Skip to main content Accessibility help

Imaging of Vascular Smooth Muscle Cells with Soft X-Ray Spectromicroscopy

  • Julia Sedlmair (a1), Sophie-Charlotte Gleber (a2), Semra Öztürk Mert (a3), Michael Bertilson (a4), Olov von Hofsten (a4), Jürgen Thieme (a5) and Thomas Pfohl (a3) (a6)...


Using X-ray microscopy and spectromicroscopy, vascular smooth muscle cells (VSMCs) were imaged, prepared without using additional embedding material or staining, but by applying simple, noncryo fixation techniques. The cells were imaged with a compact source transmission X-ray microscope and a scanning transmission X-ray microscope (STXM). With the STXM, spectromicroscopy was performed at the C K-edge and the Ca LIII,II-edges. VSMCs were chosen because of their high amount of actin stress fibers, so that the actin cytoskeleton should be visible. Other parts of the cell, such as the nucleus and organelles, were also identified from the micrographs. Both in the spectra and the images, the effects of the different preparation procedures were observable. Furthermore, Ca hotspots were detected and their density is determined.


Corresponding author

Corresponding author. E-mail:


Hide All
Bâldea, I., Schimmelpfennig, B., Plaschke, M., Rothe, J., Schirmer, J., Trofimov, A.B. & Fanghänel, T. (2007). C 1s near edge X-ray absorption fine structure (NEXAFS) of substituted benzoic acids—A theoretical and experimental study. J Electron Spectrosc 154, 109118.
Beetz, T. (2004). Soft X-ray diffraction imaging with and without lenses and radiation damage studies. PhD thesis. Stony Brook, NY: Stony Brook University.
Beetz, T. & Jacobsen, C. (2003). Soft X-ray radiation-damage studies in PMMA using a cryo-STXM. J Synchrotron Rad 10(3), 280283.
Benzerara, K., Yoon, T., Tyliszczak, T., Constantz, B., Spormann, A. & Brown, G.B. Jr. (2004). Scanning transmission X-ray microscopy study of micro-bial calcification. Geobiol 2(4), 249259.
Berglund, M., Rymell, L., Peuker, M., Wilhein, T. & Hertz, H.M. (2000). Compact water-window transmission X-ray microscopy. J Microsc 197(3), 268273.
Bertilson, M.C., von Hofsten, O., Thieme, J., Lindblom, M., Holmberg, A., Takman, P.A.C, Vogt, U. & Hertz, H.M. (2009). First application experiments with the Stockholm compact soft X-ray microscope. J Phys Conf Ser 186, 012025.
Bertsch, P.M. & Hunter, D.B. (2001). Applications of synchrotron-based X-ray microprobes. Chem Rev 101, 18091842.
Betzig, E., Patterson, G.H., Sougrat, R., Lindwasser, O.W., Olenych, S., Bonifacino, J.S., Davidson, M.W., Lippincott-Schwartz, J. & Hess, H.F. (2006). Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(1642), 810813.
Buckley, C.J. (1995). Measuring and mapping in mineralized tissues by absorption difference imaging. Rev Sci Instrum 66(2), 13181321.
Chao, W., Kim, J., Rakewa, S., Fischer, P. & Anderson, E.H. (2009). Demonstration of 12 nm resolution fresnel zone plate lens based soft X-ray microscopy. Opt Express 17(20), 1766917677.
Cheng, H. & Lederer, W. (2008). Calcium sparks. Physiol Rev 88(4), 14911545.
CXRO (2011). X-Ray Interactions with Matter. Berkeley, CA: Center for X-Ray Optics, Lawrence Berkeley National Laboratory (retrieved Jan. 2011).
Egner, A. & Hell, S.W. (2005). Fluorescence microscopy with super-resolved optical sections. Trends Cell Biol 15(4), 207215.
Fleet, M.E. & Liu, X. (2009). Calcium L2, 3-edge XANES of carbonates, carbonate apatite, and oldhamite (CaS). Am Mineral 94(8-9), 12351241.
Gleber, S.-C. (2009). Soft X-ray stereo microscopy for investigation of dynamics and elemental distribution of colloidal systems from the environment. PhD thesis. Gottingen, Germany: Georg-August-University.
Greco, T., Hodara, R., Parastatidis, I., Heijnen, H., Dennehy, M., Liebler, D. & Ischiropoulos, H. (2006). Identification of s-nitrosylation motifs by site-specific mapping of the s-nitrosocysteine proteome in human vascular smooth muscle cells. Proc Nat Acad Sci 103(19), 74207425.
Guttmann, P., Heim, S., Werner, S., Niemann, B., Follath, R. & Schneider, G. (2007). X-ray microscopy at the new U41-FSGM beam line. BESSY—Annual Report 2007, pp. 297300.
Hitchcock, A., Morin, C, Zhang, X. & Araki, T. (2005). Soft X-ray spectromicroscopy of biological and synthetic polymer systems. J Electron Spectrosc 144147, 259269.
Hitchcock, A.P., Dynes, J.J., Lawrence, J.R., Obst, M., Swerhone, G.D.W., Korber, D.R. & Leppard, G.G. (2009). Soft X-ray spectromicroscopy of nickel sorption in a natural river biofilm. Geobiol 7(4), 432453.
Huang, B., Wang, W., Bates, M. & Zhuang, X. (2008). Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319(5864), 810813.
Huang, X., Nelson, J., Kirz, J., Lima, E., Marchesini, S., Miao, H., Neiman, A., Shapiro, D., Steinbrenner, J., Stewart, A., Turner, J. & Jacobsen, C. (2009). Soft X-ray diffraction microscopy of a frozen hydrated yeast cell. Phys Rev Lett 103(19), 198101.
Jacobsen, C, Wirick, S., Flynn, G. & Zimba, C. (2000). Soft X-ray spec-troscopy from image sequences with sub-100 nm spatial resolution. J Microsc 197(2), 173184.
Janiak, R., Wilson, S., Montague, S. & Hume, J. (2001). Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells. Am J Physiol 280(1), C22C33.
Kirz, J., Jacobsen, C. & Howells, M. (2009). Soft X-ray microscopes and their biological applications. Q Rev Biophys 28(1), 33130 (reprint of 1995).
Kolczewski, C., Püttner, R., Martins, M., Schlachter, A.S., Snell, G., Santanna, M.M., Hermann, K. & Kaindl, G. (2006). Spectroscopic analysis of small organic molecules: A comprehensive near-edge X-ray-absorption fine-structure study of C6-ring-containing molecules. J Chem Phys 124(3), 034302.
Lange, K. & Gartzke, J. (2006). F-actin-based Ca signaling—A critical comparison with the current concept of Ca signaling. J Cell Physiol 209(2), 270287.
Larabell, C.A. & Le Gros, M.A. (2004). X-ray tomography generates 3-D reconstructions of the yeast, Saccharomyces cerevisiae, at 60 nm resolution. Mol Biol Cell 15(3), 957962.
Lawrence, J., Swerhone, G., Leppard, G., Araki, T., Zhang, X., West, M. & Hitchcock, A.P. (2003). Scanning transmission X-ray, laser scanning, and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms. Appl Environ Microbiol 69(9), 5543.
Lerotic, M., Jacobsen, C., Schäfer, T. & Vogt, S. (2004). Cluster analysis of soft X-ray spectromicroscopy data. Ultramicroscopy 100, 3557.
McDermott, G., Le Gros, M.A., Knoechel, C.G., Uchida, M. & Larabell, C.A. (2009). Soft X-ray tomography and cryogenic light microscopy: The cool combination in cellular imaging. Trends Cell Biol 19(11), 587595.
McGeown, G. (2010). Gl brown lecture 2010: Seeing is believing: Imaging ca2+-signalling events in living cells. Exp Physiol 95(11), 10491060.
Meyer-Ilse, W., Hamamoto, D., Nair, A., Leliévre, S.A., Denbeaux, G., Johnson, L., Pearson, A.L., Yager, D., Le Gros, M.A. & Larabell, C.A. (2001). High resolution protein localization using soft X-ray microscopy. J Microsc 201(3), 395403.
Mitrea, G., Thieme, J., Guttmann, P., Heim, S. & Gleber, S. (2008). X-ray spectromicroscopy with the scanning transmission X-ray microscope at BESSY II. J Synchrotron Rad 15(1), 2635.
Neu, T.R., Manz, B., Volke, F., Dynes, J.J., Hitchcock, A.P. & Lawrence, J.R. (2010). Advanced imaging techniques for assessment of structure, composition and function in biofilm systems. FEMS Microbiol Ecol 72, 121.
Norlund, K.L.I., Southam, G., Tyliszcak, T., Hu, Y., Karunkaran, C., Obst, M., Hitchcock, A.P. & Warren, L.A. (2009). Microbial architecture of environmental sulfur processes: A novel syntrophic sulfur-metabolizing consortia. Org Geochem 43(23), 87818786.
Parker, I., Choi, J. & Yao, Y. (1996). Elementary events of insp3-induced ca2+ liberation in xenopus oocytes: Hot spots, puffs and blips. Cell Calcium 20(2), 105121.
Parkinson, D.Y., McDermott, G., Etkin, L.D., Le Gros, M.A. & Larabell, C.A. (2008). Quantitative 3-D imaging of eukaryotic cells using soft X-ray tomography. J Struct Biol 162(3), 380386.
Resch, G.P., Goldie, K.N., Krebs, A., Hoenger, A. & Small, J.V. (2002). Visualisation of the actin cytoskeleton by cryo-electron microscopy. J Cell Sci 115(9), 18771882.
Rust, M.J., Bates, M. & Zhuang, X. (2006). Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10), 793796.
Schneider, G., Guttmann, P., Heim, S., Rehbein, S., Mueller, F., Nagashima, K., Heymann, J.B., Müller, W.G. & McNally, J. (2010). Three-dimensional cellular ultrastructure resolved by X-ray microscopy. Nat Methods 7(12), 985987.
Small, J.V., Auinger, S., Nemethova, M., Koestler, S., Goldie, K.N., Hoenger, A. & Resch, G.P. (2008). Unravelling the structure of the lamellipodium. J Microsc 231(3), 479485.
Smith, A.P., Urquhart, S.G., Winesett, D.A., Mitchell, G. & Ade, H. (2001). Use of near edge X-ray absorption fine structure spectromicroscopy to characterize multicomponent polymeric systems. Appl Spectrosc 55(12), 16761681.
Solomon, D., Lehmann, J., Kinyangi, J., Liang, B., Heymann, K., Dathe, L., Hanley, K., Wirick, S. & Jacobsen, C. (2009). Carbon (1s) NEXAFS spectroscopy of biogeochemically relevant reference organic compounds. Soil Sci Soc Am J 73(6), 1817.
Song, C., Jiang, H., Mancuso, A., Amirbekian, B., Peng, L., Sun, R., Shah, S.S., Zhou, Z.H., Ishikawa, T. & Miao, J. (2008). Quantitative imaging of single, unstained viruses with coherent X rays. Phys Rev Lett 101(8), 158101.
Stöhr, J. (1992). NEXAFS Spectroscopy. Berlin, Germany: Springer.
Studer, D., Humbel, B. & Chiquet, M. (2008). Electron microscopy of high pressure frozen samples: Bridging the gap between cellular ultrastructure and atomic resolution. Histochem Cell Biol 130(5), 877889.
Takman, P.A.C., Stollberg, H., Johansson, G., Holmberg, A., Lindblom, M. & Hertz, H.M. (2007). High-resolution compact X-ray microscopy. J Microsc 226(2), 175181.
Thieme, J., Sedlmair, J., Gleber, S.-C, Prietzel, J., Coates, J., Eusterhues, K., Abbt-Braun, G. & Salome, M. (2010). X-ray spectromicroscopy in soil and environmental sciences. J Synchrotron Rad 17(2), 149157.
Thompson, A.C, Attwood, D.T., Gullikson, E.M., Howells, M.R., Kim, K.J., Kirz, J., Kortright, J.B., Lindau, I., Pianatta, P. & Robinson, A.L. (2001). X-ray Data Booklet, 2nd ed., Thompson, A.C. & Vaughan, D. (Eds.). Berkeley, CA: Lawrence Berkeley National Laboratory.
Thyberg, J. (2000). Differences in caveolae dynamics in vascular smooth muscle cells of different phenotypes. Lab Invest 80(6), 915929.
Wang, J., Morin, C., Li, L., Hitchcock, A.P., Scholl, A. & Doran, A. (2009). Radiation damage in soft X-ray microscopy. J Electron Spectrosc 170(1-3), 2536.
Wiesemann, U. (2003). The scanning transmission X-ray microscope at BESSY II. PhD thesis. Göttingen, Germany: Institute for X-Ray Physics, University of Göttingen.
Wiesemann, U., Thieme, J., Früke, R. & Guttmann, P. (2001). Construction of a scanning transmission X-ray microscope at the undulator U-41 at BESSY II. Nucl Instrum Methods A 467468, 861863.
Willig, K., Rizzoli, S., Westphal, V., Jahn, R. & Hell, S. (2006). STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935939.
Yamamoto, A., Masaki, R., Guttmann, P., Schmahl, G. & Kihara, H. (1998). Studies on intracellular structures of COS cells by X-ray microscopy. J Synchrotron Rad 5(3), 11051107.
Zenisek, D., Davila, V., Wan, L. & Almers, W. (2003). Imaging calcium entry sites and ribbon structures in two presynaptic cells. J Neurosc 23(7), 25382548.


Imaging of Vascular Smooth Muscle Cells with Soft X-Ray Spectromicroscopy

  • Julia Sedlmair (a1), Sophie-Charlotte Gleber (a2), Semra Öztürk Mert (a3), Michael Bertilson (a4), Olov von Hofsten (a4), Jürgen Thieme (a5) and Thomas Pfohl (a3) (a6)...


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed