Digital Fluorescence Microscopy

Hans J. Tanke1

1 Leiden University, Leiden, The Netherlands
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 2.5
DOI:  10.1002/0471142956.cy0205s00
Online Posting Date:  May, 2001
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Abstract

Fluorescence microscopy is a valuable tool for biomedical research, providing good sensitivity and high multiplicity. Specialized techniques (anisotropy measurement, resonance energy transfer) can provide information on cell spatial arrangement or label microenvironment. The unit discusses camera selection strategy, microscope hardware, and image acquisition and processing.

     
 
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Table of Contents

  • Background Information
  • Choice of Camera for Image‐Plane Scanners
  • Microscope Hardware
  • Acquisition and Processing of Images
  • Literature Cited
     
 
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Materials

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Figures

Videos

Literature Cited

Literature Cited
   Aikens, R.S. 1990. CCD cameras for video microscopy. In Optical Microscopy for Biology (B. Herman and K. Jacobsen, eds.) pp. 207‐218. Wiley‐Liss, New York.
   Arndt‐Jovin, D.J., Robert‐Nicoud, M., Kaufman, S.J., and Jovin, J.M. 1985. Fluorescence digital imaging microscopy (DIM) in cell biology. Science 230:247‐256.
   Dauwerse, J.G., Wiegant, J., Raap, A.K., Breuning, M.H., and Van Ommen, G.J.B. 1992. Multiple colors by fluorescence in situ hybridization using ratio‐labelled DNA probes create a molecular karyotype. Hum. Mol. Genet. 1:593‐598.
   Florijn, R.J., Slats, J., Tanke, H.J., and Raap, A.K. 1995. Analysis of antifading reagents for fluorescence microscopy. Cytometry 19:177‐182
   Florijn, R.J., Bonnet, J., Vrolijk, J., Raap, A.K., and Tanke, H.J. 1996. The effect of chromatic errors in microscopy on the visualization of multi‐colour fluorescence in situ hybridization. Cytometry 23:8‐14.
   Hiraoka, Y., Sedat, J.W., and Agard, D.A. 1987. The use of a charge‐coupled device for quantitative optical microscopy of biological structures. Science 238:36‐41.
   Kallioniemi, A., Kallioniemi, O.‐P., Sudar, D., Rutovitz, D., Gray, J.W., Waldman, F., and Pinkel, D. 1992. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258:818‐821.
   Nederlof, P.M., van der Flier, S., Wiegant, J., Raap, A.K., Tanke, H.J., Ploem, J.S., and Van der Ploeg, M. 1990. Multiple fluorescence in situ hybridization. Cytometry 11:126‐131.
   Ried, T., Baldini, A., Rand, T., and Ward, D.C. 1992. Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. Proc. Natl. Acad. Sci. U.S.A. 89:1388‐1392.
   Shotton, D. 1993. Electronic Light Microscopy. Wiley‐Liss, New York.
   Song, L., Hennink, E.J., Young, I.T., and Tanke, H.J. 1995. Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy. Biophys. J. 68:2588‐2600.
   Tanke, H.J. 1989. Does light microscopy have a future? J. Microsc. 155:405‐418.
   Vrolijk, J., Sloos, W.C.R., Verwoerd, N.P., and Tanke, H.J. 1994. The applicability of a non‐cooled video‐rated CCD camera for the detection of fluorescence in situ hybridization signals. Cytometry 15:2‐11.
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