Single-molecule tracking technologies for quantifying the dynamics of gene regulation in cells, tissue and embryos

Abstract

For decades, we have relied on population and time-averaged snapshots of dynamic molecular scale events to understand how genes are regulated during development and beyond. The advent of techniques to observe single-molecule kinetics in increasingly endogenous contexts, progressing from in vitro studies to living embryos, has revealed how much we have missed. Here, we provide an accessible overview of the rapidly expanding family of technologies for single-molecule tracking (SMT), with the goal of enabling the reader to critically analyse single-molecule studies, as well as to inspire the application of SMT to their own work. We start by overviewing the basics of and motivation for SMT experiments, and the trade-offs involved when optimizing parameters. We then cover key technologies, including fluorescent labelling, excitation and detection optics, localization and tracking algorithms, and data analysis. Finally, we provide a summary of selected recent applications of SMT to study the dynamics of gene regulation.

Triple-color single-molecule imaging for analysis of the role of receptor oligomers in signal transduction

“…Since the depth of evanescent light produced by total internal reflection illumination can reach just the vicinity of the basal plasma membrane of the sample cell, molecules in and on the plasma membrane like receptors are suitable targets for single-molecule imaging studies. Various findings have been obtained on the mechanism of extracellular signal transduction through receptor molecules based on the analysis of receptor motility [10][11][12].…”

Deep learning for bioimage analysis in developmental biology

“…Therefore, because of scattering, light traveling from fluorescent markers can be distorted and less intense when it reaches the objective. Photobleaching and phototoxicity are also increasingly problematic deeper into the tissue, leading to low SNR as one mitigates its effect through decreased laser power and increased camera exposure or detector voltage (reviewed by Boka et al, 2021). DL has been successful at overcoming these challenges when used in the context of image restoration algorithms, which transform input images into output images with improved SNR.…”

Automated live-cell single-molecule tracking in enteroid monolayers reveals transcription factor dynamics probing lineage-determining function

“…Live-cell single-molecule tracking (SMT) allows the direct measurement of the dynamics of individual protein molecules and thus can resolve differently behaving subpopulations within the same cell. In contrast, other fluorescence-based live-cell microscopy methods determine bulk protein dynamics 39 . In SMT, individual proteins are detected, localized, and tracked over thousands of imaging frames at an interval of only a few milliseconds (fast SMT).…”