Table 1.

Practical considerations regarding labelling approaches for single-molecule localization and tracking experiments in live bacterial cells.

	Advantages	Disadvantages
Photoactivatable fluorescent protein (e.g. PAmCherry)	•Genetically encoded fluorophore (no additional labelling required, labelling specificity guaranteed) •Independently controlled photoactivation with 405 nm excitation •Very low background noise (background can be bleached before photoactivation) •Cells can be imaged during continuous growth •Protein synthesis during cell growth replenishes fluorescence •Established and well-characterised labels	•Low brightness limits spatial and temporal resolution •Observation time per molecule is limited by rapid photobleaching •Irreversible bleaching prohibits repeated measurements of the same molecules •Limited spectral range •Phototoxicity of 405 nm illumination for photoactivation •Multi-colour tracking is limited because most PA-FPs are activated by near-UV light •Quantitative localization analysis complicated by incomplete photoactivation and long maturation times •Aggregation artifacts have been reported
Protein tag labelled with synthetic fluorophore (e.g. Halo-TMR)	•Superior brightness provides enhanced localization precision and temporal resolution •Reduced photobleaching increases observation time per molecule •Available dyes span visible to far-red spectrum •Compatible with conventional fluorescence imaging •Multi-colour tracking possible using orthogonal labelling of Halo, SNAP, and CLIP tags with different dyes •Reversible blinking allows repeated measurements of the same molecules •Ability to perform sequential labelling (e.g. before and after a cell perturbation)	•Additional labelling step (laborious, may interfere with other cell treatments, time-course experiments, etc) •Removal of unbound dye requires extensive cell washing and centrifugation •Labelled protein is diluted with each cell division, causing loss of signal during prolonged time-lapse imaging •Pre-bleaching step required before single-molecule imaging •Limited control over photoswitching kinetics •Reversible blinking produces apparent clustering artifacts •Quantitative localization analysis complicated by reversible blinking, unknown labelling efficiency, and dilution of labelled proteins during cell growth •Not all Halo tag dyes are cell permeable •Not all fluorescent Halo ligands are commercially available or can be expensive