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Exciting Insights into
Cell Growth

How do cells grow? As a cell increases in size during progression of the cell cycle, how is new material added to the cell wall or the plasma membrane? Does growth occur evenly across the cell or are there specific zones where new components are deposited? Is there a pattern to addition? How is turgor pressure maintained?

The Question
EXCITING INSIGHTS INTO CELL GROWTH

Above: New cell wall growth on S. aureus, using vancomycin labeled with Alexa Fluor 532, 60x magnification and 107 nm pixel. Courtesy of Ashley Cadby, EPMM Group, University of Sheffield.

Stochastic Optical Reconstruction Microscopy

  • Excited single molecules are read out from random locations.
  • Excited fluorophores are switched off by switching to a different excited state, not necessarily the bleached state (“switched off” state is the same as the unexcited ground state).
  • Resolution at < 40 nm

STORM Conditions

  • 100-mW, 532-nm diode laser (Laser 2000)
  • 60x oil immersion objective, 1.4 NA
  • Olympus IX71 inverted optical microscope
  • 552-nm longpass dichroic filter (Semrock FF552-DI02)
  • 565(24)-nm bandpass emission filter (Semrock Brightline 565/24)
  • Focus adjusted via piezoelectric motor (Physik Instrumente)
  • Image expander—35 mm and 100 mm lenses
  • ImageEM camera (Hamamatsu)

These are fundamental questions about the biology of a cell—as fundamental as DNA replication—but many of the molecular details remain unclear.1,2

An interdisciplinary team of researchers at the University of Sheffield, including physicist Ashley Cadby, are trying to change this situation by shedding light on how gram-negative bacteria add peptidoglycan to their cell wall.2 The 40 nm resolution they achieved using STORM revealed surprising new details of this process, forcing Turner, Cadby, and colleagues to reshape their models of cell wall growth.

Read the Paper (login may be required)
Turner, R. D., Hurd, A. F., Cadby, A., Hobbs, J. K. & Foster, S. J. Cell wall elongation mode in gram-negative bacteria is determined by peptidoglycan architecture. Nat. Commun. 4, 1496 (2013).
Super-resolution with CMOS
Super-resolution microscopy has traditionally been done using EM-CCD cameras. See how Huang, et al., used a CMOS camera for video-rate super-resolution microscopy.
Helical deposition
Diffuse deposition
A
B
THE BARRIERS
EXCITING INSIGHTS INTO CELL GROWTH

A big technical hurdle in studying bacterial cell wall synthesis has been the scales that these events occur at—how do you visualize processes occurring in the low nanometer scale, but in the context of a living cell that’s >1000 times larger?

In addition, earlier fluorescence microscopy techniques have been limited by the light diffraction barrier of 200 nm (read more about breaking the light diffraction barrier in Exciting Advances Push the Limits of Visualization).

Stay Current
Scientists are using Hamamatsu cameras to address a variety of research questions. To receive bi-monthly updates on the latest imaging studies using Hamamatsu cameras, register now.
A Brief History of Super-resolution Microscopy6,7
THE SOLUTION
EXCITING INSIGHTS INTO CELL GROWTH

Turner, et al,2 used fluorescently labeled vancomycin to identify regions of peptidoglycan insertion—the antibiotic vancomycin binds to the D-ala-D-ala motif present in unmodified, and therefore nascent, material. To ensure that the labeled vancomycin entered the cell, they developed a method to fix and treat E. coli cells to allow vancomycin binding.2

Right: Vancomycin

What can camera specs tell you?
For those new to microscopy and imaging, looking through a camera specifications sheet can be like trying to decipher Greek. To learn what all the terms mean, read “Dissecting camera specifications: a field guide for biologists.”

When Turner, et al,2 imaged their samples—the gram-negative bacteria E. coli and C. crescentus—using deconvolution fluorescence microscopy, they found nascent peptidoglycan localizing to division septa, as expected, high labeling at the poles, which was unexpected, and diffuse labeling throughout the body of the cell.

Stay in Touch
Scientists are using Hamamatsu cameras to address a variety of research questions. To receive bi-monthly updates on the latest imaging studies using Hamamatsu cameras, register now.

To better understand what was happening at the nanometer scale, they turned to STORM, achieving resolution between 35 and 42 nm. What had been diffuse staining throughout the body of the cell resolved into a peppering of distinct foci randomly distributed across the surface of the cell and ranging from single motifs to 50 nm clusters. This arrangement is in surprising contrast to the helical path traveled by the cell elongation machinery.

Right: STORM images of E. coli (MG1655) sacculi showing multiple, distinct foci of insertion using fluorescent vancomycin (Alexa Fluor 532). Courtesy of Ashley Cadby, EPMM Group, University of Sheffield.

Together with the results from atomic force microscopy (AFM) studies, Turner, et al,2 propose a new model for cell wall growth, where peptidoglycan insertion happens in the least dense regions of the cell wall.

Left: Mouse over numbers to identify unique elements.

50 Frames Per Second
Turner, et al,2 turned to super-resolution microscopy to image events in the low nanometer regime. Hamamatsu’s ImageEM camera was part of the STORM setup, capturing the 35-42 nm -resolution images at 50 frames per second. Learn more about ImageEM.
ImagEM DOES more than super-resolution
THE POSSIBILITIES
EXCITING INSIGHTS INTO CELL GROWTH
Belete Ayele Desimmie, Rik Schrijvers, Jonas Demeulemeester, Doortje Borrenberghs,
Caroline Weydert, Wannes Thys, Sofie Vets, Barbara Van Remoortel, Johan Hofkens, Jan De Rijck, Jelle Hendrix, Norbert Bannert, Rik Gijsbers, Frauke Christ, and Zeger Deby
Retrovirology. 2013; 10: 57.
PMCID: PMC3671127
Jessica N. Mazerik and Matthew J. Tyska
J Biol Chem. 2012 April 13; 287(16): 13104–13115.
PMCID: PMC3339983
Martin Lehmann, Susana Rocha, Bastien Mangeat, Fabien Blanchet,
Hiroshi Uji-i, Johan Hofkens, and Vincent Piguet
PLoS Pathog. 2011 December; 7(12): e1002456.
PMCID: PMC3240612

Turner, et al,2 used super-resolution microscopy to see nanometer-scale events in the bacterial cell wall. This powerful technique—really a suite of techniques—is giving a growing number of researchers the ability to see further into biology than they ever have before.

From the basic biology of HIV to experiments into how myosin-1A binds to the membrane, researchers across the globe are using Hamamatsu’s ImagEM camera for studies at the micro- and nano-scale. See how in the selected papers above, available at PubMed Central.

References

01. McCusker, D. & Kellogg, D. R. Plasma membrane growth during the cell cycle: unsolved mysteries and recent progress. Curr. Opin. Cell Biol. 24, 845–851 (2012).

02. Turner, R. D., Hurd, A. F., Cadby, A., Hobbs, J. K. & Foster, S. J. Cell wall elongation mode in Gram-negative bacteria is determined by peptidoglycan architecture. Nat. Commun. 4, 1496 (2013).

03. Varma, A., Pedro, M. A. de & Young, K. D. FtsZ Directs a Second Mode of Peptidoglycan Synthesis in Escherichia coli. J. Bacteriol. 189, 5692–5704 (2007).

04. Pedro, M. A. de, Quintela, J. C., Höltje, J. V. & Schwarz, H. Murein segregation in Escherichia coli. J. Bacteriol. 179, 2823–2834 (1997).

05. De Pedro, M. A., Schwarz, H. & Koch, A. L. Patchiness of murein insertion into the sidewall of Escherichia coli. Microbiol. Read. Engl. 149, 1753–1761 (2003).

06. Han, J. J., Shreve, A. P. & Werner, J. H. in Charact. Mater. (John Wiley & Sons, Inc., 2002). at http://onlinelibrary.wiley.com/doi/10.1002/0471266965.com128/abstract

07. Hell, S. W. in Single Mol. Spectrosc. Chem. Phys. Biol. (Gräslund, A., Rigler, R. & Widengren, J.) 365–398 (Springer Berlin Heidelberg, 2010). at http://link.springer.com/chapter/10.1007/978-3-642-02597-6_19

08. Synge, E. H. A suggested method for extending microscopic resolution into the ultra-microscopic region. Philos Mag 6, 356 (1928).

09. Ash, E. A. & Nicholls, G. Super-resolution aperture scanning microscope. Nature 237, 510–512 (1972).

10. Pohl, D. W., Denk, W. & Lanz, M. Optical stethoscopy: Image recording with resolution λ/20. Appl. Phys. Lett. 44, 651–653 (1984).

11. Lewis, A., Isaacson, M., Harootunian, A. & Muray, A. Development of a 500 Å spatial resolution light microscope: I. light is efficiently transmitted through λ/16 diameter apertures. Ultramicroscopy 13, 227–231 (1984).

12. Hell, S. W. Improvement of lateral resolution in far-field fluorescence light microscopy by using two-photon excitation with offset beams. Opt. Commun. 106, 19–24 (1994).

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

14. Hell, S. W. & Kroug, M. Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit. Appl. Phys. B 60, 495–497 (1995).

15. Gordon, M. P., Ha, T. & Selvin, P. R. Single-molecule high-resolution imaging with photobleaching. Proc. Natl. Acad. Sci. U. S. A. 101, 6462–6465 (2004).

16. Qu, X., Wu, D., Mets, L. & Scherer, N. F. Nanometer-localized multiple single-molecule fluorescence microscopy. Proc. Natl. Acad. Sci. U. S. A. 101, 11298–11303 (2004).

17. Betzig, E. et al. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 313, 1642–1645 (2006).

18. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006).

18. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006).

19. 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).