Quantitative Fluorescence Confocal Laser Scanning Microscopy (CLSM)
Abstract
The confocal imaging geometry provides a dramatic optical advantage for fluorescence microscopy by discriminating against out-of-focus background with minimal loss of image-forming signal. Significant enhancement of both axial and lateral imaging resolution is also available but only with substantial signal loss. Because of these optical advantages, the confocal laser scanning microscope (CLSM) can clearly image thin optical sections from within thick fluorescence-labeled living specimens. A stack of optical sections is easily combined to reveal three-dimensional (3D) fluorescent marker distributions with diffraction-limited spatial resolution. When bright stable fluorophores are available, cellular dynamics can be measured by recording a time series of CLSM images.
Keywords
Objective Lens Shot Noise Spherical Aberration Axial Resolution Chromatic AberrationPreview
Unable to display preview. Download preview PDF.
References
- Axelrod, D., Koppel, D.E., Schlessinger, J., Elson, E., and Webb, W.W., 1976, Mobility measurements by analysis of fluorescence photobleaching recovery kinetics, Biophys. J. 16:1055.PubMedCrossRefGoogle Scholar
- Berlman, I.B., 1971, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed., Academic Press, San Diego.Google Scholar
- Bloom, J. A., and Webb, W.W., 1983, Lipid diffusibility of the intact erythrocyte membrane, Biophys. J. 42:295.PubMedCrossRefGoogle Scholar
- Bloom, J.A., and Webb, W.W., 1984, Photodamage to intact erythrocyte membranes at high laser intensities: Methods of assay and suppression, J. Histochem. Cytochem. 32:608.PubMedCrossRefGoogle Scholar
- Born, M., and Wolf, E., 1983a, Principles of Optics, 6th ed., A. Wheaton & Co., Oxford, England.Google Scholar
- Born, M., and Wolf, E., 1983b, Principles of Optics, 6th ed., A. Wheaton & Co., Oxford, England, p. 443.Google Scholar
- Brakenhoff, G.J, and Visscher, K., 1990, Novel confocal imaging and visualization techniques. In: Micro90: Proceedings of the Royal Microscopical Society Conference, London, July 1990 (A. Hilger and H.Y. Elder, eds.), IOL Publishing Ltd., Bristol.Google Scholar
- Cox, I.J., and Sheppard, C.J.R., 1986, Information capacity and resolution in an optical system, J. Opt. Soc. Am., 3:1152–1158.CrossRefGoogle Scholar
- Davoust, J., Devaux, P.F., and Leger, L., 1982, Fringe pattern photobleaching, a new method for the measurement of transport coefficients of biological macromolecules, EMBO J. 1:1233–1238.PubMedGoogle Scholar
- Egger, M.D., and Petráň, M., 1967, New reflected light microscope for viewing unstained brain and ganglion cells, Science 157:305–307.PubMedCrossRefGoogle Scholar
- Goldstein, S., 1989, A no-moving-parts video rate laser beam scanning type 2 confocal reflected/transmission microscope, J. Microsc. 153:1–2.CrossRefGoogle Scholar
- Gunter, W.D. Jr., 1970, Optical device to increase photocathode quantum efficiency, Appl. Opt. 9:251–257.PubMedCrossRefGoogle Scholar
- Haugland, R.P., 1985, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes Inc., Eugene, Oregon.Google Scholar
- Hell, S., and Stelzer, E.H K., 1992, Fundamental improvement of resolution with a 4Pi-confocal fluorescence microscope using two-photon excitation, Opt. Commun. 93:277–282.CrossRefGoogle Scholar
- Inoué, S. 1986, Video Microscopy, Plenum Press, New York.Google Scholar
- Jovin, T.M., and Jovin, D. A., 1987. In: Microspectrofluorometry of Single Living Cells (E. Kohen, J.S. Ploem, and J.S. Hirschberg, eds.), Academic Press, San Diego.Google Scholar
- Jovin, T.M., and Arndt-Jovin, D.J., 1989, Luminescence Digital Imaging Microscopy, Annu. Rev. Biophys. Biophys Chem. 18:271–308.PubMedCrossRefGoogle Scholar
- Kino, G.S., 1990, Intermediate optics inNipkow disk microscopes. In: Handbook of Biological Confocal Microscopy (J.B. Pawley, ed.), Plenum Press, New York, pp. 105–111.CrossRefGoogle Scholar
- Koester, C.J., 1980, A scanning mirror microscope with optical sectioning characteristics: Applications in ophthalmology, Appl. Opt. 19:1749–1757.PubMedCrossRefGoogle Scholar
- Lichtman, J.W., Sunderland, W.J., and Wilkinson, R.S., 1989, High-resolution imaging of synaptic structure with a simple confocal microscope, New Biol. 1:75–82.PubMedGoogle Scholar
- Packard, B.S., and Wolf, D.E., 1985, Fluorescence lifetimes of carbocyanine lipid analogues in phosopholipid bilayers, Biochemistry 24:5176–5181.PubMedCrossRefGoogle Scholar
- Pawley, J.B., 1994, Sources of noise in three-dimensional microscopical data sets. In: Three-dimensional Confocal Microscopy: Volume Investigation of Biological Specimens (J.K. Stevens, L.R. Mills, and J.E. Trogadis, eds.), Academic Press, San Diego, pp. 48–90.Google Scholar
- Sandison, D.R., 1993, Fluorescence Confocal Laser Scanning Microscopy in Living Biological Specimens, PhD thesis, Department of Physics, Cornell University, Ithaca, New York.Google Scholar
- Sandison, D.R., and Webb, W.W., 1994, Background rejection and signal-to-noise optimization in confocal and alternative fluorescence microscopes, Appl. Opt. 33:603–615.PubMedCrossRefGoogle Scholar
- Sandison, D.R., Piston, D.W., and Webb, W.W. 1994a, Background rejection and optimization of signal-to-noise in confocal microscopy. In: Three-dimensional Confocal Microscopy: Volume Investigation of Biological Specimens (J.K. Stevens, L.R. Mills, and J. E. Trogadis, eds.), Academic Press, San Diego, pp. 29–45.CrossRefGoogle Scholar
- Sandison, D.R., Piston, D.W., Williams, R.M., and Webb, W.W., 1994b, Quantitative comparison of background rejection, signal-to-noise ratio, and resolution in confocal and fullfield laser scanning microscopes, App. Opt. 1994, in press.Google Scholar
- Saffman, P.G., and Delbruck, M., 1975, Brownian motion in biological membranes, Proc. Natl. Acad. Sci. USA 72:3111–3113.PubMedCrossRefGoogle Scholar
- Schneider, M.B., and Webb, W.W., 1981, Measurement of submicron laser beam radii, Appl. Opt. 20:1382–1388.PubMedCrossRefGoogle Scholar
- Sheppard, C.J.R., and Matthews, H.J., 1987, Imaging in high aperture optical systems, J. Opt. Soc. Am. A4:1354–1360.CrossRefGoogle Scholar
- Sheppard, C.J.R., 1988, Depth of field in optical microscopy, J. Microsc. 149:73–75.CrossRefGoogle Scholar
- Smith, B.A., and McConnell, H.M., 1978, Determination of molecular motion in membranes using periodic pattern photobleaching, Proc. Natl. Acad. Sci. USA 75:2759–2763.PubMedCrossRefGoogle Scholar
- Tanke, H.J., 1989, Does light microscopy have a future?, J. Micros. 155: 405–418.CrossRefGoogle Scholar
- Webb, J.P., McColgin, W.C., Peterson, O.G., Stockman, D.L., and Eberly, J.H., 1970, Intersystem crossing rate and triplet state lifetime for a lasing dye, J. Chem. Phys. 53:4227–4229.CrossRefGoogle Scholar
- Wells, K.S., Sandison, D.R., Strickler, J., and Webb, W.W., 1990, Quantitative fluorescence imaging with laser scanning confocal microscopy. In: Handbook of Biological Confocal Microscopy (J.B. Pawley, ed.), Plenum Press, New York, pp. 27–39.CrossRefGoogle Scholar
- White, J.G., Amos, W.B., and Fordham, M., 1987, An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy, J. Cell Biol. 105:41–48.PubMedCrossRefGoogle Scholar
- Wilson, T., and Carlini, A.R. 1988, Three-dimensional imaging in confocal imaging systems with finite sized detectors, J. Microsc. 149:51–66.CrossRefGoogle Scholar
- Wilson, T., and Sheppard, C. 1984, Theory and Practice of Scanning Optical Microscopy, Academic Press, London.Google Scholar