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EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE
Citation:
Zucker, R M. EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE. Chapter 5, Douglass Taates (ed.), Methods in Molecular Biology: Cellular Imaging Techniques. Humana Press Incorporated, Totowa, NJ, 319:77-135, (2005).
Impact/Purpose:
to achieve better performance from confocal laser scanning microscopes
Description:
BACKGROUND. The confocal laser scanning microscope (CLSM) has enormous potential in many biological fields. Currently there is a subjective nature in the assessment of a confocal microscope's performance by primarily evaluating the system with a specific test slide provided by each user's laboratory. In order to achieve better performance from this equipment, it is necessary to run a series of tests to ensure that the equipment is performing properly. METHODS. Our ultimate research objective was to try to quantify fluorescence from a CLSM. It is essential to have a stable machine with known parameters to achieve this goal. We have devised test methods on the Leica TCS-SP and TCS-4D systems to ensure our machine was working correctly and delivering the proper performance. Tests measuring field illumination, lens performance, laser power output, dichroic reflectivity, spectral registration, axial resolution, laser stability, PMT reliability and system noise were derived. These tests should be applicable to other manufacturers? systems with slight modifications.RESULTS. We have developed some new tests in this study and perfected some standard tests to assess the functionality of the CLSM. Tests measuring field illumination, lens clarity, laser power and stability, dichroic functionality, spectral registration, axial resolution, overall machine stability, and system noise were derived in order to determine the quality of the CLSM. The ability to apply estimates of image noise (CV) provides a better way to assess the confocal system performance.
Conclusions. To achieve maximum performance from a CLSM, it is necessary to run a series of tests to ensure that the machine is functioning properly. Without these tests the confocal data may be inaccurate and lack proper resolution, sensitivity and precision. These tests have given us the tools to eliminate the subjective nature of assessing the confocal microscope by only evaluating a user-defined or histological sample.