Intensity Calibration and Flat‐Field Correction for Fluorescence Microscopes

Michael Model1

1 Department of Biological Sciences, Kent State University, Kent, Ohio
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 10.14
DOI:  10.1002/0471142956.cy1014s68
Online Posting Date:  April, 2014
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Abstract

Standardization in fluorescence microscopy involves calibration of intensity in reproducible units and correction for spatial nonuniformity of illumination (flat‐field or shading correction). Both goals can be achieved using concentrated solutions of fluorescent dyes. When a drop of a highly concentrated fluorescent dye is placed between a slide and a coverslip it produces a spatially uniform field, resistant to photobleaching and with reproducible quantum yield; it can be used as a brightness standard for wide‐field and confocal microscopes. For wide‐field microscopes, calibration can be further extended to absolute molecular units. This can be done by imaging a solution of known concentration and known depth; the latter can be prepared by placing a small spherical lens in a diluted solution of the same fluorophore that is used in the biological specimen. Curr. Protoc. Cytom. 68:10.14.1‐10.14.10. © 2014 by John Wiley & Sons, Inc.

Keywords: fluorescence microscopy; confocal microscopy; standardization; calibration; shading correction; sodium fluorescein; Acid Fuschin; Rose Bengal; Acid Blue 9

     
 
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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  • Concentrated standard solutions including:
    • Sodium fluorescein (Sigma‐Aldrich) for blue excitation, green emission
    • Rose Bengal (Sigma‐Aldrich) for green excitation, red emission
    • Acid Blue 9 (TCI America) for red excitation, far‐red emission
    • Prepare 0.25 g of the dye in 1 ml water; vortex initially, and then continue dissolving for 30 min on a shaker or in a sonication bath
  • Diluted solution of the fluorophore that was used to stain the specimen (approximately 20 μl of a 10 μM solution)
  • Widefield or confocal fluorescence microscope (upright widefield microscope for calibration in molecular units)
  • Slides and coverslips
  • Unmounted lens with at least one spherical surface (for example, half‐ball with 5 mm radius, Edmund Optics, cat. no. 45‐937)
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Figures

Videos

Literature Cited

Literature Cited
  Cardullo, R.A. and Alm, E.J. 1998. Introduction to image processing. Methods Cell Biol. 56:91‐115.
  Chacon, E., Reece, J.M., Nieminen, A.L., Zahrebelski, G., Herman, B., and Lemasters, J.J. 1994. Distribution of electrical potential, pH, free Ca2+, and volume inside cultured adult rabbit cardiac myocytes during chemical hypoxia: A multiparameter digitized confocal microscopic study. Biophys. J. 66:942‐952.
  Dee, R.J. 2011. A concentrated fluorescent dye solution as a standard for use in quantification of fluorescence in a microfluidic platform. http://www.ucl.ac.uk/∼ucbprjd/CP/Final%20Report.pdf.
  Dogsa, I., Brloznik, M., Stopar, D., and Mandic‐Mulec I. 2013. Exopolymer diversity and the role of levan in Bacillus subtilis biofilms. PLoS One 8:e62044.
  Galush, W.J., Nye, J.A., and Groves, J.T. 2008. Quantitative fluorescence microscopy using supported lipid bilayer standards. Biophys. J. 95:2512‐2519.
  Garsha, K. 2008. Quantitative fluorescence microscopy: Considerations and controls. Springer Ser. Fluoresc. 6:55‐88.
  Halter, M., Tona, A., Bhadriraju, K., Plant, A.L., and Elliott, J.T. 2007. Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts. Cytometry A 71:827‐834.
  Halter, M., Sisan, D.R., Chalfoun, J., Stottrup, B.L, Cardone, A., Dima, A.A., Tona, A., Plant, A.L., and Elliott, J.T. 2011. Cell cycle dependent TN‐C promoter activity determined by live cell imaging. Cytometry A 79:192‐202.
  Han, F. and Zhang, B. 2013. Characterizing cell‐cell interactions induced spatial organization of cell phenotypes: Application to density‐dependent protein nucleocytoplasmic distribution. Cell Biochem. Biophys. 65:163‐172.
  Model, M.A. 2012. Imaging the cell's third dimension. Microsc. Today 20:32‐37.
  Model, M.A. and Healy, K.E. 2000. Quantification of the surface density of a fluorescent label with a microscope. J. Biomed. Mater. Res. 50:90‐99.
  Model, M.A. and Burkhardt, J.K. 2001. A standard for calibration and shading correction of a fluorescence microscope. Cytometry 44:309‐316.
  Model, M.A. and Blank, J.L. 2006. Intensity calibration of a laser scanning confocal microscope based on concentrated dyes. Quant. Anal. Quant. Cytol. Histol. 28:253‐261.
  Model, M.A., Reese, J.L., and Fraizer, G.C. 2009. Measurement of wheat germ agglutinin binding with a fluorescence microscope. Cytometry A 75:874‐881.
  Nedoma, J. and Vrba, J. 2006. Specific activity of cell‐surface acid phosphatase in different bacterioplankton morphotypes in an acidified mountain lake. Environ. Microbiol. 8:1271‐1279.
  Oberholzer, M., Ostreicher, M., Christen, H., and Bruhlmann, M. 1996. Methods in quantitative image analysis. Histochem. Cell Biol. 105:333‐355.
  Pawley, J. 2000. The 39 steps: A cautionary tale of quantitative 3‐D fluorescence microscopy. Biotechniques 28:884‐888.
  Peterson, A.W., Halter, M., Tona, A., Bhadriraju, K., and Plant, A.L. 2009. Surface plasmon resonance imaging of cells and surface‐associated fibronectin. BMC Cell Biol. 10:16.
  Resch‐Genger, U., Hoffmann, K., Nietfeld, W., Engel, A., Neukammer, J., Nitschke, R., Ebert, B., and Macdonald, R. 2005. How to improve quality assurance in fluorometry: Fluorescence‐inherent sources of error and suited fluorescence standards. J. Fluorescence 15:337‐362.
  Rost, F.W.D. 1991. Quantitative Fluorescence Microscopy. Cambridge University Press, Cambridge, U.K.
  Russ, J.C. 1999. The Image Processing Handbook, Third Edition. CRC Press/IEEE Press, Boca Raton, Fla.
  Schelshorn, D.W., Schneider, A., Kuschinsky, W., Weber, D., Krüger, C., Dittgen, T., Bürgers, H.F., Sabouri, F., Gassler, N., Bach, A., and Maurer, M.H. 2009. Expression of hemoglobin in rodent neurons. J. Cereb. Blood Flow Metab. 29:585‐595.
  Shaw, S.L. 2006. Imaging the live plant cell. Plant J. 45:573‐598.
  Sisken, J.E. 1989. Fluorescent standards. Methods Cell Biol. 30:113‐126.
  Wang, L., Calcedo, R., Bell, P., Lin, J., Grant, R.L., Siegel, D.L., and Wilson, J.M. 2011. Impact of pre‐existing immunity on gene transfer of nonhuman primate liver with adeno‐associated virus 8 vectors. Hum. Gene Ther. 22:1369‐1401.
  Zwier, J.M., Oomen, L., Brocks, L., Jalink, K., and Brakenhoff, G.J. 2008. Quantitative image correction and calibration for confocal fluorescence microscopy using thin reference layers and SIPchart‐based calibration procedures. J. Microsc. 231:59‐69.
  Zucker, R.M. and Price, O. 2001. Evaluation of confocal microscopy system performance. Cytometry 44:273‐294.
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