible light leads to decarboxylation of glutamate 222. Nat. Struct Biol. 9:37-41.
12.) Ando, R., H. Hama, M. Yamamoto-Hino, H. Mizuno, and A. Miyawaki. 2002. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. USA 99:12651-12656.
13.) Wiedenmann, J., S. Ivanchenko, F. Oswald, F. Schmitt, C. Rocker, A. Salih, K.D. Spindler, and G.U. Nienhaus. 2004. EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc. Natl. Acad. Sci. USA 101:15905-15910.
14.) Tsutsui, H., S. Karasawa, H. Shimizu, N. Nukina, and A. Miyawaki. 2005. Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep. 6:233-238.
15.) Gurskaya, N.G., V.V. Verkhusha, A.S. Shcheglov, D.B. Staroverov, T.V. Chepurnykh, A.F. Fradkov, S. Lukyanov, and K.A. Lukyanov. 2006. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat. Biotechnol. 24:461-465.
16.) Lukyanov, K.A., A.F. Fradkov, N.G. Gurskaya, M.V. Matz, Y.A. Labas, A.P. Savitsky, M.L. Markelov, A.G. Zaraisky. 2000. Natural animal coloration can be determined by a non-fluorescent green fluorescent protein homolog. J. Biol. Chem. 275:25879-25882.
17.) Chudakov, D.M., V.V. Belousov, A.G. Zaraisky, V.V. Novoselov, D.B. Staroverov, D.B. Zorov, S. Lukyanov, and K.A. Lukyanov. 2003. Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21:191-194.
18.) Chudakov, D.M., A.V. Feofanov, N.N. Mudrik, S. Lukyanov, and K.A. Lukyanov. 2003. Chromophore environment provides clue to “kindling fluorescent protein” riddle. J. Biol. Chem. 278:7215-7219.
19.) Wilmann, P.G., J. Petersen, R.J. Devenish, M. Prescott, and J. Rossjohn. 2005. Variations on the GFP chromophore: a polypeptide fragmentation within the chromophore revealed in the 2.1-A crystal structure of a nonfluorescent chromoprotein from Anemonia sulcata. J. Biol. Chem. 280:2401-2404.
20.) Quillin, M.L., D.M. Anstrom, X. Shu, S. O'Leary, K. Kallio, D.M. Chudakov, and S.J. Remington. 2005. Kindling fluorescent protein from Anemonia sulcata: dark-state structure at 1.38 A resolution. Biochemistry 44:5774-5787.
21.) Yampolsky, I.V., S.J. Remington, V.I. Martynov, V.K. Potapov, S. Lukyanov, and K.A. Lukyanov. 2005. Synthesis and properties of the chromophore of the asFP595 chromoprotein from Anemonia sulcata. Biochemistry 44:5788-5793.
22.) Schafer, L.V., G. Groenhof, A.R. Klingen, G.M. Ullmann, M. Boggio-Pasqua, M.A. Robb, and H. Grubmuller. 2007. Photoswitching of the fluorescent protein asFP595: mechanism, proton pathways, and absorption spectra. Angew. Chem. Int. Ed. Engl. 46:530-536.
23.) Grigorenko, B., A. Savitsky, I. Topol, S. Burt, and A. Nemukhin. 2006. Ground-state structures and vertical excitations for the kindling fluorescent protein asFP595. J. Phys. Chem. B Condens. Matter Mater. Surf. Interfaces Biophys. 110:18635-18640.
24.) Amat, P., G. Granucci, F. Buda, M. Persico, and V. Tozzini. 2006. The chromophore of asFP595: A theoretical study. J. Phys. Chem. B Condens. Matter Mater. Surf. Interfaces Biophys. 110:9348-9353.
25.) Andresen, M., M.C. Wahl, A.C. Stiel, F. Grater, L.V. Schafer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmuller, S.W. Hell, and S. Jakobs. 2005. Structure and mechanism of the reversible photoswitch of a fluorescent protein. Proc. Natl. Acad. Sci. USA 102:13070-13074.
26.) Ando, R., H. Mizuno, and A. Miyawaki. 2004. Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306:1370-1373.
27.) Stiel, A.C., S. Trowitzsch, G. Weber, M. Andresen, C. Eggeling, S.W. Hell, S. Jakobs, and M.C. Wahl. 2006. 1.8 A bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants. Biochem. J. 402:35-42.
28.) Wilmann, P.G., K. Turcic, J.M. Battad, M.C. Wilce, R.J. Devenish, M. Prescott, and J. Rossjohn. 2006. The 1.7 a crystal structure of Dronpa: a photoswitchable green fluorescent protein. J. Mol. Biol. 364:213-224.
29.) Habuchi, S., P. Dedecker, J. Hotta, C. Flors, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens. 2006. Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching. Photochem. Photobiol. Sci. 5:567-576.
30.) Dedecker, P., J. Hotta, R. Ando, A. Miyawaki, Y. Engelborghs, and J. Hofkens. 2006. Fast and reversible photoswitching of the fluorescent protein Dronpa as evidenced by fluorescence correlation spectroscopy. Biophys. J. 91:L45-L47.