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Optimization and the Evaluation of Confocal System Performance to Quantify Fluorescence

Published online by Cambridge University Press:  02 July 2020

Robert M. Zucker  and
Owen T. Price
Robert M. Zucker
Affiliation:
USEPA, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory. Research Triangle Park, NC. 27711
Owen T. Price
Affiliation:
USEPA, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory. Research Triangle Park, NC. 27711
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Extract

The laser scanning confocal microscope (LSCM) has enormous potential in many fields of biology. Quality assurance is essential for the equipment to function correctly and realize its fullest potential. Currently the assessment of a confocal microscope’s performance is subjective primarily by evaluating the system with a specific test slide provided by the user. To achieve better performance, it is necessary to run a series of validation tests. These tests that were applied to measure field illumination, lens, clarity, dichroic functioning, objective spectral alignment, axial resolution, laser power and stability, photomultiplier tube (PMT) functioning, and system noise. Although th?se tests were derived on a Leica TCS-SP system and Leica TCD4D system, they should be applicable to other manufacturers’ systems.

For optimal images, it is important to control the variables of laser power, PMT tube voltage, averaging, and bleaching. The relationship between PMT voltage, laser power, and averaging was determined using a Spherotech Rainbow 10 μm diameter test bead. It was demonstrated that the noise (coefficient of variation of bead intensity, CV) in an image greatly increases as the PMT increases. The system variables that affected the precision and accuracy of the confocal system will be discussed.

Type
Recent Advances in Confocal Microscopy
Information
Microscopy and Microanalysis , Volume 5 , Issue S2: Proceedings: Microscopy & Microanalysis '99, Microscopy Society of America 57th Annual Meeting, Microbeam Analysis Society 33rd Annual Meeting, Portland, Oregon August 1-5, 1999 , August 1999 , pp. 464 - 465
Copyright
Copyright © Microscopy Society of America

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References

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