A novel spectral microscope system: application in quantitative pathology

In this paper, a novel spectral microscope system is presented together with a method for the quantitative assessment of the uptake by histologic samples of stains used in pathology to label tissue features of diagnostic importance. The critical component of the microscope is a variable interference filter-based monochromator. The system is capable of performing real-time spectral imaging in a plurality of spectral bands and micro-spectroscopy in any image pixel, in the spectral range 400-1000 nm. The wavelength-tuning step is 2.4-2.6 nm, while the full-width at half maximum in each step is about 1.5% of the operating central wavelength. The developed system integrates algorithms and calibration procedures for the calculation of the stain-uptake by the tissue. The acquired spectra from both stained tissue and calibration stain solutions enable the calculation of the concentration maps of the stains, even if the latter are multiple and overlap spatially and spectrally. The system was used for the quantitative mapping of the expression of estrogen and progesterone receptors in breast cancer cells. In this particular case, model validation shows that although two stains are employed, capturing of their transmittance at more than ten wavelengths is required in order to obtain an acceptable accuracy. These findings highlight the need for the development and implementation of spectral microscopy in pathology and its potential to introduce novel more reliable diagnostic criteria.