- Single-exposure on-line (SEOL) digital holography is a recently proposed technique for monitoring, visualization, and recognition of three-dimensional (3D) objects. In contrast to traditional multi-exposure on-line digital holography, it uses only one exposure, which makes it particularly suitable for imaging and recognizing moving micro-organisms. However, the cost of using only one exposure is the superposition of a conjugate image on the desired reconstructed image. The influence of the conjugate image on the visualization and recognition performance is investigated. The conditions for which the cross-talk noise induced by the conjugate image is negligible are derived. It is demonstrated that with conditions common in imaging of microscopic 3D biological objects, SEOL digital holography is highly tolerant of cross-talk noise induced by the conjugate image.