Abstract
We present a noninvasive method for characterizing the refractive index (RI) and thickness distribution in biological tissues using a combined multiphoton microscopy (MPM) and optical coherence tomography (OCT) system. Tissue layers are distinguished by the MPM and OCT images, and the RI and thickness of each layer are determined by analyzing the co-registered MPM and OCT images. The precision of this method is evaluated on four standard samples which are water, air, immersion oil and cover glass. Precision of within ~1% error compared to reference values is obtained. Biological tissue measurement is demonstrated on fish cornea. Three layers are detected, which are identified as the epithelium and stroma I and II of the cornea. The corresponding RI of each layer is measured to be ~1.446–1.448, 1.345–1.372, and 1.392–1.436, respectively. The difference of RI in the three layers correlates with the tissue compositions including cells in epithelium, large collagen fiber bundles in stroma I, and small collagen fibers in stroma II. The combined MPM/OCT technique is shown to be able to distinguish tissue layers through biochemically specific contrasts and measure RI and thickness of tissue layers at different depths.
Highlights
The refractive index (RI) of biological tissue is a key parameter for characterizing the lighttissue interactions [1,2]
RI was obtained by taking the ratio between the optical pathlength measured by optical coherence tomography (OCT) and the focus shift resulted from translating the focus of an objective lens inside biological tissues [1]
The high resolution en face two-photon excited fluorescence (TPEF) and SHG images show the compositions of each tissue layer which can provide information on why RI varies among the different layers
Summary
The refractive index (RI) of biological tissue is a key parameter for characterizing the lighttissue interactions [1,2]. One method was based on total internal reflection where it used a semicylindrical lens to determine the critical angle and the RI of biological tissues [10,11,12] It required direct contact with sample and only the RI of the layer in contact with the optics was measured. RI was obtained by taking the ratio between the optical pathlength measured by OCT and the focus shift resulted from translating the focus of an objective lens inside biological tissues [1] This method required focusing the laser beam onto the front and rear surfaces of a sample. We present a new method which can measure the RI and thickness of multiple tissue layers simultaneously in thick biological tissues noninvasively It is based on a combined multimodal multiphoton microscopy (MPM) and OCT system.
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