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CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY
Citation:
ZUCKER, R. M. AND J. M. LERNER. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY. Presented at International Society of Analytical Cytology, Quebec, QC, CANADA, May 19 - 24, 2006.
Description:
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 correct alignment with stable laser power and spectral registration. This evaluation can not be made using a histological slide to create a "pretty picture" which appears to be the most common method to check the performance of a CLSM system. We have developed tests to replace this subjective approach with objective measurements of field illumination, lens function, total laser power, laser stability, dichroic reflectance, axial resolution, galvanometer 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. (3, 4)
The spectral characterization test is well suited to all wavelength dispersive CLSM systems including the Leica SP, Zeiss Meta, Olympus FV 1000, and Nikon C1 confocal microscopes. We used a multi-ion discharge lamp (MIDL) (LightForm, Inc., Hillsborough NJ) containing mercury ions and inorganic fluorophores as a light source because it emits stable reference peaks between 400 and 650 nm. The spectral features of the MIDL lamp include peaks at 436, 485, 545 586 and 611nm. The signature of peaks and valleys is a measure of the accurate representation of wavelengths of light by the CLSM. The lamp is simply positioned on the microscope stage above the objective lens (3). 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 is reliable and 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. Confocal microscope misalignment and instability in spectral imaging systems can be assessed using this MIDL reference standard (4).
References
1. Zucker RM and Price OT: Cytometry 44:273-294 2001
2. Zucker RM and Price OT: Cytometry 44:295-308 2001
3. Lerner JL Zucker RM: Cytometry 62A:8-34 2004