Optical Considerations at Ultraviolet Wavelengths in Confocal Microscopy

  • A. Christyne Bliton
  • James D. Lechleiter


This chapter addresses the use of ultraviolet (UV) light in confocal microscopy. We discuss both realized and potential advantages of UV-visible confocal imaging, as well as the special optical problems caused by the extension of the microscope’s wavelength range beyond the visible spectrum. Although this technology is new, a number of papers have appeared describing the use of both beam-scanning UV-confocal microscopes (Bliton et al., 1993; Fricker and White, 1992; Read et al., 1992; Lechleiter and Clapham, 1992; Kuba et al., 1991; Montag et al., 1991; Schubert, 1991; Ulfhake et al., 1991) and stage-scanning UV-confocal microscopes (Himpens et al., 1992).


Modulation Transfer Function Antireflection Coating Chromatic Aberration Conventional Microscope Ultraviolet Wavelength 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Adams, S.R., and Tsien, R.Y., 1993, Controlling cell chemistry with caged compounds, Annu. Rev. Physiol. 55:755–84.PubMedCrossRefGoogle Scholar
  2. Adams, S., Harootunian, A., Buechler, Y., and Tsien, R., 1991, Fluorescent ratio imaging of cyclic AMP in single cells, Nature 349:694–697.PubMedCrossRefGoogle Scholar
  3. Agard, D.A., Hiraoka, Y., Shaw, P., and Sedat, J.W., 1989, Fluorescence microscopy in three dimensions, Methods Cell Biol. 30:353–377.PubMedCrossRefGoogle Scholar
  4. Betero, M., Boccacci, P., Brakenhoff, G.J., Malfanti, F., and van der Voort, H.T.M., 1990, Three-dimensional image restoration and super-resolution in fluorescence confocal microscopy, J. Microsc. 157:3–20.CrossRefGoogle Scholar
  5. Bliton, C., Lechleiter, J., and Clapham, D.E., 1993, Optical modifications enabling simultaneous confocal imaging with dyes excited by ultra-violet and visible-wavelength light, J. Microsc. 169:15–26.CrossRefGoogle Scholar
  6. Born, M., and Wolf, E., 1980, Principles of Optics, 6th ed. Pergamon Press, Elmsford, New York.Google Scholar
  7. Boyde, A., Jones, S.J., Taylor, M.L., Wolfe, L.A., and Watson, T.F., 1990, Fluorescence in the tandem scanning microscope, J. Microsc. 157:1, 39–49.PubMedCrossRefGoogle Scholar
  8. Brakenhoff, G.J., Binnerts, J.S., and Woldringh, C.L., 1980, Developments in high resolution confocal scanning light microscopy (CSLM). In: Scanned Image Microscopy (E.A. Ash, ed.), Academic Press, New York, pp. 183–200.Google Scholar
  9. Carlsson, K., 1991, The influence of specimen refractive index, detector signal integration, and non-uniform scan speed on the imaging properties in confocal microscopy, J. Microsc. 163:167–178.CrossRefGoogle Scholar
  10. Cogswell, C.J., and Sheppard, C.J.R., 1992, Confocal differential interference contrast (DIC) microscopy including a theoretical analysis of conventional and confocal DIC imaging, J. Microsc. 165:81–101.CrossRefGoogle Scholar
  11. Czaban, B.B., and Forer, A., 1991, Visualization of ultraviolet microbeam irradiation, J. Microsc. 164:61–65.CrossRefGoogle Scholar
  12. Denk, W., Strickler, J.H., and Webb, W.W., 1990, Two-photon laser scanning fluorescence in microscopy, Science 248:73–76.PubMedCrossRefGoogle Scholar
  13. Ellis-Davies, G.C., and Kaplan, J.H., 1995, Caged compound review, Am. J. Physiol, in preparation.Google Scholar
  14. Fricker, M.D., and White, N.S., 1992, Wavelength considerations in confocal microscopy of botanical specimens, J. Microsc. 166:29–42.CrossRefGoogle Scholar
  15. Greulich, K.O., and Weber, G., 1992, The light microscope on its way from an analytical to a preparative tool, J. Microsc. 167:127–151.CrossRefGoogle Scholar
  16. Hell, S., Witting, S., Schickfus, M.V., Wijnaendts van Resand, R.W., Hunklinger, S., Smolka, E., and Neiger, M., 1991, A confocal beam-scanning white light microscope, J. Microsc. 163:179–187.CrossRefGoogle Scholar
  17. Hell, S., Reiner, G., Cremer, C., and Stelzer, E.H.K., 1993, Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index, J. Microsc. 169:391–405.CrossRefGoogle Scholar
  18. Himpens, B., DeSmedt, H., Droogmans, G., and Casteels, R., 1992, Differences in regulation between nuclear and cytoplasmic Ca2+ in cultured smooth muscle cells, Am. J. Physiol. 263:C95–C105.Google Scholar
  19. Hobbs, P.C.D., and Kino, G.S., 1990, Generalizing the confocal microscope via heterodyne interferometry and digital filtering, J. Microsc. 160:245–264.CrossRefGoogle Scholar
  20. Inoué, S., 1986, Microscope image formation. In: Video Microscopy (S. Inoué, ed.), Plenum Press, New York, Chapter 5.Google Scholar
  21. Kaplan, J.H., 1990, Special topic: Caged compounds in cellular physiology, Annu. Rev. Physiol 52:853–855.CrossRefGoogle Scholar
  22. Kapuscinski, J., and Darzinkiewicz, Z., 1990, Spectral properties of fluoro-chromes used in flow cytometry, Methods Cell Biol 33:665–668.Google Scholar
  23. Kingslake, R., 1978, Lens Design Fundamentals, Academic Press, New York.Google Scholar
  24. Kingslake, R., 1983, Optical System Design, Academic Press, New York.Google Scholar
  25. Kuba, K., Hua, S.Y., and Nohmi, M., 1991, Spatial and dynamic changes in intracellular Ca2+ measured by confocal laser scanning microscopy in bullfrog sympathetic ganglion cells, Neurosci. Res. 10:245–246.PubMedCrossRefGoogle Scholar
  26. Laser Institute of America, 1991, Laser Safety Guide (J.A. Smith, ed.), Orlando, Florida.Google Scholar
  27. Lasers & Optronics, 1992, 1992 Buying Guide Industry and Product Directory, Elsevier Communications, Morris Plains, New Jersey.Google Scholar
  28. Lechleiter, J.D., and Clapham, D.E., 1992, Molecular mechanisms of intracellular calcium excitability in X. laevis oocytes, Cell 69:283–294.PubMedCrossRefGoogle Scholar
  29. Macias-Garza, F., Diller, K.R., Bovik, A.C., and Aggarwal, S.J., 1988, Digital reconstruction of three-dimensional serially sectioned optical images, IEEE Trans. 36:1067–1075.Google Scholar
  30. Macias-Garza, F., Diller, K.R., Bovik, A.C., and Aggarwal, S.J., 1989, Improvement in the resolution of three-dimensional data sets collected using optical serial sectioning, J. Microsc. 153:205–221.CrossRefGoogle Scholar
  31. Mercado, R.I., 1992, Design of achromats and superachromats. In: Lens Design (W.J. Smith, ed.), SPIE Optical Engineering Press, Vol. CR41, pp. 270–296.Google Scholar
  32. Mercado, R.I., and Robb, P.N., 1991, Color-corrected optical systems, U.S. Patent 5,020,889, June 4, 1991.Google Scholar
  33. Montag, M., Kukulies, J., Jorgens, R., Gundlach, H., Trendelenburg, M.F., and Spring, H., 1991, Work with the confocal scanning UV-laser microscope: Specific DNA localization at high sensitivity and multiple-parameter fluorescence, J. Microsc. 163:201–210.PubMedCrossRefGoogle Scholar
  34. Photonics, 1992, Book 2: The Photonics Buyers’ Guide to Products and Manufacturers, Laurin Publishing Co., Pittsfield, Massachusetts.Google Scholar
  35. Read, N.D., Allan, W.T.G., Knight, H., Knight, M.R., Malho, R., Russel, A., Schacklock, P.S., and Trewavas, A.J., 1992, Imaging and meaurement of cytosolic free calcium in plant and fungal cells, J. Microsc. 166:57–86.CrossRefGoogle Scholar
  36. Robb, P., 1993, Commercial applications of liquid optical systems, SPIE, Vol. 2018, Proceedings, Passive Materials for Optical Elements II.Google Scholar
  37. Schubert, W., 1991, Triple immunofluorescence confocal laser scanning microscopy: Spatial correlation of novel cellular differentiation markers in human muscle biopsies, Eur. J. Cell Biol 55:272–285.PubMedGoogle Scholar
  38. Shao, Z., Baumann, O., and Somlyo, A.P., 1991, Axial resolution of confocal microscopes with parallel beam detection, J. Microsc. 164:13–19.PubMedCrossRefGoogle Scholar
  39. Shaw, P. J., and Rawlins, D.J., 1991, PSF of a confocal microscope: Its measurement and use in deconvolution of 3-D data, J. Microsc. 163:151–165.CrossRefGoogle Scholar
  40. Sheppard, C.J.R., Gu, M., and Roy, M., 1992, Signal-to-noise ratio in confocal microscope systems, J. Microsc. 168:209–218.CrossRefGoogle Scholar
  41. Sigler, R.D., 1990, Color-corrected optical systems with liquid lens elements, U.S. Patent number 4,915,483, April 10.Google Scholar
  42. Smith, W.J., 1989, Optical component specifications. In: Book 3: The Photonics Design and Applications Handbook, Laurin Publishing Co., Pittsfield, Massachusetts, pp. 377–378.Google Scholar
  43. Smith, W.J., 1990, Modern Optical Engineering, McGraw-Hill, New York.Google Scholar
  44. Smith, W.J., and Genesee Optics Software, Inc., 1992, Modern Lens Design, McGraw-Hill, New York.Google Scholar
  45. Su, F., 1993, An interview with Jim Janesick, Jet Propulsion Laboratory, Large-area scientific CCDs—From memory device to imager, OE Reports SPIE, 110.Google Scholar
  46. Takamatsu, T., Minamikawa, T., Kawachi, H., and Fujita, S., 1991, Imaging of calcium wave propagation in guinea-pig ventricular cell pairs by confocal laser scanning microscopy, Cell Struct. Funct. 16(4):341–346.PubMedCrossRefGoogle Scholar
  47. Tower, J.R., 1992, Silicon based imaging: Extending performance on all fronts, Photonics Spectra April: 165–167.Google Scholar
  48. Tsien, R.Y., 1989, Fluorescent probes of cell signalling, Annu. Rev. Neurosci. 12:227–253.PubMedCrossRefGoogle Scholar
  49. Ulfhake, B., Carlsson, K., Mossberg, K., Arvidsson, U., and Helm, P.J., 1991, Imaging of fluorescent neurons labeled with fluor-gold and axon terminal labeled with AMCA conjugated antiserum using a UV-laser confocal scanning microscope, J. Neurosci. Methods 40:39–48.PubMedCrossRefGoogle Scholar
  50. Veldkamp, W.B., and McHugh, T.J., 1992, Binary optics, Sci. Am. May:92–97.Google Scholar
  51. Visser, T.D., Oud, J.L., and Brakenhoff, G.J., 1992, Refractive index and axial distance measurements in 3-D microscopy, Optik 90(1): 17–19.Google Scholar
  52. Walker, B., 1989, Surface tolerances. In: Book 3: The Photonics Design and Applications Handbook, Laurin Publishing Co., Pittsfield, Massachusetts, pp. 379–380.Google Scholar
  53. Willis, B., Roysam, B., Turner, J.N., and Holmes, T.J., 1993, Iterative, constrained 3-D image reconstruction of transmitted light bright-field micrographs based on maximum likelihood estimation, J. Microsc. 169:347–361.PubMedCrossRefGoogle Scholar
  54. Wilson, T., 1989, Optical sectioning in confocal fluorescent microscopes, J. Microsc. 154:143–156.CrossRefGoogle Scholar
  55. Young, M.R., Jiang, S.H., Davies, R.E., Walker, J.G., Pike, E.R., and Bertero, M., 1992, Super-resolution in coherent confocal scanning microscopy using optical masks, J. Microsc. 165:131–138.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • A. Christyne Bliton
    • 1
  • James D. Lechleiter
    • 2
  1. 1.Meridian InstrumentsOkemosUSA
  2. 2.Department of NeuroscienceUniversity of Virginia Health Science CenterCharlottesvilleUSA

Personalised recommendations