Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

Abstract

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ,30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.B iomineralization is a widespread biological phenomenon occurring in living organisms ranging from single cells to complex multicellular organisms. Biomineralization of inorganic materials in single cell organisms is an ideal system for studying fundamental mechanisms of biomineralization 1 . Model systems range from prokaryotic organisms, such as magnetite formation in magnetotactic bacteria 2-4 , to eukaryotic organisms, such as silica biomineralization in diatoms 5,6 . Understanding biomineralization in these organisms in terms of crystal nucleation and growth, as well as the involvement of biological macromolecules and cellular processes, is of fundamental interest to scientists as similar principles can be used to develop synthetic nanomaterials.Magnetite biomineralization by magnetotactic bacteria is a topic of great interest in nanotechnology 7-12 , functional materials [11][12][13][14][15][16] , and astrobiology 17 . Magnetotactic bacteria take up soluble iron species that they use to biomineralize chains of magnetite nanocrystals, known as magnetosomes, in intracellular membrane vesicles 2 . The nanocrystals have nearly perfect mineral crystal structures with consistent species-specific morphologies, leading to well-defined magnetic properties 2,3,9,12,18,19 . As a result, magnetotactic bacteria are one of the best model systems for investigating the molecular mechanisms of biomineralization. Magnetite biomineralization in these bacteria is a complex process involving a number of steps including cellular uptake and reduction of ferric ions, complexation of the iron with membrane proteins, an...

Opportunities and challenges in liquid cell electron microscopy

“…Correlative liquid cell STEM and fluorescence microscopy (Fig. 4A) explore magnetosome structure in the natural cellular environment (96). These images are static, but dynamic information could in principle be obtained if the dose can be kept low.…”

“…The dose delivered in one image is typically above the lethal dose. There is much ongoing interest in establishing whether tolerable doses exist for in vivo liquid cell microscopy (96,103); it may be possible to study certain biochemical processes, as it appears that the speed of cell death depends on the region of the cell that is imaged.…”

A Journey Towards Understanding Biology Holistically at the Nanoscale

“…Both bubble formation in cryo-EM and cell shrinking in LC-TEM has been described in literature (116,193), supporting the observation of distinct differences between damage manifestation between these two techniques.…”

“…LC-TEM has also been demonstrated for biological samples from proteins (111)(112)(113)(114) to whole cells (115). Both prokaryotes (74,116,117) and eukaryotes (118) have been imaged with LC-TEM, and although mammalian cells have been imaged the large fluid thicknesses necessitated by their size results in images which are low in contrast (119)(120)(121). As electron beam damage is a concern for biological samples, many reports of cells imaged with LC-TEM are fixed with a crosslinking agent such as glutaraldehyde in order to stabilize the cell from the effects of secondary damage (121).…”

Visualizing Electron Beam-Capping Ligand Reactions for Controlled Nanoparticle Imaging with Liquid Phase Transmission Electron Microscopy

“…Electron beam induced reactions during LP-TEM are only perceptible if visible changes occur in the material being imaging, e.g., growth/etching of nanoparticles, [41][42][43][44][45][46] dissolution of metal organic frameworks, 47 pitting of carbon nanotubes, 48 shrinkage of bacterial cells, 49 or formation of visible polymeric nanoparticles. 50 Nanoparticle surface ligands are not observed during LP-TEM due to their small size (typically < 2 nm ligand layer thickness) and low image contrast compared to inorganic nanoparticles.…”