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CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR MEASUREMENTS, QUANTITATION AND SPECTROSCOPY
Zucker, R M. AND J. M. Lerner. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR MEASUREMENTS, QUANTITATION AND SPECTROSCOPY. Presented at Microscopy & Microanalysis, Savannah, GA, August 01 - 05, 2004.
The confocal laser-scanning microscopy (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 the emitted signal. The accuracy of these measurements demands that the system be in alignment with stable laser power and spectral registration. For many applications it is useful to confirm the system's spatial resolution, sensitivity and precision prior to acquiring image data. The characterization of FRET, "unmixing" co-localized mixed fluorescence spectra and the use of deconvolution necessitates that the system be correctly configured and be operating optimally. 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 method with objective measurements of field illumination, lens function and clarity, spectral registration, total laser power, laser stability, dichroic reflectance, axial resolution, scanning stability, overall machine stability, and system noise (1-2). We 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 series (SP), the Zeiss LSM510 Meta (Meta) and Olympus FV1000 confocal microscopes. We used an inexpensive, eye-safe, battery operated, multi-ion discharge lamp (MIDL) (LightForm, Inc., Hillsborough NJ) containing mercury ions and inorganic fluorophores as an absolute reference light source because it emits stable, reproducible, peaks between 400 and 650 nm. The lamp is simply positioned on the microscope stage above (or below) the objective lens. The characteristics of an acquired spectrum enable us to measure wavelength accuracy, spectral sensitivity, contrast, wavelength ratios and spectral resolution. Significantly, it can also be used to contrast and compare the performance of one instrument against another in a different location. Figure 1 illustrates the spectral features of the MIDL lamp as presented by an Olympus FV1000 "lambda scan". The MIDL characterization was accurate and showed significant detail in the 611 nm spectral feature. Figure 2 shows the same spectrum presented by a Zeiss LSM 510 Meta system. Clearly the 545 nm line was bisected by two WDP. The wavelength accuracy is as expected for a WDP with a spectral sampling increment of ~10.7 nm.