Hyperspectral imaging and spectral unmixing of stained tissue sections using a spectrally programmable light engine

A series of hyperspectral transmission images of hernatoxylin and eosin stained tissue sections from cervical biopsies were acquired at 10 nm intervals and assembled into a hyperspectral image cube. Custom software providing extraction of spectra at each pixel allows selection of images with maximum contrast for determination of selected features and differentiation of tissue features. Illumination profiles were created using a spectrally and temporally programmable light engine based on a spatial light modulator that can dynamically create any narrow or broadband spectral profile was used to select illumination wavelengths. Images were acquired with a monochrome CCD camera. Several methods of combining images from individual or composite spectral bands to recreate color images for pathologist review are shown. Unlike current "mechanical" illumination systems employing optical filters, filter wheels, motors, shutters and multiple control interfaces, the light engine integrates the lamp, wavelength control, intensity control and exposure control in a simple MEMS based system, where the only moving part is the lamp cooling fan. Illumination can now be programmed dynamically with digital control of all illumination parameters allowing wavelengths and intensities to be changed much faster than with filter wheels, and providing exposure control orders of magnitude more precise than mechanical shutters. This system can be integrated with digital imaging systems. Digitally controlled illumination is bit additive with image data providing high dynamic range imaging with monochrome or with color imaging devices. Performance of image analysis software for nuclear morphometric and tissue architecture analysis are compared for different wavelength regions.