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Visualization of the Microtubules of Glutaraldehyde-Fixed Cells by Reflection-Enhanced Backscatter Confocal Microscopy

Published online by Cambridge University Press:  09 December 2005

Charles H. Keith  and
Mark A. Farmer
Charles H. Keith
Affiliation:
Department of Cellular Biology, The University of Georgia, 724 Biological Sciences Building, Athens, GA 30602
Mark A. Farmer
Affiliation:
Department of Cellular Biology, The University of Georgia, 724 Biological Sciences Building, Athens, GA 30602 Center for Advanced Ultrastructural Research, The University of Georgia, 151 Barrow Hall, Athens, GA 30602
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Abstract

Performing reflection-mode (backscatter-mode) confocal microscopy on cells growing on reflective substrates gives images that have improved contrast and are more easily interpreted than standard reflection-mode confocal micrographs (Keith et al., 1998). However, a number of factors degrade the quality of images taken with the highest-resolution microscope objectives in this technique. We here describe modifications to reflection-enhanced backscatter confocal microscopy that (partially) overcome these factors. With these modifications of the technique, it is possible to visualize structures the size—and refractility—of individual microtubules in intact cells. Additionally, we demonstrate that this technique, in common with fluorescence techniques such as standing wave widefield fluorescence microscopy and 4-Pi confocal microscopy, offers improved resolution in the Z-direction.

Keywords

imaginghyperresolutionopaque substrates
Type
BIOLOGICAL APPLICATIONS
Information
Microscopy and Microanalysis , Volume 12 , Issue 2 , April 2006 , pp. 113 - 123
Copyright
© 2006 Microscopy Society of America

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References

REFERENCES

Bailey, B., Farkas, D.L., Taylor, D.L., & Lanni, F. (1993). Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation. Nature 366, 44–48.Google Scholar
Bajer, A.S., Sato, H., & Mole-Bajer, J. (1986). Video microscopy of colloidal gold particles and immuno-gold labelled microtubules in improved rectified DIC and epi-illumination. Cell Struct Funct 11, 317–330.Google Scholar
Brenner, M.S.S., Fellers, T.J., & Davidson, M.W. (2004). Water immersion objectives. (Nikon MicroscopyU, http://www.microscopyu.com).
Colicos, M.A., Collins, B.E., Sailor, M.J., & Goda, Y. (2001). Remodeling of synaptic actin induced by photoconductive stimulation. Cell 107, 605–616.Google Scholar
Freimann, R., Pentz, S., & Horler, H. (1997). Development of a standing-wave fluorescence microscope with high nodal plane flatness. J Microsc 187 (Pt 3), 193–200.Google Scholar
Frohn, J.T., Knapp, H.F., & Stemmer, A. (2000). True optical resolution beyond the Rayleigh limit achieved by standing wave illumination. Proc Natl Acad Sci USA 97, 7232–7236.Google Scholar
Hell, S.W., Schrader, M., & van der Voort, H.T. (1997). Far-field fluorescence microscopy with three-dimensional resolution in the 100-nm range. J Microsc 187 (Pt 1), 1–7.Google Scholar
Hell, S.W. & Stelzer, E.H.K. (1992). Properties of a 4Pi confocal fluorescence microscope. J Opt Sci Am A 9, 2159–2166.Google Scholar
Howell, B., Deacon, H., & Cassimeris, L. (1999). Decreasing oncoprotein 18/stathmin levels reduces microtubule catastrophes and increases microtubule polymer in vivo. J Cell Sci 112, 3713–3722.Google Scholar
Kalisch, W.E., Whitmore, T., & Siegel, A. (1985). Laser scanning microscopy of surface spread polytene chromosomes. J Microsc 137, 217–224.Google Scholar
Katsumi, A., Milanini, J., Kiosses, W.B., del Pozo, M.A., Kaunas, R., Chien, S., Hahn, K.M., & Schwartz, M.A. (2002). Effects of cell tension on the small GTPase Rac. J Cell Biol 158, 153–164.Google Scholar
Keith, C.H., Bird, G.J., & Farmer, M.A. (1998). Coherent backscatter enhances reflection confocal microscopy. Biotechniques 25, 858–860, 862–864, 866.Google Scholar
Lanni, F. & Bailey, B. (1994). Standing-wave excitation for fluorescence microscopy. Trends Cell Biol 4, 262–265.Google Scholar
Murnane, A.C., Brown, K., & Keith, C.H. (2002). Preferential initiation of PC12 neurites in directions of changing substrate adhesivity. J Neurosci Res 67, 321–328.Google Scholar
Nagorni, M. & Hell, S.W. (2001). Coherent use of opposing lenses for axial resolution increase in fluorescence microscopy. I. Comparative study of concepts. J Opt Soc Am A Opt Image Sci Vis 18, 36–48.Google Scholar
Norwood, D.E. & Gilmour, A. (2001). The differential adherence capabilities of two Listeria monocytogenes strains in monoculture and multispecies biofilms as a function of temperature. Lett Appl Microbiol 33, 320–324.Google Scholar
Osborn, M., Born, T., Koitsch, H.J., & Weber, K. (1978). Stereo immunofluorescence microscopy: I. Three-dimensional arrangement of microfilaments, microtubules and tonofilaments. Cell 14, 477–488.Google Scholar
Sato, H., Ellis, G.W., & Inoue, S. (1975). Microtubular origin of mitotic spindle form birefringence. Demonstration of the applicability of Wiener's equation. J Cell Biol 67, 501–517.Google Scholar
Schrader, M., Bahlmann, K., Giese, G., & Hell, S.W. (1998). 4Pi-confocal imaging in fixed biological specimens. Biophys J 75, 1659–1668.Google Scholar
Schrader, M. & Hell, S.W. (1996). 4Pi-confocal images with axial superresolution. J Microsc 183, 189–193.Google Scholar
Sheppard, C.J., Roy, M., & Sharma, M.D. (2004). Image formation in low-coherence and confocal interference microscopes. Appl Opt 43(7), 1493–1502.Google Scholar
Sheppard, C.J.R. & Gong, Y. (1991). Improvement in axial resolution by interference confocal microscopy. Optik 87, 129–132.Google Scholar
Slayter, E.M. & Slayter, H.S. (1992). Light and Electron Microscopy. Cambridge, New York: Cambridge University Press.
Tanaka, E. & Kirschner, M.W. (1995). The role of microtubules in growth cone turning at substrate boundaries. J Cell Biol 128, 127–137.Google Scholar
Walker, R.A., Pryer, N.K., & Salmon, E.D. (1991). Dilution of individual microtubules observed in real time in vitro: Evidence that cap size is small and independent of elongation rate. J Cell Biol 114, 73–81.Google Scholar
Wang, L. & Brown, A. (2001). Rapid intermittent movement of axonal neurofilaments observed by fluorescence photobleaching. Mol Biol Cell 12, 3257–3267.Google Scholar