Imaging Prokaryotic Cell Outer Membranes with the Double Helix Point Spread Function

July 8th, 2021 by Double Helix Optics

Double Helix Optics would like to thank Stephen Upton for his contributions to, and co-authoring of, this post.

Reconstruction of bacteria imaged with PALM methodology using DHO's SPINDLE module and DH2 phase mask.

Figure 1. Reconstruction of bacteria imaged with PALM methodology with DHO’s SPINDLE module and DH2 Phase Mask. Image data is analyzed, processed, and rendered using DHO software, 3DTRAX®.

This post highlights the work of researchers from the lab of Marcelo Sousa at the University of Colorado at Boulder and their 3D super-resolution study of prokaryotic cell outer membranes utilizing DHO’s SPINDLE® imaging module and DH2 phase mask. This blog showcases some of their recent (unpublished) results.

The DH2 phase mask is designed for high-precision 3D localization and tracking applications where the objects of interest are around 1 µm in depth, or less. Sousa’s lab chose to use the DH2 phase mask to study outer membrane protein organization in E. Coli, which are about 1 µm in depth.

Why study prokaryotic cell outer membranes?

Bacteria, a type of prokaryote, are an essential part of the Earth’s ecosystem. There are several bacteria types, however, that are extremely harmful or deadly to other organisms, including humans. Naturally, there is a great desire to fully understand how bacteria “work”.

Bacteria possess an outer membrane that provides structure and protection to the cell, even against antibiotics. The outer membrane contains outer membrane proteins (OMPs). OMPs serve a variety of functions for bacteria including nutrient transportation, protein secretion, cell structure, and adhesion. These proteins are formed within the cell and migrate to the outer membrane. To localize the proteins of interest and understand how and where these proteins organize on the cell, the Sousa Lab required visualization with super-resolution sensitivity both laterally and axially.

Recent results using Double Helix’s SPINDLE with DH2 engineered PSF Phase Mask

Utilizing the DH2 within a Double Helix Optics SPINDLE® attached to a STORM super-resolution microscope at the University of Colorado at Boulder’s BioFrontiers Advanced Imaging Core, the team captured 3D localization images of bacteria OMPs using a fusion protein consisting of an OMP tagged with a photoactivatable fluorophore. They performed a 40-minute PALM experiment with snapshots of localizations in individual and dividing cells. The images generated during the PALM experiment were analyzed, drift corrected, processed, and rendered in 3D using Double Helix’s 3DTRAX® software.

OMP localizations in individual and dividing bacteria

OMP emitters are localized and color coded with regard to their respective depth (axial) position.

Figure 1 (above) contains a widefield image of OMP localization reconstructions for several bacteria. OMPs were localized in all three dimensions of the ~1 µm thick bacteria.  Figure 2 contains an ROI of three bacteria at different stages of cell division. Using the DH2 mask with 3DTRAX® enabled visualization of OMP clustering, membrane shape, and deformation during division. While the bacteria size is ~1 µm deep, the DH2 mask offered 1.5 µm depth-of-field. This enabled localizing emitters on bacteria that drifted with loss of clear data capture. All images were acquired using a 100X/1.49NA microscope objective.

Figure 2. ROI of 3 bacteria of interest, all in different stages of division. Note that the full colorbar depth range is not represented in the image, as the bacteria size are less than the 1.5 µm depth provided by the DH2. This allows the sample to drift in z without a loss of information.

Comments from the researchers…

“The work thus far presents some beautiful depictions of individual bacterial cells and their outer membrane proteins. Despite the relatively dim nature of fluorescent proteins, these data display the remarkable ability of DH2 masks to generate a DH-PSF with minimal photon loss and a unique imaging feat as a whole. Overall, the use of DH-PSF technology has the potential to help us learn more about novel nanoscale processes in bacteria and inform future antibiotic strategic decisions. ”

Double Helix Optics carries a wide range of PSF engineered phase masks designed to meet the variety of 3D imaging requirements. Contact us to discuss how to unlock true 3D imaging with your system.