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Three-dimensional optical microscopy is often complicated by a refractive index mismatch between the sample and objective lens. This mismatch causes focal shift, a difference between sample motion and focal-plane motion, that hinders the accuracy of 3D reconstructions. We present two methods for measuring focal shift using fluorescent beads of different sizes and ring-stained fluorescent beads. These simple methods are applicable to most situations, including total internal reflection objectives and samples very close to the interface. For distances 0–1.5  μ m into an aqueous environment, our 1.49-NA objective has a relative focal shift of 0.57 ± 0.02, significantly smaller than the simple  n   2 / n   1  approximation of 0.88. We also expand on a previous sub-critical angle theory by means of a simple polynomial extrapolation. We test the validity of this extrapolation by measuring the apparent focal shift in samples where the refractive index is between 1.33 and 1.45 and with objectives with numerical apertures between 1.25 and 1.49.

Simple Experimental Methods for Determining the Apparent Focal Shift in a Microscope System