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In this review, we discuss multi-color single-molecule imaging and tracking strategies for studying microbial cell biology. We first summarize and compare the methods in a detailed literature review of published studies conducted in bacteria and fungi. We then introduce a guideline on which factors and parameters should be evaluated when designing a new experiment, from fluorophore and labeling choices to imaging routines and data analysis. Finally, we give some insight into some of the recent and promising applications and developments of these techniques and discuss the outlook for this field.

Visualizing the inner life of microbes: practices of multi-color single-molecule localization microscopy in microbiology