Estimating the effects of lens distortion on serial section electron microscopy images

Section to section alignment is a preliminary step to the creation of three dimensional reconstructions from serial section electron micrographs. Typically, the micrograph of one section is aligned to its neighbors by analyzing a set of fiducial points to calculate an appropriate polynomial transform. This transform is then used to map all of the pixels of the micrograph into alignment. Such transforms are usually linear or piecewise linear in order to limit the accumulation of small errors, which may occur with the use of higher–order approximations. Linear alignment is unable to correct common higher order geometric distortions, such as lens distortion in the case of TEM, and scan distortion in the case of transmission-mode SEM. Here, we attempt to show that standard calibration replicas may be used to calculate a high order distortion model despite the irregularities that are often present in them. We show that SEM scan distortion has much less of an effect than TEM lens distortion; however, the effect of TEM distortion on prior geometric measurements made over three-dimensional reconstructions of dendrites, axons, and synapses and their subcellular compartments is negligible.