Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging

Key Points

  • Single-molecule imaging of fluorescent fusion proteins can be used to track individual biomolecules that are moving in bacterial cells and to probe their diffusion or their directed or confined motion, which provides insights into various dynamic cellular processes.

  • Single-molecule imaging, when combined with a method that actively controls the concentration of emitting fluorescent proteins, can be used to achieve super-resolution. Thus, it is now possible to visualize previously hidden structural details of protein localization patterns with a resolution that is no longer limited by the diffraction of light.

  • Single-molecule and single-particle tracking have shown that the actin homologue MreB moves in a circumferential pattern around the bacterial cell, driven by cell wall synthesis. Other cytoskeletal protein structures, such as the FtsZ ring and PopZ nanodomains, have been characterized at subdiffraction spatial resolution.

  • Super-resolution imaging has shown that some nucleoid-associated proteins bind DNA and form well-defined organizational regions, whereas others localize more randomly throughout the nucleoid. These data provide information about the spatial organization of the chromosome.

  • The activities of DNA repair enzymes in live cells have been revealed by a combination of single-molecule tracking and single-molecule super-resolution imaging, which show a clear increase in repair rates under conditions of increased DNA damage.

  • The spatial organization of transcription and translation has been investigated using single-particle tracking and super-resolution imaging of ribosomes and RNA polymerase, revealing clear differences between model organisms.

  • Single-molecule imaging has shown that individual transcription factors search for their target DNA sequence by a combination of one-dimensional sliding along DNA and three-dimensional diffusive hopping between DNA stands.

Abstract

The ability to detect single molecules in live bacterial cells enables us to probe biological events one molecule at a time and thereby gain knowledge of the activities of intracellular molecules that remain obscure in conventional ensemble-averaged measurements. Single-molecule fluorescence tracking and super-resolution imaging are thus providing a new window into bacterial cells and facilitating the elucidation of cellular processes at an unprecedented level of sensitivity, specificity and spatial resolution. In this Review, we consider what these technologies have taught us about the bacterial cytoskeleton, nucleoid organization and the dynamic processes of transcription and translation, and we also highlight the methodological improvements that are needed to address a number of experimental challenges in the field.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Principles of single-molecule tracking and imaging.
Figure 2: Cytoskeletal and structural proteins imaged by single-molecule methods.
Figure 3: Nucleoid organization in model organisms observed by single-molecule methods.
Figure 4: Chromosome integrity and partitioning observed by single-molecule approaches.
Figure 5: Transcription and translation are spatially uncoupled in Escherichia coli.

References

  1. 1

    Moerner, W. E. & Kador, L. Optical detection and spectroscopy of single molecules in a solid. Phys. Rev. Lett. 62, 2535–2538 (1989).

    CAS  PubMed  Google Scholar 

  2. 2

    Orrit, M. & Bernard, J. Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal. Phys. Rev. Lett. 65, 2716–2719 (1990).

    CAS  PubMed  Google Scholar 

  3. 3

    Ambrose, W. P. & Moerner, W. E. Fluorescence spectroscopy and spectral diffusion of single impurity molecules in a crystal. Nature 349, 225–227 (1991).

    CAS  Google Scholar 

  4. 4

    Dickson, R. M., Cubitt, A. B., Tsien, R. Y. & Moerner, W. E. On/off blinking and switching behavior of single molecules of green fluorescent protein. Nature 388, 355–358 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Deich, J., Judd, E. M., McAdams, H. H. & Moerner, W. E. Visualization of the movement of single histidine kinase molecules in live Caulobacter cells. Proc. Natl Acad. Sci. USA 101, 15921–15926 (2004). This paper provides the first evidence that single copies of fluorescent protein fusions can be imaged and analysed in bacteria.

    CAS  PubMed  Google Scholar 

  6. 6

    Shapiro, L., McAdams, H. & Losick, R. Generating and exploiting polarity in bacteria. Science 298, 1942–1946 (2002).

    CAS  PubMed  Google Scholar 

  7. 7

    Shapiro, L., McAdams, H. H. & Losick, R. Why and how bacteria localize proteins. Science 326, 1225–1228 (2009). This review describes the central role that protein localization patterns have in bacterial cell biology, which should be investigated using advanced imaging methods.

    CAS  PubMed  Google Scholar 

  8. 8

    Niu, L. & Yu, P. Investigating intracellular dynamics of FtsZ cytoskeleton with photoactivation single-molecule tracking. Biophys. J. 95, 2009–2016 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nature Methods 5, 155–157 (2008).

    CAS  PubMed  Google Scholar 

  10. 10

    Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    CAS  Google Scholar 

  11. 11

    Hess, S. T., Girirajan, T. P. K. & Mason, M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3, 793–796 (2006). References 10, 11, and 12 are the first reports of super-resolution imaging by single-molecule localization and active control of emitter concentrations.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Xie, X. S., Choi, P. J., Li, G. W., Lee, N. K. & Lia, G. Single-molecule approach to molecular biology in living bacterial cells. Annu. Rev. Biophys. 37, 417–444 (2008). This review covers bacterial single-molecule studies up to 2008.

    CAS  Google Scholar 

  14. 14

    Reyes-Lamothe, R., Sherratt, D. J. & Leake, M. C. Stoichiometry and architecture of active, DNA replication machinery in Escherichia coli. Science 328, 498–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Lia, G., Michel, B. & Allemand, J. Polymerase exchange during Okazaki fragment synthesis observed in living cells. Science 335, 328–331 (2012).

    CAS  PubMed  Google Scholar 

  16. 16

    Robinson, A. & van Oijen, A. M. Bacterial replication, transcription and translation: mechanistic insights from single-molecule biochemical studies. Nature Rev. Microbiol. 11, 303–315 (2013).

    CAS  Google Scholar 

  17. 17

    Typas, A., Banzhaf, M., Gross, C. A. & Vollmer, W. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nature Rev. Microbiol. 10, 123–136 (2012). This review describes the dynamics of MreB and FtsZ and their role in cell wall synthesis.

    CAS  Google Scholar 

  18. 18

    Adams, D. W. & Errington, J. Bacterial cell division: assembly, maintenance and disassembly of the Z ring. Nature Rev. Microbiol. 7, 642–653 (2009).

    CAS  Google Scholar 

  19. 19

    Gerdes, K., Howard, M. & Szardenings, F. Pushing and pulling in prokaryotic DNA segregation. Cell 141, 927–942 (2010).

    CAS  PubMed  Google Scholar 

  20. 20

    Bi, E. & Lutkenhaus, J. FtsZ ring structure associated with division in Escherichia Coli. Nature 354, 161–164 (1991).

    CAS  PubMed  Google Scholar 

  21. 21

    Jones, L. J. F., Carballido-López, R. & Errington, J. Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis. Cell 104, 913–922 (2001).

    CAS  PubMed  Google Scholar 

  22. 22

    van den Ent, E., Amos, L. A. & Loewe, J. Prokaryotic origin of the actin cytoskelecton. Nature 413, 39–44 (2001).

    CAS  PubMed  Google Scholar 

  23. 23

    Celler, K., Koning, R. I., Koster, A. J. & van Wezel, G. P. Multidimensional view of the bacterial cytoskeleton. J. Bacteriol. 195, 1627–1636 (2013).

    PubMed  PubMed Central  Google Scholar 

  24. 24

    Cabeen, M. T. & Jacobs-Wagner, C. The bacterial cytoskeleton. Annu. Rev. Genet. 44, 365–392 (2010).

    CAS  PubMed  Google Scholar 

  25. 25

    Figge, R. M., Divakaruni, A. V. & Gober, J. W. MreB, the cell shape-determining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus. Mol. Microbiol. 51, 1321–1332 (2004).

    CAS  PubMed  Google Scholar 

  26. 26

    Gitai, Z., Dye, N. & Shapiro, L. An actin-like gene can determine cell polarity in bacteria. Proc. Natl Acad. Sci. USA 101, 8643–8648 (2004).

    CAS  PubMed  Google Scholar 

  27. 27

    Gitai, Z., Dye, N. A., Reisenauer, A., Wachi, M. & Shapiro, L. MreB actin-mediated segregation of a specific region of a bacterial chromosome. Cell 120, 329–341 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Divakaruni, A. V., Loo, R. R. O., Xie, Y., Loo, J. A. & Gober, J. W. The cell-shape protein MreC interacts with extracytoplasmic proteins including cell wall assembly complexes in Caulobacter cresentus. Proc. Natl Acad. Sci. USA 102, 18602–18607 (2005).

    CAS  PubMed  Google Scholar 

  29. 29

    Iwai, N. et al. Structure–activity relationship of s-benzylisothiourea derivatives to induce spherical cells in Escherichia coli. Biosci. Biotechnol. Biochem. 68, 2265–2269 (2004).

    CAS  PubMed  Google Scholar 

  30. 30

    Bean, G. J. et al. A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a low-affinity state in MreB. Biochemistry 48, 4852–4857 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Carballido-Lopez, R. The bacterial actin-like cytoskeleton. Microbiol. Mol. Biol. Rev. 70, 888–909 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Shaevitz, J. W. & Gitai, Z. The structure and function of bacterial actin homologs. Cold Spring Harb. Perspect. Biol. 2, a000364 (2010).

    PubMed  PubMed Central  Google Scholar 

  33. 33

    Takacs, C. N. et al. MreB drives de novo rod morphogenesis in Caulobacter crescentus via remodeling of the cell wall. J. Bacteriol. 192, 1671–1684 (2010).

    CAS  PubMed  Google Scholar 

  34. 34

    Dye, N. A., Pincus, Z., Theriot, J. A., Shapiro, L. & Gitai, Z. Two independent spiral structures control cell shape in Caulobacter. Proc. Natl Acad. Sci. USA 102, 18608–18613 (2005).

    CAS  PubMed  Google Scholar 

  35. 35

    Divakaruni, A. V., Baida, C., White, C. L. & Gober, J. W. The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes. Mol. Microbiol. 66, 174–188 (2007).

    CAS  PubMed  Google Scholar 

  36. 36

    den Blaauwen, T., de Pedro, M. A., Nguyen-Disteche, M. & Ayala, J. A. Morphogenesis of rod-shaped sacculi. FEMS Microbiol. Rev. 32, 321–344 (2008).

    CAS  PubMed  Google Scholar 

  37. 37

    Kim, S. Y., Gitai, Z., Kinkhabwala, A., Shapiro, L. & Moerner, W. E. Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus. Proc. Natl Acad. Sci. USA 103, 10929–10934 (2006). This study shows that single copies of MreB move in a slow, directed, circumferential fashion around the cell periphery in C. crescentus.

    CAS  PubMed  Google Scholar 

  38. 38

    Biteen, J. S. et al. Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP. Nature Methods 5, 947–949 (2008). This study introduces eYFP protein fusion constructs as a suitable label for super-resolution imaging in live bacteria.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Garner, E. C. et al. Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis. Science 333, 222–225 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Dominguez-Escobar, J. et al. Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria. Science 333, 225–228 (2011).

    CAS  PubMed  Google Scholar 

  41. 41

    van Teeffelen, S. et al. The bacterial actin MreB rotates, and rotation depends on cell-wall assembly. Proc. Natl Acad. Sci. USA 108, 15822–15827 (2011).

    CAS  PubMed  Google Scholar 

  42. 42

    Swulius, M. T. et al. Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria. Biochem. Biophys. Res. Commun. 407, 650–655 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Swulius, M. T. & Jensen, G. J. The helical MreB cytoskeleton in Escherichia coli MC1000/pLE7 is an artifact of the N-terminal yellow fluorescent protein tag. J. Bacteriol. 194, 6382–6386 (2012). This study shows that the helical shape of MreB superstructure is an artefact of the fluorescent protein fusion construct that was used.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Bendezu, F. O., Hale, C. A., Bernhardt, T. G. & de Boer, P. A. J. RodZ (YfgA) is required for proper assembly of the MreB actin cytoskeleton and cell shape in E. coli. EMBO J. 28, 193–204 (2009).

    CAS  PubMed  Google Scholar 

  45. 45

    Salje, J., van den Ent, F., de Boer, P. & Loewe, J. Direct membrane binding by bacterial actin MreB. Mol. Cell 43, 478–487 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    White, C. L., Kitich, A. & Gober, J. W. Positioning cell wall synthetic complexes by the bacterial morphogenetic proteins MreB and MreD. Mol. Microbiol. 76, 616–633 (2010).

    CAS  PubMed  Google Scholar 

  47. 47

    Erickson, H. P., Anderson, D. E. & Osawa, M. FtsZ in bacterial cytokinesis: cytoskeleton and force generator all in one. Microbiol. Mol. Biol. Rev. 74, 504–528 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Huang, K., Durand-Heredia, J. & Janakiraman, A. FtsZ ring stability: of bundles, tubules, crosslinks, and curves. J. Bacteriol. 195, 1859–1868 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Osawa, M., Anderson, D. E. & Erickson, H. P. Reconstitution of contractile FtsZ rings in liposomes. Science 320, 792–794 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Li, Z., Trimble, M. J., Brun, Y. V. & Jensen, G. J. The structure of FtsZ filaments in vivo suggests a force-generating role in cell division. EMBO J. 26, 4694–4708 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Fu, G. et al. In vivo structure of the E. coli FtsZ-ring revealed by photoactivated localization microscopy (PALM). PLoS ONE 5, e12680 (2010).

    PubMed  PubMed Central  Google Scholar 

  52. 52

    Ben-Yehuda, S. & Losick, R. Asymmetric cell division in B. subtilis involves a spiral-like intermediate of the cytokinetic protein FtsZ. Cell 109, 257–266 (2002).

    CAS  PubMed  Google Scholar 

  53. 53

    Thanedar, S. & Margolin, W. FtsZ exhibits rapid movement and oscillation waves in helix-like patterns in Escherichia coli. Curr. Biol. 14, 1167–1173 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Thanbichler, M. & Shapiro, L. MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter. Cell 126, 1–16 (2006).

    Google Scholar 

  55. 55

    Peters, P. C., Migocki, M. D., Thoni, C. & Harry, E. J. A new assembly pathway for the cytokinetic Z ring from a dynamic helical structure in vegetatively growing cells of Bacillus subtilis. Mol. Microbiol. 64, 487–499 (2007).

    CAS  PubMed  Google Scholar 

  56. 56

    Anderson, D. E., Guieros-Filho, F. J. & Erickson, H. P. Assembly dynamics of FtsZ rings in Bacillus subtilis and Escherichia coli and effects of FtsZ-regulating proteins. J. Bacteriol. 186, 5775–5781 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Biteen, J. S., Goley, E. D., Shapiro, L. & Moerner, W. E. Three-dimensional super-resolution imaging of the midplane protein FtsZ in live Caulobacter crescentus cells using astigmatism. ChemPhysChem 13, 1007–1012 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58

    Eswaramoorthy, P. et al. Cellular architecture mediates DivIVA ultrastructure and regulates Min activity in Bacillus subtilis. mBio 2, e00257-11 (2011).

    PubMed  PubMed Central  Google Scholar 

  59. 59

    Strauss, M. P. et al. 3D-SIM super resolution microscopy reveals a bead-like arrangement for FtsZ and the division machinery: implications for triggering cytokinesis. PLoS Biol. 10, e1001389 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Jennings, P. C., Cox, G. C., Monahan, L. G. & Harry, E. J. Super-resolution imaging of the bacterial cytokinetic protein FtsZ. Micron 42, 336–341 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61

    Bowman, G. R. et al. A polymeric protein anchors the chromosomal origin/ParB complex at a bacterial cell pole. Cell 134, 945–955 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62

    Bowman, G. R. et al. Caulobacter PopZ forms a polar subdomain dictating sequential changes in pole composition and function. Mol. Microbiol. 76, 173–189 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63

    Ebersbach, G., Briegel, A., Jensen, G. J. & Jacobs-Wagner, C. A self-associating protein critical for chromosome attachment, division, and polar organization in Caulobacter. Cell 134, 956–968 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64

    Gahlmann, A. et al. Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in 3D. Nano Lett. 13, 987–993 (2013). This paper introduces a method of two-colour, super-resolution imaging to accurately colocalize single molecules that are labelled with different fluorophores.

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65

    Wery, M., Woldringh, C. L. & Rouvière-Yaniv, J. HU-GFP and DAPI co-localize on the Escherichia coli nucleoid. Biochimie 83, 193–200 (2001).

    CAS  PubMed  Google Scholar 

  66. 66

    Llopis, P. M. et al. Spatial organization of the flow of genetic information in bacteria. Nature 466, 77–81 (2010).

    CAS  PubMed Central  Google Scholar 

  67. 67

    Dillon, S. C. & Dorman, C. J. Bacterial nucleoid-associated proteins, nucleoid structure and gene expression. Nature Rev. Microbiol. 8, 185–195 (2010).

    CAS  Google Scholar 

  68. 68

    Sarkar, T., Vitoc, I., Mukerji, I. & Hud, N. V. Bacterial protein HU dictates the morphology of DNA condensates produced by crowding agents and polyamines. Nucleic Acids Res. 35, 951–961 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69

    Lee, S. F., Thompson, M. A., Schwartz, M. A., Shapiro, L. & Moerner, W. E. Super-resolution imaging of the nucleoid-associated protein HU in Caulobacter crescentus. Biophys. J. 100, L31–L33 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. 70

    Illian, J., Penttinen, A., Stoyan, H. & Stoyan, D. Statistical Analysis and Modelling of Spatial Point Patterns. (John Wiley & Sons, 2008).

    Google Scholar 

  71. 71

    Wang, W., Li, G., Chen, C., Xie, X. S. & Zhuang, X. Chromosome organization by a nucleoid-associated protein in live bacteria. Science 333, 1445–1449 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. 72

    Dekker, J., Rippe, K., Dekker, M. & Kleckner, N. Capturing chromosome conformation. Science 295, 1306–1311 (2002).

    CAS  Google Scholar 

  73. 73

    Badrinarayanan, A., Reyes-Lamothe, R., Uphoff, S., Leake, M. C. & Sherratt, D. J. In vivo architecture and action of bacterial structural maintenance of chromosome proteins. Science 338, 528–531 (2012). This paper determines the minimal functional complex of MukBEF in living cells using single-molecule imaging and stoichimetry analysis.

    CAS  PubMed  Google Scholar 

  74. 74

    Kleine Borgmann, L. A., Ries, J., Ewers, H., Ulbrich, M. H. & Graumann, P. L. The bacterial SMC complex displays two distinct modes of interaction with the chromosome. Cell Rep. 3, 1483–1492 (2013).

    CAS  PubMed  Google Scholar 

  75. 75

    Niki, H. et al. Escherichia coli MukB protein involved in chromosome partition forms a homodimer with a rod-and-hinge structure having DNA-binding and ATP/GTP binding activities. EMBO J. 11, 5101–5109 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. 76

    Yamanaka, K., Ogura, T., Niki, H. & Hiraga, S. Identification of two new genes, mukE and mukF, involved in chromosome partitioning in Escherichia coli. Mol. Gen. Genet. 250, 241–251 (1996).

    CAS  PubMed  Google Scholar 

  77. 77

    Wang, X., Llopis, P. M. & Rudner, D. Z. Organization and segregation of bacterial chromosomes. Nature Rev. Genet. 14, 191–203 (2013).

    CAS  PubMed  Google Scholar 

  78. 78

    den Blaauwen, T., Lindqvist, A., Lowe, J. & Nanninga, N. Distribution of the Escherichia coli structural maintenance of chromosomes (SMC)-like protein MukB in the cell. Mol. Microbiol. 42, 1179–1188 (2001).

    CAS  PubMed  Google Scholar 

  79. 79

    Lindow, J., Kuwano, M., Moriya, S. & Grossman, A. Subcellular localization of the Bacillus subtilis structural maintenance of chromosomes (SMC) protein. Mol. Microbiol. 46, 997–1009 (2002).

    CAS  PubMed  Google Scholar 

  80. 80

    Mascarenhas, J., Soppa, J., Strunnikov, A. & Graumann, P. Cell cycle-dependent localization of two novel prokaryotic chromosome segregation and condensation proteins in Bacillus subtilis that interact with SMC protein. EMBO J. 21, 3108–3118 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. 81

    Leake, M. C. et al. Stoichiometry and turnover in single, functioning membrane protein complexes. Nature 443, 355–358 (2006).

    CAS  PubMed  Google Scholar 

  82. 82

    Uphoff, S., Reyes-Lamothe, R., Garza de Leon, F., Sherratt, D. J. & Kapanidis, A. N. Single-molecule DNA repair in live bacteria. Proc. Natl Acad. Sci. USA 110, 8063–8068 (2013).

    CAS  PubMed  Google Scholar 

  83. 83

    Ptacin, J. L. et al. A spindle-like apparatus guides bacterial chromosome segregation. Nature Cell Biol. 12, 791–798 (2010). This study shows that the chromosome-partitioning protein ParB in C. crescentus moves along a receding ParA structure during chromosome segregation.

    CAS  PubMed  Google Scholar 

  84. 84

    Miller, O. L., Hamkalo, B. A. & Thomas, C. A. Visualization of bacterial genes in action. Science 169, 392–395 (1970).

    PubMed  Google Scholar 

  85. 85

    Robinow, C. & Kellenberger, E. The bacterial nucleoid revisited. Microbiol. Rev. 58, 211–232 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. 86

    Woldringh, C. L. The role of co-transcriptional translation and protein translocation (transertion) in bacterial chromosome segregation. Mol. Microbiol. 45, 17–29 (2002).

    CAS  PubMed  Google Scholar 

  87. 87

    Norris, V. & Madsen, M. Autocatalytic gene expression occurs via transertion and membrane domain formation and underlies differentiation in bacteria: a model. J. Mol. Biol. 253, 739–748 (1995).

    CAS  PubMed  Google Scholar 

  88. 88

    Bakshi, S., Siryaporn, A., Goulian, M. & Weisshaar, J. C. Superresolution imaging of ribosomes and RNA polymerase in live Escherichia coli cells. Mol. Microbiol. 85, 21–38 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. 89

    Nevo-Dinur, K., Nussbaum-Shochat, A., Ben-Yehuda, S. & Amster-Choder, O. Translation-independent localization of mRNA in E. coli. Science 331, 1081–1084 (2011).

    CAS  PubMed  Google Scholar 

  90. 90

    Yu, J., Xiao, J., Ren, X., Lao, K. & Xie, X. S. Probing gene expression in live cells, one protein molecule at a time. Science 311, 1600–1603 (2006). This paper uses single-molecule imaging to directly observe gene expression dynamics in live cells.

    CAS  PubMed  Google Scholar 

  91. 91

    Choi, P. J., Cai, L., Frieda, K. & Xie, X. S. A. Stochastic single-molecule event triggers phenotype switching of a bacterial cell. Science 322, 442–446 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. 92

    Taniguchi, Y. et al. Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329, 533–538 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. 93

    Elf, J., Li, G. W. & Xie, X. S. Probing transcription factor dynamics at the single-molecule level in a living cell. Science 316, 1191–1194 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. 94

    von Hippel, P. H. & Berg, O. G. Facilitated target location in biological systems. J. Biol. Chem. 264, 675–678 (1989).

    CAS  PubMed  Google Scholar 

  95. 95

    Sletten, E. M. & Bertozzi, C. R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew. Chem. Int. Edn Engl. 48, 6974–6998 (2009).

    CAS  Google Scholar 

  96. 96

    Sauer, M. Localization microscopy coming of age: from concepts to biological impact. J. Cell. Sci. 126, 3505–3513 (2013). This paper provides a comprehensive review of single-molecule localization microscopy, with a thorough discussion of current challenges.

    CAS  PubMed  Google Scholar 

  97. 97

    Plass, T. et al. Amino acids for Diels–Alder reactions in living cells. Angew. Chem. Int. Edn Engl. 51, 4166–4170 (2012).

    CAS  Google Scholar 

  98. 98

    Plass, T., Milles, S., Koehler, C., Schultz, C. & Lemke, E. A. Genetically encoded copper-free click chemistry. Angew. Chem. Int. Edn Engl. 50, 3878–3881 (2011).

    CAS  Google Scholar 

  99. 99

    Lang, K. et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nature Chem. 4, 298–304 (2012).

    CAS  Google Scholar 

  100. 100

    Lang, K. et al. Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels–Alder reactions. J. Am. Chem. Soc. 134, 10317–10320 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  101. 101

    Charbon, G. et al. Subcellular protein localization by using a genetically encoded fluorescent amino acid. ChemBioChem 12, 1818–1821 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. 102

    Lukinavicius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nature Chem. 5, 132–139 (2013).

    CAS  Google Scholar 

  103. 103

    Puthenveetil, S., Liu, D. S., White, K. A., Thompson, S. & Ting, A. Y. Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase. J. Am. Chem. Soc. 131, 16430–16438 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  104. 104

    Fernandez-Suarez, M. & Ting, A. Y. Fluorescent probes for super-resolution imaging in living cells. Nature Rev. Mol. Cell Biol. 9, 929–943 (2008). This paper reviews fluorescent probes for super-resolution imaging.

    CAS  Google Scholar 

  105. 105

    Uttamapinant, C. et al. A fluorophore ligase for site-specific protein labeling inside living cells. Proc. Natl Acad. Sci. USA 107, 10914–10919 (2010).

    CAS  PubMed  Google Scholar 

  106. 106

    Liu, D. S. et al. Diels–Alder cycloaddition for fluorophore targeting to specific proteins inside living cells. J. Am. Chem. Soc. 134, 792–795 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  107. 107

    Siegrist, M. S. et al. D-amino acid chemical reporters reveal peptidoglycan dynamics of an intracellular pathogen. ACS Chem. Biol. 8, 500–505 (2013).

    CAS  PubMed  Google Scholar 

  108. 108

    Kuru, E. et al. In situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent d-amino acids. Angew. Chem. Int. Edn Engl. 51, 12519–12523 (2012).

    CAS  Google Scholar 

  109. 109

    Szent-Gyorgyi, C. et al. Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nature Biotech. 26, 235–240 (2008).

    CAS  Google Scholar 

  110. 110

    Yushchenko, D. A., Zhang, M., Yan, Q., Waggoner, A. S. & Bruchez, M. P. Genetically targetable and color-switching fluorescent probe. ChemBioChem 13, 1564–1568 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  111. 111

    Benke, A., Olivier, N., Gunzenhaeuser, J. & Manley, S. Multicolor single molecule tracking of stochastically active synthetic dyes. Nano Lett. 12, 2619–2624 (2012).

    CAS  PubMed  Google Scholar 

  112. 112

    Lee, H. D. et al. Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores. J. Am. Chem. Soc. 132, 15099–15101 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. 113

    Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).

    CAS  Google Scholar 

  114. 114

    Hofmann, M., Eggeling, C., Jakobs, S. & Hell, S. W. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc. Natl Acad. Sci. USA 102, 17565–17569 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  115. 115

    Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  116. 116

    Gustafsson, M. G. L. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl Acad. Sci. USA 102, 13081–13086 (2005).

    CAS  PubMed  Google Scholar 

  117. 117

    Moerner, W. E. New directions in single-molecule imaging and analysis. Proc. Natl Acad. Sci. USA 104, 12596–12602 (2007).

    CAS  PubMed  Google Scholar 

  118. 118

    Hell, S. W. Microscopy and its focal switch. Nature Methods 6, 24–32 (2009).

    CAS  PubMed  Google Scholar 

  119. 119

    Coltharp, C. & Xiao, J. Superresolution microscopy for microbiology. Cell. Microbiol. 14, 1808–1818 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  120. 120

    Lippincott-Schwartz, J. & Patterson, G. H. Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol. 19, 555–565 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  121. 121

    Heilemann, M., Dedecker, P., Hofkens, J. & Sauer, M. Photoswitches: key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification. Laser Photon. Rev. 3, 180–202 (2009).

    CAS  Google Scholar 

  122. 122

    Moerner, W. E. Microscopy beyond the diffraction limit using actively controlled single molecules. J. Microsc. 246, 213–220 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. 123

    Cordes, T. et al. Resolving single-molecule assembled patterns with superresolution blink-microscopy. Nano Lett. 10, 645–651 (2010).

    CAS  PubMed  Google Scholar 

  124. 124

    Schoen, I., Ries, J., Klotzsch, E., Ewers, H. & Vogel, V. Binding-activated localization microscopy of DNA structures. Nano Lett. 11, 4008–4011 (2011).

    CAS  Google Scholar 

  125. 125

    Lee, M. K., Williams, J., Twieg, R. J., Rao, J. & Moerner, W. E. Enzymatic activation of nitro-aryl fluorogens in live bacterial cells for enzymatic turnover-activated localization microscopy. Chem. Sci. 4, 220–225 (2013).

    CAS  Google Scholar 

  126. 126

    Sharonov, A. & Hochstrasser, R. M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl Acad. Sci. USA 103, 18911–18916 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  127. 127

    Sahl, S. J. & Moerner, W. E. Super-resolution fluorescence imaging with single molecules. Curr. Opin. Struct. Biol. 23 778–787 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  128. 128

    Huang, F. et al. Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms. Nature Methods 10, 653–658 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  129. 129

    Ondrus, A. E. et al. Fluorescent saxitoxins for live cell imaging of single voltage-gated sodium ion channels beyond the optical diffraction limit. Chem. Biol. 19, 902–912 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  130. 130

    Jones, S. A., Shim, S., He, J. & Zhuang, X. Fast, three-dimensional super-resolution imaging of live cells. Nature Methods 8, 499–505 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  131. 131

    Lill, Y. et al. Single-molecule study of molecular mobility in the cytoplasm of Escherichia coli. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 86, 021907 (2012).

    PubMed  Google Scholar 

  132. 132

    Shaner, N. C., Steinbach, P. A. & Tsien, R. Y. A guide to choosing fluorescent proteins. Nature Methods 2, 905–909 (2005).

    CAS  PubMed  Google Scholar 

  133. 133

    Subach, F. V. et al. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nature Methods 6, 153–159 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  134. 134

    Patterson, G. H. & Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  135. 135

    Gurskaya, N. G. et al. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nature Biotech. 24, 461–465 (2006).

    CAS  Google Scholar 

  136. 136

    Chudakov, D. M., Lukyanov, S. & Lukyanov, K. A. Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2. Nature Protoc. 2, 2024–2032 (2007).

    CAS  Google Scholar 

  137. 137

    McKinney, S. A., Murphy, C. S., Hazelwood, K. L., Davidson, M. W. & Looger, L. L. A bright and photostable photoconvertible fluorescent protein. Nature Methods 6, 131–133 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  138. 138

    Biteen, J. S., Thompson, M. A., Tselentis, N. K., Shapiro, L. & Moerner, W. E. Superresolution imaging in live Caulobacter crescentus cells using photoswitchable enhanced yellow fluorescent protein. Nature Methods 5, 947–949 (2009).

    Google Scholar 

  139. 139

    Gunewardene, M. et al. Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells. Biophys. J. 101, 1522–1528 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  140. 140

    Landgraf, D., Okumus, B., Chien, P., Baker, T. A. & Paulsson, J. Segregation of molecules at cell division reveals native protein localization. Nature Methods 9, 480–482 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  141. 141

    Li, G. & Elf, J. Single molecule approaches to transcription factor kinetics in living cells. FEBS Lett. 583, 3979–3983 (2009).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors gratefully acknowledge fruitful collaborations and stimulating discussions with L. Shapiro and members of her laboratory over the years, as well as many current and former members of the Moerner laboratory. The authors specifically thank M. K. Lee, A. R. von Diezmann and J. L. Ptacin for critical reading of the manuscript. This work was supported in part by the US National Institute of General Medical Sciences Grant No. R01GM086196 (W.E.M) and a Swiss National Science Foundation Postdoctoral Fellowship PA00P2_145310 (A.G.).

Author information

Affiliations

Authors

Corresponding author

Correspondence to W. E. Moerner.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

PowerPoint slides

Glossary

Centroid estimation

In the context of localization microscopy, a method to determine the location of a fluorescent emitter or a group of several closely packed emitters (referred to as a single-particle) by calculating the spatial arithmetic mean of all pixel positions, which are weighted by their intensity.

Photoactivation

The process by which fluorophores that are initially in a non-fluorescent (dark) state can be converted to a fluorescent (bright) state by illumination with short-wavelength light.

Photoswitching

The process by which photoswitchable fluorophores can be turned 'on' or 'off' by an active control mechanism, such as illumination with a specific wavelength of light.

Photoinduced blinking

Using certain illumination intensities (or, in some cases, by adding chemical additives), fluorophores can reversibly enter a non-fluorescent (dark) state. From this state, they can spontaneously recover and become fluorescent (bright) again, which gives the appearance of blinking.

Epifluorescence microscopy

A standard wide-field fluorescence microscopy technique, in which the same objective lens is used to illuminate the entire specimen and to collect emitted fluorescence.

Cryo-electron tomography

(CET). A technique in transmission electron microscopy, in which a vitrified specimen is imaged from different angles at cryogenic temperatures. From the resulting electron micrograph tilt series, a three-dimensional tomogram can be computationally reconstructed.

Total internal reflection fluorescence microscopy

(TIRF microscopy). A technique in which only fluorophores that are in close proximity to the glass–water interface are excited by an evanescent wave that is generated by total internal reflection of the excitation light at this interface. The large reduction of the excitation volume in the axial direction (from 700 nm to 100 nm) results in more selective excitation of the sample and lower background fluorescence compared with epifluorescence illumination.

Fluorescence recovery after photobleaching

(FRAP). An optical technique that is used to estimate the diffusion of fluorescently labelled molecules by determining the timescale of fluorescence recovery after high-intensity light has been applied to a well-defined region of the specimen to photobleach many of the fluorophores in its footprint.

Astigmatic point spread function

(Astigmatic PSF). A cylindrical lens can be inserted in the fluorescence collection path to offset the x and y focus position along the optical axis. An imaging system that has an astigmatic PSF can be used to determine the z-position of a single-molecule emitter by calibrating the change in elliptical shape of the PSF as a function of defocus.

Double-helix point spread function

(Double-helix PSF). Optical phase manipulation in the Fourier plane of the fluorescence emission path can be used to produce a double-helix PSF. An imaging system that has a double-helix PSF can be used to determine the z-position of a single-molecule emitter by calibrating the amount of angular rotation of the PSF as a function of defocus.

Chromosome conformation capture

(3C). A molecular biology technique that is based on crosslinking and analysis of which DNA segments are closely associated, to determine the spatial organization of the chromosome.

Fluorescence in situ hybridization

(FISH). An optical technique that is used to detect and locate specific DNA (or RNA) sequences. A FISH probe, which consists of a fluorophore linked to a single-stranded DNA (or RNA) sequence, binds to its complementary target DNA (or RNA) site after being introduced into fixed and permeabilized cells.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Gahlmann, A., Moerner, W. Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging. Nat Rev Microbiol 12, 9–22 (2014). https://doi.org/10.1038/nrmicro3154

Download citation

Further reading

Search

Quick links

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing