Background
Confocal spectral imaging (CSI) microscopic systems currently on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectral characterizations. However, some CSI systems have been found to present inconsistent spectral profiles of reference spectra within a particular system and between related and unrelated instruments. This variability confirms that there is a need for a standardized, objective calibration and validation protocol.

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 from the MIDL data to accurately predict the spectral resolution, ratio of wavelength to wavelength, contrast, and aliasing parameters of any CSI system. We were also able to predict and confirm the influence of pinhole diameter on spectral profiles.

Results
Using this simulation, we determined that there was good agreement between observed and theoretical expectations, thus enabling us to identify malfunctioning subsystems. We examined eight CSI systems and one nonconfocal 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 been otherwise recognized.

Conclusions
We found that using a primary light source that emits an absolute standard reference spectrum enabled us to diagnose instrumental errors and measure accuracy and reproducibility under normalized conditions. With this information, a CSI operator can determine whether a CSI system is working optimally and 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 CSI system.