An online calibration method for astigmatism-based 3D particle tracking in complex living tissue

In this paper we present an online calibration method that adjusts astigmatism-based 3D particle tracking techniques to the aberrations induced by the refractive index mismatch, so that they are applicable to complex tissue. As opposed to existing techniques, our method determines the aberration directly from the acquired 2D image stream and requires no prior calibration. We analyze the influence of the refractive index mismatch on fluorescent beads immobilized in Mowiol and establish an axial model that describes the induced aberrations. By exploiting particle movement and the redundancy introduced by the astigmatism we show that it is possible to estimate the current axial model independently of the imaging depth. We evaluate our algorithm on semi-synthetic image streams and demonstrate that the bias, due to the refractive index mismatch, can be decreased. We obtain our results using a spinning disk confocal setup. However, our method also applies to wide-field imaging without limitations.