Refractive index of tissue measured with confocal microscopy

Abstract

Refractive index of tissue is an essential parameter in many bio-optical experiments, yet little data can be found in literature. Several methods have been proposed to measure refractive index in tissue samples, but all have specific limitations, such as low accuracy, the need for large amounts of tissue, or the complexity of the measurement setup. We propose a new method using a standard confocal microscope and requiring only small tissue samples. A thin slice of tissue is put next to a layer of immersion fluid of exactly the same thickness. The actual thickness of the fluid layer is directly measured with the microscope, as there is no refractive index mismatch. A difference between index of refraction of the tissue and of the immersion medium causes an axial scaling factor. The optical thickness of the specimen is thus measured with the microscope, and as its actual thickness equals the known thickness of the fluid layer, the axial scaling factor is readily determined. From this factor, we calculate the refractive index of the tissue. We use a diffraction model to take the point spread function (PSF) of the microscope into account, so we can determine the index of refraction to a very high accuracy. We demonstrate the method on bovine muscle tissue and find a value of n=1.382+/-0.004, at 592 nm.