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 ϩ , Ar ϩ , 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 non-

We investigated the use of high resolution hyperspectral imaging microscopy to detect abnormalities in skin tissue using hematoxylin eosin stained preparations of normal and abnormal skin, benign nevi and melanomas. A goal of this study was to provide objective data that could be utilized by any researcher; and form the beginnings of a reference spectral data base. All spectral characterizations were acquired in percent transmission, and absorption, with contiguous wavelength acquisition between 400 and 800 nm; and a spectral resolution of approximately 1 nm. Biopsy sections were characterized with varying sample thickness, staining and magnification in order to determine their impact on spectral characterizations. Spectra were classified using spectral waveform cross correlation analysis, an algorithm that is linearity invariant. Classified spectra were incorporated into spectral libraries; and all spectra acquired from the field of view were correlated with library spectra to a quantified, user determined, confidence threshold (minimum correlation coefficient). The results revealed that all skin conditions in our initial data sets could be objectively differentiated providing that staining and section thickness was controlled. We also demonstrated that it is likely that a reference spectral library database could be created to include bioinformatics and cluster analysis. This would assist multiple laboratories to participate in the input and retrieval of target spectral information.

Confocal spectral imaging (CSI) microscope systems now on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectral characterizations. However, we find that some CSI present inconsistent spectral profiles of reference spectra within a particular system as well as between related and unrelated instruments. We also find evidence of instability that, if not diagnosed, could lead to inconsistent data. This variability confirms the need for diagnostic tools to provide a standardized, objective means of characterizing instability, evidence of misalignment, as well as performing calibration and validation functions. 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. An MIDL characterization verifies the accuracy and consistency of a CSI system and validates acquisitions of biological samples. We examined a total of 10 CSI systems, all of which displayed spectral inconsistencies, enabling us to identify malfunctioning subsystems. Only one of the 10 instruments met its optimal performance expectations. We have found that using a primary light source that emits an absolute standard "reference spectrum" enabled us to diagnose instrument errors and measure accuracy and reproducibility under normalized conditions. Using 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. It is evident that if CSI systems of a similar make and model were standardized to reveal the same spectral profile from a standard light source, then researchers could be confident that real-life experimental findings would be repeatable on any similar system.