Published In

Frontiers in Imaging

Document Type

Article

Publication Date

5-9-2024

Subjects

Microbial Imaging

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.

Rights

© 2024 Riekeles, Albalkhi, Dubay, Nadeau and Lindensmith. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

DOI

10.3389/fimag.2024.1393314

Persistent Identifier

https://archives.pdx.edu/ds/psu/41783

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