Method for the refractive index of various tissues based on fluorescence microscopy

Abstract

Refractive index is an important optical constant that characterizes the interaction between light and specimen. A difference in refractive index between specimen and immersion medium introduces the imaging aberration and leads to a problem that the direct thickness measurement of a specimen by optical microscopy is not accurate. However, this aberration correction still requires the exact information of the refractive index of specimen and immersion medium. Herein, we propose an imaging strategy to estimate the refractive index for an unknown specimen. A simplified diffraction model is generated to obtain the relationship between axial scaling factor and refractive index. Then regular fluorescence microscopy is performed to measure the actual axial scaling factors of specimens from mouse muscle and tumor xenograft. Referring to our theoretical plot of axial scaling factor versus refractive index, the refractive index of tissue specimen is determined. For example, we obtain a mean refractive index (n) value of 1.36 for normal muscle tissues, and 1.41 for tumor xenografts. We demonstrate that this diffraction model-based estimation method is an alternative to the current techniques, improving the accurate measurement for refractive index of tissue specimen. The simple instrument requirement with an easy specimen preparation for this estimation method of refractive index may increase the image quality on tissue specimens with less aberration.