Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging

Key Points

  • Single-molecule imaging of fluorescent fusion proteins can be used to track individual biomolecules that are moving in bacterial cells and to probe their diffusion or their directed or confined motion, which provides insights into various dynamic cellular processes.

  • Single-molecule imaging, when combined with a method that actively controls the concentration of emitting fluorescent proteins, can be used to achieve super-resolution. Thus, it is now possible to visualize previously hidden structural details of protein localization patterns with a resolution that is no longer limited by the diffraction of light.

  • Single-molecule and single-particle tracking have shown that the actin homologue MreB moves in a circumferential pattern around the bacterial cell, driven by cell wall synthesis. Other cytoskeletal protein structures, such as the FtsZ ring and PopZ nanodomains, have been characterized at subdiffraction spatial resolution.

  • Super-resolution imaging has shown that some nucleoid-associated proteins bind DNA and form well-defined organizational regions, whereas others localize more randomly throughout the nucleoid. These data provide information about the spatial organization of the chromosome.

  • The activities of DNA repair enzymes in live cells have been revealed by a combination of single-molecule tracking and single-molecule super-resolution imaging, which show a clear increase in repair rates under conditions of increased DNA damage.

  • The spatial organization of transcription and translation has been investigated using single-particle tracking and super-resolution imaging of ribosomes and RNA polymerase, revealing clear differences between model organisms.

  • Single-molecule imaging has shown that individual transcription factors search for their target DNA sequence by a combination of one-dimensional sliding along DNA and three-dimensional diffusive hopping between DNA stands.


The ability to detect single molecules in live bacterial cells enables us to probe biological events one molecule at a time and thereby gain knowledge of the activities of intracellular molecules that remain obscure in conventional ensemble-averaged measurements. Single-molecule fluorescence tracking and super-resolution imaging are thus providing a new window into bacterial cells and facilitating the elucidation of cellular processes at an unprecedented level of sensitivity, specificity and spatial resolution. In this Review, we consider what these technologies have taught us about the bacterial cytoskeleton, nucleoid organization and the dynamic processes of transcription and translation, and we also highlight the methodological improvements that are needed to address a number of experimental challenges in the field.

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Figure 1: Principles of single-molecule tracking and imaging.
Figure 2: Cytoskeletal and structural proteins imaged by single-molecule methods.
Figure 3: Nucleoid organization in model organisms observed by single-molecule methods.
Figure 4: Chromosome integrity and partitioning observed by single-molecule approaches.
Figure 5: Transcription and translation are spatially uncoupled in Escherichia coli.


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The authors gratefully acknowledge fruitful collaborations and stimulating discussions with L. Shapiro and members of her laboratory over the years, as well as many current and former members of the Moerner laboratory. The authors specifically thank M. K. Lee, A. R. von Diezmann and J. L. Ptacin for critical reading of the manuscript. This work was supported in part by the US National Institute of General Medical Sciences Grant No. R01GM086196 (W.E.M) and a Swiss National Science Foundation Postdoctoral Fellowship PA00P2_145310 (A.G.).

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PowerPoint slides


Centroid estimation

In the context of localization microscopy, a method to determine the location of a fluorescent emitter or a group of several closely packed emitters (referred to as a single-particle) by calculating the spatial arithmetic mean of all pixel positions, which are weighted by their intensity.


The process by which fluorophores that are initially in a non-fluorescent (dark) state can be converted to a fluorescent (bright) state by illumination with short-wavelength light.


The process by which photoswitchable fluorophores can be turned 'on' or 'off' by an active control mechanism, such as illumination with a specific wavelength of light.

Photoinduced blinking

Using certain illumination intensities (or, in some cases, by adding chemical additives), fluorophores can reversibly enter a non-fluorescent (dark) state. From this state, they can spontaneously recover and become fluorescent (bright) again, which gives the appearance of blinking.

Epifluorescence microscopy

A standard wide-field fluorescence microscopy technique, in which the same objective lens is used to illuminate the entire specimen and to collect emitted fluorescence.

Cryo-electron tomography

(CET). A technique in transmission electron microscopy, in which a vitrified specimen is imaged from different angles at cryogenic temperatures. From the resulting electron micrograph tilt series, a three-dimensional tomogram can be computationally reconstructed.

Total internal reflection fluorescence microscopy

(TIRF microscopy). A technique in which only fluorophores that are in close proximity to the glass–water interface are excited by an evanescent wave that is generated by total internal reflection of the excitation light at this interface. The large reduction of the excitation volume in the axial direction (from 700 nm to 100 nm) results in more selective excitation of the sample and lower background fluorescence compared with epifluorescence illumination.

Fluorescence recovery after photobleaching

(FRAP). An optical technique that is used to estimate the diffusion of fluorescently labelled molecules by determining the timescale of fluorescence recovery after high-intensity light has been applied to a well-defined region of the specimen to photobleach many of the fluorophores in its footprint.

Astigmatic point spread function

(Astigmatic PSF). A cylindrical lens can be inserted in the fluorescence collection path to offset the x and y focus position along the optical axis. An imaging system that has an astigmatic PSF can be used to determine the z-position of a single-molecule emitter by calibrating the change in elliptical shape of the PSF as a function of defocus.

Double-helix point spread function

(Double-helix PSF). Optical phase manipulation in the Fourier plane of the fluorescence emission path can be used to produce a double-helix PSF. An imaging system that has a double-helix PSF can be used to determine the z-position of a single-molecule emitter by calibrating the amount of angular rotation of the PSF as a function of defocus.

Chromosome conformation capture

(3C). A molecular biology technique that is based on crosslinking and analysis of which DNA segments are closely associated, to determine the spatial organization of the chromosome.

Fluorescence in situ hybridization

(FISH). An optical technique that is used to detect and locate specific DNA (or RNA) sequences. A FISH probe, which consists of a fluorophore linked to a single-stranded DNA (or RNA) sequence, binds to its complementary target DNA (or RNA) site after being introduced into fixed and permeabilized cells.

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Gahlmann, A., Moerner, W. Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging. Nat Rev Microbiol 12, 9–22 (2014). https://doi.org/10.1038/nrmicro3154

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