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Confocal Microscopy System Performance: Axial Resolution 1

  • Robert M. Zucker (a1)

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The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. When tests are made to evaluate the performance of a CLSM, the usual subjective assessment is accomplished by using a histological test slide to create a “pretty picture.” Without the use of functional tests many of the machines may be working at sub-optimal performance levels, delivering sub optimum performance, and possibly misleading data. In order to replace the subjectivity in evaluating a confocal microscope, tests were derived or perfected that measure field illumination, lens clarity, laser power, laser stability, dkhroic functionality, spectral registration, axial resolution, scanning stability, PMT quality, overall machine stability, and system noise (1-3). It is anticipated by using this type of test data, performance standards for confocal microscopes will be obtained and the current subjectivity in evaluating CLSM performance will be eliminated.

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2

To whom all correspondence and reprint requests should be addressed: U.S. Environmental Protection Agency, Reproductive Toxicology Division (MD- 72), National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC 27711; Phone: (919) 541-1585; FAX: (919) 541-4017 e-mail: zucke robert@.epa.gov

1

The research described in this article has been reviewed and approved for publication as an EPA document. Approval does not necessarily signify that the contents reflects the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use

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References

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1. Zucker, R.M., O.T., Price, Evaluation of confocal system performance Cytometry, 44:273294, 2001.
2. Zucker, R.M., Price, O.T., Statistical evaluation of confocal microscopy images, Cytornetry, 44:295308, 2001.
3. Zucker, RM and Price, O.T., Practical confocal microscopy and the evaluation of system performance. Methods, 18: 447458, 1999,
4. Centroze, V, and Pawley, J: Tutorial on practical confocal microscopy and use of the confocal test specimen. In Handbook of Biological Confocal Microscopy, second edition. (Pawley J., Ed) Plenum Press. New York 559567, 1995.
5. Sheppard, C.J.R. and Shotton, D.M.: Confocal Laser Scanning Microscopy, Bios Scientific Publishing, New York, 1997.
6. Marjlof, L. and Forsgren, P.O.: Accurate imaging in confocal microscopy in Methods of cell biology, 79-95, ed. Matsumato, B., Academic Press, San Diego, California, 1993.
7. Carter, D.: Practical considerations for collecting confocal images. Methods Mol Biol. Confocal Microscopy Methods and Protocols, edited by Paddock, S., Humana Press, Totowa NJ. 122:3557 1999.
8 Pawley, J.; Fundamental limits in confocal Microscopy (chapter 2) in Handbook of Biological Confocal Microscopy, second edition. (Pawley J., Ed) Plenum Press, New York 1936, 1995.

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