Nice GCaMP movie

One of the labs we did in this year’s QB3/UCSF Microscopy course was to image cells transfected with a labeled β2-adrenergic receptor and with the calcium reporter GCaMP. Addition of a β2-receptor agonist triggers signaling, leading to calcium influx, reported by GCaMP, and receptor internalization, which can be seen as clustering of the labelled receptors.  This experiment generated some very pretty movies. I’m showing one here, a time lapse acquired on a Spectral Applied Research Diskovery system, operated in spinning disk confocal mode. The first movie is the full field of view, as captured on an Andor Zyla 4.2. The first movie shows the full 2k x 2k field of view, downsampled. The second movie show a full resolution crop from the movie.

Paper Roundup – July 2014

  • A new, very bright lancelet GFP. It is unfortunately dimeric, however. [1]
  • A nice review of total internal reflection imaging, focusing on the theory and optical properties [2]
  • A modified fluorescence fluctuation spectroscopy approach for performing fluorescence brightness analysis in the plasma membrane to quantify protein-protein interactions [3]
  • A temporal focusing multiphoton excitation microscope that also performs structured illumination imaging [4]
  • Multi-color STORM imaging using a single dye pair and repeat labeling of the specimen [5]
  • A review of super-resolution probes for STED microscopy and for emitter-switching approches [6]
  • A clever approach to measuring ciliary beating and swimming of Chlamydomonas using video microscopy and image analysis of the cell position [7]
  • A split mEos3.2 for bimolecular fluorescence complementation PALM [8]
  • An optimized approach for structured illumination imaging to achieve resolution enhancement and optical sectioning [9]
  • A review of optogenetic tools for probing cell signaling [10]
  • Imaging of single unlabeled proteins by their scattering [11]
  • A fast light-sheet system for imaging the beating zebrafish heart [12]
  • Spontaneously blinking dyes by intramolecular cyclization for super-resolution imaging [13]
  • All optical electrophysiology using improved voltage sensors and an orthogonal channelrhodopsin [14]
  • Translocation reporters for kinase activity [15]

References

  1. E.K. Bomati, J.E. Haley, J.P. Noel, and D.D. Deheyn, "Spectral and structural comparison between bright and dim green fluorescent proteins in Amphioxus", Scientific Reports, vol. 4, 2014. http://dx.doi.org/10.1038/srep05469
  2. A.E. Knight, "Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)", Pure and Applied Chemistry, vol. 86, 2014. http://dx.doi.org/10.1515/pac-2012-0605
  3. E. Smith, P. Macdonald, Y. Chen, and J. Mueller, "Quantifying Protein-Protein Interactions of Peripheral Membrane Proteins by Fluorescence Brightness Analysis", Biophysical Journal, vol. 107, pp. 66-75, 2014. http://dx.doi.org/10.1016/j.bpj.2014.04.055
  4. L. Cheng, C. Lien, Y. Da Sie, Y.Y. Hu, C. Lin, F. Chien, C. Xu, C.Y. Dong, and S. Chen, "Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device", Biomedical Optics Express, vol. 5, pp. 2526, 2014. http://dx.doi.org/10.1364/BOE.5.002526
  5. J. Tam, G.A. Cordier, J.S. Borbely, . Sandoval Álvarez, and M. Lakadamyali, "Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore", PLoS ONE, vol. 9, pp. e101772, 2014. http://dx.doi.org/10.1371/journal.pone.0101772
  6. T.J. Chozinski, L.A. Gagnon, and J.C. Vaughan, "Twinkle, twinkle little star: Photoswitchable fluorophores for super-resolution imaging", FEBS Letters, vol. 588, pp. 3603-3612, 2014. http://dx.doi.org/10.1016/j.febslet.2014.06.043
  7. S. Fujita, T. Matsuo, M. Ishiura, and M. Kikkawa, "High-Throughput Phenotyping of Chlamydomonas Swimming Mutants Based on Nanoscale Video Analysis", Biophysical Journal, vol. 107, pp. 336-345, 2014. http://dx.doi.org/10.1016/j.bpj.2014.05.033
  8. Z. Liu, D. Xing, Q.P. Su, Y. Zhu, J. Zhang, X. Kong, B. Xue, S. Wang, H. Sun, Y. Tao, and Y. Sun, "Super-resolution imaging and tracking of protein–protein interactions in sub-diffraction cellular space", Nature Communications, vol. 5, 2014. http://dx.doi.org/10.1038/ncomms5443
  9. K. O’Holleran, and M. Shaw, "Optimized approaches for optical sectioning and resolution enhancement in 2D structured illumination microscopy", Biomedical Optics Express, vol. 5, pp. 2580, 2014. http://dx.doi.org/10.1364/BOE.5.002580
  10. D. Tischer, and O.D. Weiner, "Illuminating cell signalling with optogenetic tools", Nature Reviews Molecular Cell Biology, vol. 15, pp. 551-558, 2014. http://dx.doi.org/10.1038/nrm3837
  11. M. Piliarik, and V. Sandoghdar, "Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites", Nature Communications, vol. 5, 2014. http://dx.doi.org/10.1038/ncomms5495
  12. M. Mickoleit, B. Schmid, M. Weber, F.O. Fahrbach, S. Hombach, S. Reischauer, and J. Huisken, "High-resolution reconstruction of the beating zebrafish heart", Nature Methods, vol. 11, pp. 919-922, 2014. http://dx.doi.org/10.1038/nmeth.3037
  13. S. Uno, M. Kamiya, T. Yoshihara, K. Sugawara, K. Okabe, M.C. Tarhan, H. Fujita, T. Funatsu, Y. Okada, S. Tobita, and Y. Urano, "A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging", Nature Chemistry, 2014. http://dx.doi.org/10.1038/nchem.2002
  14. D.R. Hochbaum, Y. Zhao, S.L. Farhi, N. Klapoetke, C.A. Werley, V. Kapoor, P. Zou, J.M. Kralj, D. Maclaurin, N. Smedemark-Margulies, J.L. Saulnier, G.L. Boulting, C. Straub, Y.K. Cho, M. Melkonian, G.K. Wong, D.J. Harrison, V.N. Murthy, B.L. Sabatini, E.S. Boyden, R.E. Campbell, and A.E. Cohen, "All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins", Nature Methods, vol. 11, pp. 825-833, 2014. http://dx.doi.org/10.1038/nmeth.3000
  15. S. Regot, J. Hughey, B. Bajar, S. Carrasco, and M. Covert, "High-Sensitivity Measurements of Multiple Kinase Activities in Live Single Cells", Cell, vol. 157, pp. 1724-1734, 2014. http://dx.doi.org/10.1016/j.cell.2014.04.039