Hyperspectral imaging has shown great promise for diagnostic applications, particularly in cancer surgery. However, non-bulk tissue-related spectral variations complicate the data analysis. Common techniques, such as standard normal variate normalization, often lead to a loss of amplitude and scattering information. This study investigates a novel approach to address these spectral variations in hyperspectral images of optical phantoms and excised human breast tissue. Our method separates surface and volume reflectance, hypothesizing that spectral variability arises from significant variations in surface reflectance across pixels. An illumination setup was developed to measure samples with a hyperspectral camera from different axial positions but with identical zenith angles. This configuration, combined with a novel data analysis approach, allows for the estimation and separation of surface reflectance for each direction and volume reflectance across all directions. Validated with optical phantoms, our method achieved an 83% reduction in spectral variability. Its functionality was further demonstrated in excised human breast tissue. Our method effectively addresses variations caused by surface reflectance or glare while conserving surface reflectance information, which may enhance sample analysis and evaluation. It benefits samples with unknown refractive index spectra and can be easily adapted and applied across a wide range of fields where hyperspectral imaging is used.
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