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Intensity Calibration and Shading Correction for Fluorescence Microscopes

Michael A. Model¹

¹Kent State University, Kent, Ohio

Publication Name:

Current Protocols in Cytometry

Unit Number:

Unit 10.14

DOI:

10.1002/0471142956.cy1014s37

Online Posting Date:

August, 2006

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Abstract

Standardization in image cytometry involves intensity calibration and shading correction. This unit presents a method using concentrated solutions of fluorophores. A drop of highly concentrated dye solution placed between a slide and a coverslip produces a spatially uniform fluorescent sample with reproducible quantum yield and resistance to photobleaching. The technique has a number of practical features that make it inexpensive, reproducible, and straightforward. Descriptions are given for both wide-field and confocal scanning fluorescence microscopy.

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

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Basic Protocol
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Figures

Figure 10.14.1

Example of shading correction. Images were acquired with an Axiovert TV100 microscope (Carl Zeiss) through a Plan Neofluar 20/0.5 air objective using illumination from a 100-W Hg lamp. (A) Image of a sodium fluorescein slide (Standard). There is a more than two-fold difference in the intensity between the brightest area below the center and the dark upper corners. (B) Original image (Specimen) of Jurkat cells stained with anti-phosphotyrosine Alexa 488 (for details see Model and Burkhardt, 2001). (C) Image Corrected. Note the significant brightening of cells in the upper left corner. The background level was determined by measuring the empty areas in Specimen; it is uniform throughout the image with intensity an order of magnitude less than that of the average cell.

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Literature Cited

Literature Cited
	Cardullo, R.A. and Alm, E.J. 1998. Introduction to image processing. Methods Cell Biol. 56:91-115.
	Likar, B., Maintz, J.B.A., Viergever, M.A., and Pernus, F. 2000. Retrospective shading correction based on entropy minimization. J. Microsc. 197:285-295.
	Model, M.A. and Blank, J.L. Intensity calibration of a laser scanning confocal microscope based on concentrated dyes. Quant. Anal. Quant. Cytol. Histol. Accepted for publication.
	Model, M.A. and Burkhardt, J.K. 2001. A standard for calibration and shading correction of a fluorescence microscope. Cytometry 44:309-316.
	Oberholzer, M., Ostreicher, M., Christen, H., and Bruhlmann, M. 1996. Methods in quantitative image analysis. Histochem. Cell Biol. 105:333-355.
	Resch-Genger, U., Hoffmann, K., Nietfeld, W., En gel, 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.
	Sandison, D.R., Williams, R.M., Wells, K.S., Strickler, J., and Webb, W.W. 1995. Quantitative fluorescence confocal laser scanning microscopy (CLSM). In Handbook of Biological Confocal Microscopy, 2nd ed. (J.B. Pawley ed.) pp. 39-53. Plenum Press, New York.
	Sisken, J.E. 1989. Fluorescent standards. Methods Cell Biol. 30:113-126.
	Swedlow, J.R., Hu, K., Andrews, P.D., Roos, D.S., and Murray, J.M. 2002. Measuring tubulin content in Toxoplasma gondii: A comparison of laser-scanning confocal and wide-field fluorescence microscopy. Proc. Natl. Acad. Sci. U.S.A. 99:2014-2019.
	Taylor, D.L. and Salmon, E.D. 1989. Basic fluorescence microscopy. Methods Cell Biol. 29:207-237.
	Wilson, T. 1995. The role of the pinhole in confocal imaging system. In Handbook of Biological Confocal Microscopy, 2nd ed. (J.B. Pawley ed.) pp. 167-182. Plenum Press, New York.
	Wu, H.-X. and Ji, L. 2005. Fully automated intensity compensation for confocal microscopic images. J. Microsc. 220:9-19.
	Zucker, R.M. and Price, O. 2001. Evaluation of confocal microscopy system performance. Cytometry 44:273-294.
Key References
	Model and Burkhardt, 2001. See above.
	Model and Blank, in press. See above.
Detailed description of calibration based on concentrated solutions for wide-field (Model and Burkhardt, 2001) and confocal scanning (Model and Blank, in press) fluorescence microscopy.