Bacteria at super-resolution

A new method that couples microfluidics and super-resolution microscopy to image live bacteria at unsurpassed spatial and temporal resolutions has been developed. Uniquely, this technology considerably improves cell stability, allows for long time-lapse imaging and completely eliminates chromatic aberrations.

HFSP Career Development Award holder Marcelo Nollmann and Young Investigator Grant holder Tam Mignot and colleagues
authored on Tue, 11 February 2014

Bacteria have evolved complex, highly-coordinated, multi-component cellular engines to achieve high degrees of efficiency in processes such as chromosome segregation during cell division and sporulation. Studying the cellular localization, dynamics and architecture of the molecular complexes involved in these processes is essential for understanding their function and mechanism. Conventional fluorescence microscopy techniques enable non-invasive observation of protein organization in live cells with high specificity. However, due to intrinsic limitations introduced by the diffraction of light, the maximum resolution achievable with these techniques (~250nm) remains comparable to the size of bacteria cell (typically 1-2µm). Probing the localization and architecture of bacterial machineries that are only several nanometers large is therefore impossible.

Recent advances in fluorescence microscopy have led to the development of several conceptually independent ‘super-resolution’ techniques with higher resolution than the diffraction limit. Among them, single-molecule based super-resolution techniques (smSRM) such as PALM or STORM achieve the highest resolution (20-40nm) and are compatible with in-vivo imaging, making them ideal to investigate bacterial machineries at the nanometer precision. These methods rely on the stochastic photo-activation and localization of thousands of single emitters in order to reconstruct a high-resolution image. The acquisition process being rather long (~min), smSRM require long-term stability of samples and high signal-to-noise-ratios, difficult conditions to meet with traditional immobilization methods such as agarose pads.

Figure: Bacteria at super-resolution

To address this problem, we developed a simple, easy to implement and inexpensive method in which cells are functionalized to a microfluidics device and fluorophores are injected and imaged sequentially. This method has several advantages over traditional methods (ie. Agarose pads), as it permits the long-term immobilization of cells and proper correction of drift with a precision below 5nm over several tens of minutes. Chromatic aberrations caused by the use of different filter sets can be avoided using the sequential injection of fluorescent dye. And the flat immobilization of cells on the surface is ensured by the combination of proper surface chemistry (poly-L-Lysine or Chitosan) and the application of constant flow force thanks to the fluidics. In addition, different surface chemistries can be used to immobilize bacteria and image them at different time-scales to follow structural changes such as cell division. At last, we introduce an automated cell detection and image analysis procedure that can be used to obtain cell-to-cell, single-molecule localization and dynamic heterogeneity as well as average properties at the super-resolution level.

This microfluidics device was successfully used to study the localization and assembly of the DNA transporter SpoIIIE during Bacillus Subtilis sporulation. Moreover, we demonstrate that it is perfectly suited to other microscopy techniques such as Total Internal Reflection Microscopy (TIRM), Structured Illumination (3D-SIM) and two-photon microscopy.     

Reference

Super-resolution imaging of bacteria in a microfluidics device. Cattoni DI, Fiche JB, Valeri A, Mignot T, Nöllmann M. Plos ONE. 2013 doi: 10.1371/journal.pone.0076268. eCollection 2013.

Pubmed link