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CALIBRATION AND VALIDATION OF CONFOCAL SPECTRAL IMAGING SYSTEMS
Citation:
Lerner, J. M. AND R M. Zucker. CALIBRATION AND VALIDATION OF CONFOCAL SPECTRAL IMAGING SYSTEMS. CYTOMETRY 62A:8-34, (2004).
Impact/Purpose:
To improve the performance of confocal spectral imaging systems
Description:
Confocal spectral imaging (CSI) microscope systems now on the market can perform spectral characterization of biological specimens containing fluorescent proteins, labels or dyes. Some CSI have been found to present inconsistent spectral characterizations within a particular system as well as between related and unrelated instruments. This variability confirms that there is a need for a standardized, objective calibration and validation protocol. We have found that using a primary light source that emits an absolute standard 'reference spectrum' will characterize instrumental errors, accuracy and reproducibility under normalized conditions. Methods: Our protocol uses an inexpensive multi-ion discharge lamp (MIDL) that contains Hg, Ar and inorganic fluorophores that emit distinct, stable spectral features in place of a sample. We derived reference spectra (DRS) from the MIDL data to accurately predict the spectral resolution, wavelength-to-wavelength ratio, contrast and aliasing for each CSI system. We were also able to predict and confirm the influence of pinhole diameter on spectral profiles. Using this simulation, we determined that there was good agreement between observed versus theoretical expectations, enabling us to identify malfunctioning sub-systems. Results: We examined a total of seven CSI systems and one non-confocal spectral system, all of which displayed spectral inconsistencies. No instrument met its optimal performance expectations. In two systems we established the need for factory realignment that had not otherwise been recognized. Conclusions: Spectral characterization using a standardized light source enables a CSI operator to determine the operational health of a system and to make objective comparisons with the performance of other CSI systems. We determined that if CSI systems were standardized to produce the same spectral profile of a MIDL lamp, researchers could be confident that the same experimental findings would be obtained on any similar CSI system. It would also be unsurprising that spectral data acquired on a standardized system may differ from that acquired on non-standardized CSI systems.