[PDF]    http://dx.doi.org/10.3952/lithjphys.45301

Open access article / Atviros prieigos straipsnis

Lith. J. Phys. 45, 207–211 (2005)


SCANNING NEAR-FIELD OPTICAL MICROSCOPY OF LIVE CELLS IN LIQUID
R. Januškevičiusa, V. Vaičikauskasa, D.J. Arndt-Jovinb, and T.M. Jovinb
aDepartment of Nonlinear Optics and Spectroscopy, Institute of Physics, Savanorių 231, LT-02300 Vilnius, Lithuania
bDepartment of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany

Received 14 March 2005

A scanning near-field optical microscope (SNOM) is applied to fluorescence imaging of biological samples in liquid, including live cells. The SNOM is mounted on a Zeiss Axiovert 135 TV fluorescence microscope. For feedback we use tuning fork shear force method. The scanning tip is produced from a 125 μm optical fibre (8.3 μm core diameter) in a commercial Sutter P-2000 pipette puller and is coated with aluminium. Other commercial tips have also been used. Coarse z-axis adjustment is hydraulic, and fine positioning is accomplished with piezoelectric tube units. We have constructed the original liquid chamber, which allows long term stability of scanning and highest values of the Q factor (300 or more). The depth of liquid layer was less than 40 μm. Near-field images – the topography and distribution of membrane fluorescence of live human epithelial A431 cells, stably transfected with an EGFP fusion protein of the epidermal growth factor transmembrane receptor protein (EGFR, erbB1), were obtained in liquid.
Keywords: scanning near-field optical microscopy, fluorescence microscopy, sub-diffraction limit, live cells
PACS: 07.79.Fc, 87.64.Xx


GYVŲ LĄSTELIŲ SKENUOJANTI ARTIMO LAUKO MIKROSKOPIJA SKYSTYJE
R. Januškevičiusa, V. Vaičikauskasa, D.J. Arndt-Jovinb, T.M. Jovinb
aFizikos institutas, Vilnius, Lietuva
bMakso Planko Biofizinės chemijos institutas, Gėtingenas, Vokietija
Sukurtas ir sukonstruotas optinis artimo lauko mikroskopas pritaikytas gyvų ląstelių ir kitų biologinių objektų, esančių tirpaluose, fluorescenciniam signalui fiksuoti. Mikroskopas sumontuotas ant fluorescencinio Zeiss Axiovert 135 mikroskopo. Skenavimui naudotos adatos, pagaminamos kaitinant ir tempiant šviesolaidį, kurios vėliau metalizuojamos. Sukonstruota speciali skystinė kiuvetė, pritaikyta biologiniams objektams skenuoti tirpale. Mažas tirpalo gylis kiuvetėje minimaliai įtakoja adatos jautrį skenuojant bandinio paviršių ir nekritiškai sumažina adatos rezonanso Q faktorių. Tai leidžia skenuoti lengvai pažeidžiamą gyvų ląstelių paviršių. Gauti eksperimentiniai gyvų žmogaus epitelio A431 ląstelių, žymėtų EGFP baltymu, artimo lauko fluorescencijos ir topografijos
vaizdai.


References / Nuorodos

[1] V. Subramaniam, A.K. Kirsch, and T.M. Jovin, Cell biological applications of scanning near-field optical microscopy (SNOM), Cell. Mol. Biol. 44, 689–700 (1998)
[2] P. Nagy, A. Jenei, A.K. Kirsch, J. Szöllösi, S. Damjanovich, and T.M. Jovin, Activation dependent clustering of the erbB2 receptor tyrosine kinase detected by scanning near-field optical microscopy, J. Cell Sci. 112, 1733–1741 (1999),
http://dx.doi.org/10.1242/jcs.112.11.1733
[3] J. Hwang, L.A. Gheber, L. Margolis, and M. Edidin, Domains in cell plasma membranes investigated by near-field scanning optical microscopy, Biophys. J. 74, 2184–2190 (1998),
http://dx.doi.org/10.1016/S0006-3495(98)77927-5
[4] H. Muramatsu, N. Chiba, K. Homma, and K. Nakajima, Near-field optical microscopy in liquids, Appl. Phys. Lett. 66, 3245–3247 (1995),
http://dx.doi.org/10.1063/1.113392
[5] L.A. Gheber, J. Hwang, and M. Edidin, Design and optimization of a near-field scanning optical microscope for imaging biological samples in liquid, Appl. Opt. 37, 3574–3581 (1998),
http://dx.doi.org/10.1364/AO.37.003574
[6] P.J. Moyer and S.B. Kämmer, High-resolution imaging using near-field scanning optical microscopy and shear force feedback in water, Appl. Phys. Lett. 68, 3380–3382 (1996),
http://dx.doi.org/10.1063/1.116510
[7] T.H. Keller, T. Rayment, D. Klenerman, and R.J. Stephenson, Scanning near-field microscopy in reflection mode imaging in liquid, Rev. Sci. Instrum. 68, 1448–1454 (1997),
http://dx.doi.org/10.1063/1.1147937
[8] T.H. Keller, T. Rayment, and D. Klenerman, Optical chemical imaging of tobacco mosaic virus in solution at 60-nm resolution, Biophys. J. 73, 653–658 (1998),
http://dx.doi.org/10.1016/S0006-3495(98)77914-7
[9] A. Karrai and R. Grober, Piezo-electric tuning fork tip–sample distance control for near field optical microscopes, Ultramicroscopy 61, 197–205 (1995),
http://dx.doi.org/10.1016/0304-3991(95)00104-2
[10] P. Lambelet, M. Pfeffer, A. Sayah, and F. Marquis-Weible, Reduction of tip–sample interaction forces for scanning near-field optical microscopy in a liquid environment, Ultramicroscopy 71, 117–121 (1998),
http://dx.doi.org/10.1016/S0304-3991(97)00055-7
[11] W.H. Rensen and N.F. Hulst, Imaging soft samples in liquid with tuning fork based shear force microscopy, Appl. Phys. Lett. 77, 1557–1559 (2000),
http://dx.doi.org/10.1063/1.1308058
[12] A.P. Sommer and R.P. Franke, Hydrophobic optical elements for near-field optical analysis (NOA) in liquid environment – a preliminary study, Micron 33, 227–231 (2002),
http://dx.doi.org/10.1016/S0968-4328(01)00021-X
[13] A.H. Mannelquist, H.I. Iwamoto, G. Szabo, and Z. Shao, Near-field optical microscopy in aqueous solution: Implementation and characterization of a vibrating probe, J. Microsc. 205, 53–60 (2002),
http://dx.doi.org/10.1046/j.0022-2720.2001.00965.x
[14] M. Koopman, A. Cambi, B.I. de Bakker, B. Joosten, C. Figdor, N.F. van Hulst, and M. Garcia-Parajo, Near-field scanning optical microscopy in liquid for high resolution single molecule detection on dentritic cells, FEBS Lett. 573, 6–10 (2004),
http://dx.doi.org/10.1016/j.febslet.2004.07.035
[15] M. Koopman, B.I. de Bakker, M. Garcia-Parajo, and N.F. van Hulst, Shear force imaging of soft samples in liquid using a diving bell concept, Appl. Phys. Lett. 83, 5083–5085 (2003),
http://dx.doi.org/10.1063/1.1634385
[16] R.T. Doyle, M.J. Szulzcewski, and P.G. Haydon, Extraction of near-field fluorescence from composite signals to provide high resolution images of glial cells, Biophys. J. 80, 2477–2482 (2001),
http://dx.doi.org/10.1016/S0006-3495(01)76216-9