You are here:
CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: QA TESTS, QUANTITATION AND SPECTROSCOPY
Citation:
Zucker, R M. AND J. M. Lerner. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: QA TESTS, QUANTITATION AND SPECTROSCOPY. Presented at Microscopy & Microanalysis, Philadelphia, PA, April 04 - 07, 2004.
Description:
Confocal Microscopy System Performance: QA tests, Quantitation and Spectroscopy.
Robert M. Zucker 1 and Jeremy M. Lerner 2,
1Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
2 LightForm, Inc., 601 Route 206, Ste 26-479, Hillsborough New Jersey 08844
E-mail: Zucker.Robert@epa.gov
Key Words: Confocal microscope, Quality assurance, spectroscopy, Calibration
The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of emitted signals. The accuracy of these measurements demands that the system be in alignment with stable laser power and spectral registration, however the most common method to check the performance of a CLSM system is to characterize a histological slide to create a ?pretty picture?. We have developed tests to replace this subjective approach with objective measurements of field illumination, lens function and clarity, total laser power, laser stability, dichroic reflectance, axial resolution, scanning stability, overall machine stability, and system noise (1-2). We have developed additional tests to measure spectral performance to serve as guidelines for investigators to assess both the performance of their instruments as well as the quality of their data.
The spectral characterization test is well suited to all wavelength dispersive CLSM systems including the Leica SP and Zeiss Meta confocal microscopes. We used a multi-ion emission lamp (MIE) containing mercury ions and inorganic fluorophores as a light source because it emits stable reference peaks between 400 and 650 nm. Figure 1 illustrates the spectral features of the MIE lamp as presented by a Leica SP1 "lambda scan". The lamp was simply positioned on the microscope stage above the objective lens. The characteristics of the acquired spectrum enable us to measure spectral sensitivity, contrast, wavelength ratios and spectral resolution. Since the wavelength position of the emission peaks are absolutely reproducible it is possible to compare the performance of all three PMT?s in one Leica system and the difference in spectral response between similar systems in different laboratories.
References
1. Zucker RM and Price OT: Cytometry 44:273-294 2001
2. Zucker RM and Price OT: Cytometry 44:295-308 2001
This abstract of a proposed presentation does not necessarily reflect EPA policy