Using the SPINDLE® imaging module with a double-helix phase mask, the Sousa Lab at CU Boulder has advanced the quest for new antibiotics by imaging bacterial cell outer membranes in 3D with super resolution.
A brightfield image of outer membrane protein (OMP) localization reconstructions for several ~1 µm thick E. coli bacteria. OMPs are proteins that are embedded in the outer cell membrane and facilitate crucial transmembrane cellular processes. Images were acquired using a 100x/1.49NA microscope objective and emitter localizations are color coded by axial depth.
Close-up of E. coli bacteria at different stages of cell division. OMPS are inserted and cluster together as cells separate.
Why this is important
While bacteria are an essential part of the Earth’s ecosystem, many species are extremely harmful or deadly to other organisms, including humans. Historically, the medical profession has used an arsenal of antibiotics to fight bacterial infections, but several varieties of bacteria have evolved in recent decades to quickly bypass traditional antibiotic mechanisms. As a result, aggressive, blunt-force antibiotics must be used to combat severe infections, often causing harm to patients. When bacteria develop resistance to these drugs of last resort it is a serious problem because there are then no effective treatment options.
Doing their part in the movement to develop new antibiotics, the Sousa Lab has initiated studies of crucial proteins exposed on the cellular surface of key bacteria.
Gram-negative bacteria possess a cell envelope that is composed of two membranes: an outer membrane that provides structure and protection to the cell and a simpler phospholipid inner membrane. The outer membrane is unique to these phyla of bacteria and contains transmembrane outer membrane proteins (OMPs). OMPs serve a variety of functions for bacteria, including nutrient transportation, protein secretion, cell-structure scaffolding, and adhesion.
These proteins are formed within the cell and migrate to the outer membrane through a largely unknown process. Understanding how this process occurs in bacteria is a critical step along the path of developing new antibiotics.
To illuminate the the organization and dynamics of OMPs and their interacting proteins, the team captured images of OMPs in E. coli bacteria using 3D photoactivatable light microscopy (3D PALM). In this experiment, fluorescent proteins were cyclically activated, imaged, and photobleached to build up a complete picture of all tagged OMPs.
With quantitative 3D localization data in hand, the Sousa Lab were able to compare the spatial patterns of important OMPs and identify clusters in dividing cells, providing insights into where new OMPs are inserted into bacterial cell membranes.
How Double Helix Optics made it possible
Bacteria can be shorter than 1 µm in length, so conventional fluorescence microscopy, with its resolution limit of ~200 nm, isn’t a feasible method to study their intricate details. Furthermore, 3D information collection reduces the risk of incorrectly co-localizing objects or underestimating diffusion coefficients. Therefore, the Sousa Lab employed 3D single molecule localization microscopy (3D SMLM) with the double-helix PSF to obtain the most precise and reliable data possible. DHO’s DH2 phase mask was chosen for this purpose, as it is optimized for imaging and tracking objects that are 1 µm in depth or less, allowing for an optimal signal-to-noise ratio and maximum localization precision to be maintained when studying small bacterial cells.
The DH2 phase mask was used in combination with a Double Helix Optics SPINDLE® module, attached as a straightforward upgrade to a Nikon N-STORM super-resolution microscope at the University of Colorado at Boulder’s BioFrontiers Advanced Imaging Core. Double Helix Optics 3DTRAX® software provided all the capabilities required to localize, drift correct, analyze, and visualize captured images. Using the DH2 mask with 3DTRAX® enabled the visualization of protein clustering, membrane shape, and deformation during cellular division.