Alignment of the individual images of a tilt series is a critical step in obtaining high-quality electron microscope reconstructions. We report on general methods for producing good alignments, and utilizing the alignment data in Subsequent reconstruction steps. Our alignment techniques utilize bundle adjustment. Bundle adjustment is the simultaneous calculation of the position of distinguished markers in the object space and the transforms of these markers to their positions in the observed images, along the bundle of particle trajectories along which the object is projected to each EM image. Bundle adjustment techniques are general enough to encompass the Computation of linear, projective or nonlinear transforms for backprojection, and can compensate for curvilinear trajectories through the object, sample warping, and optical aberration, We will also report on new reconstruction codes and describe our results using these codes. (c) 2006 Elsevier Inc. All rights reserved.