Label-Free Imaging and Refractive Index Mapping of Biological Tissues by Hyperspectral SPR Microscopy

Staining of tissue sections and subsequent microscopic imaging are two key steps in conducting conventional histopathological analysis, but these processes are complex and time-consuming, and the image analysis results largely depend on the experience of the operator. To overcome these limitations, this work proposes a label-free quantitative histopathological analysis method based on the hyperspectral surface plasmon resonance microscopy (HSPRM) system. The HSPRM system consists of a Kretschmann-type spectral SPR sensor and a hyperspectral microscope, which are optically connected through an imaging lens. The HSPRM system with an equilateral triangular prism coupler made of high-index glass can capture wavelength-scanning SPR images and pixel-resolved resonance wavelengths (RWs) of tissue sections, and allows for two-dimensional (2D) mapping of the refractive index (RI) of the tissue in a wide dynamic range by fitting the measured RWs pixel by pixel with the multilayer Fresnel model. As a result, the HSPRM system can easily distinguish the cancerous region from the normal region based on the measured 2D distribution of the RI of the tissue sample. This outstanding capability of the HSPRM system was verified through comparative tests of tumor tissue and normal tissue from mouse mammary glands. The experimental results indicated that the average RI of the tumor tissue is higher than that of the normal tissue. Multiple measurements demonstrated that the prerequisite for label-free quantitative histopathological analysis with the HSPRM system is to ensure gapless adhesion of the tissue section to the SPR sensor chip.