;Confocal microscopes have been widely used to produce a 3D image by acquiring stacks of 2D images. Yet to have accurate 3D distance measurement between features based on images, it is necessary to calibrate pixel-to-micron conversation, which is especially difficult along the optical axis (z direction). When there is a refractive index difference between immersion medium of a sample and an objective, there are problems such as focus shift, aberrations, absorption and scattering which contribute to dramatic reduction in signal intensity as the focus gets deeper into the sample. In this thesis, I will look into the effects of refractive index on 3D imaging with the emphasis on z distance calibration. We employ a simple device which consists of a 2D periodic and fluorescent pattern mounted at 60 degrees with respect to z direction. We can easily compare the interval distance between the original and z-projected image to compute x, y, and z conversion factors and also intensity patterns. We systematic change the immersion medium index and find the relationship between z-conversion factor, intensity drop, and index-mismatch.