Motion history images: a new method for tracking microswimmers in 3D

Abstract

Quantitative tracking of rapidly moving micron-scale objects remains an elusive challenge in microscopy due to low signal-to-noise. This paper describes a novel method for tracking micron-sized motile organisms in off-axis Digital Holographic Microscope (DHM) raw holograms and/or reconstructions. We begin by processing the microscopic images with the previously reported Holographic Examination for Life-like Motility (HELM) software, which provides a variety of tracking outputs including motion history images (MHIs). MHIs are stills of videos where the frame-to-frame changes are indicated with color time-coding. This exposes tracks of objects that are difficult to identify in individual frames at a low signal-to-noise ratio. The visible tracks in the MHIs are superior to tracks identified by all tested automated tracking algorithms that start from object identification at the frame level, particularly in low signal-to-noise ratio data, but do not provide quantitative track data. In contrast to other tracking methods, like Kalman filter, where the recording is analyzed frame by frame, MHIs show the whole time span of particle movement at once and eliminate the need to identify objects in individual frames. This feature also enables post-tracking identification of low-SNR objects. We use these tracks, rather than object identification in individual frames, as a basis for quantitative tracking of Bacillus subtilis by first generating MHIs from X, Y, and t stacks (raw holograms or a projection over reconstructed planes), then using a region-tracking algorithm to identify and separate swimming pathways. Subsequently, we identify each object's Z plane of best focus at the corresponding X, Y, and t points, yielding ap full description of the swimming pathways in three spatial dimensions plus time. This approach offers an alternative to object-based tracking for processing large, low signal-to-noise datasets containing highly motile organisms.