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.

TEM and Associated Techniques

“…Liquid cell STEM enables the visualization of many dynamic physical, 14.5 In Situ Analysis with a Liquid Cell 337 chemical, and structural nanoscale phenomena taking place in liquids, as evidenced by a growing number of reports on fluid cell TEM and STEM studies [176,[198][199][200][201][202][203][204][205][206][207][208][209][210][211][212][213][214][215]. This technique could tremendously enhance the current understanding of the fundamental processes involved in formation and assembly of nanoparticles by revealing the generally lacking dynamics at the nanoscale.…”

“…The liquid specimens are then examined with a Continuous Flow Fluid Cell TEM Holder Platform, with the in situ fluid delivery carried out with a syringe pump operating with the variable pumping speed (0.5-10 μl min −1 ). As a special note, obtaining time-resolved data of iron oxide biomineralization in vivo [208,209] might become a reality. Exploring new analytical approaches for the combination of imaging fully hydrated species with HAADF opened up new ways for the study of dynamics of processes at the single-cell level and the nanometer-scale resolution.…”

“…Exploring new analytical approaches for the combination of imaging fully hydrated species with HAADF opened up new ways for the study of dynamics of processes at the single-cell level and the nanometer-scale resolution. Recently, Woehl and coauthors [208,209] reported on the new correlative technique for imaging of magnetotactic bacteria in liquid using a Fluid Cell TEM Holder Platform, with the bacterial cells sealed between the two thin electron-transparent silicon nitride window membranes. Figure 14 in magnetosome chains in the bacteria allowed their visualization with sufficient contrast.…”

“…Correlative HAADF-STEM and fluorescence microscopy (FM) were used to characterize the bacterial specimen in situ under the conditions most closely resembling the natural growth environment of the microorganism. This was the first successful report on use of HAADF-STEM in the fluid cell for the monitoring of cellular dynamics in situ [209]. Liquid cell analysis of iron oxides is a very young field, currently involving just a handful of research groups.…”

Considerations for imaging thick, low contrast, and beam sensitive samples with liquid cell transmission electron microscopy

“…Recent advances in instrumentation has allowed for electron microscopy of liquid samples (4), a technique that may permit visualizing physiological structures (5,6) or dynamics (7) at high resolutions and in real-time. Liquid cell transmission electron microscopy (LC-TEM) has also been used to image whole cells in a fully hydrated state, where eukaryotes (8)(9)(10)(11) and prokaryotes (12)(13)(14) have been imaged using both TEM and STEM. However, sample thickness is a consistent issue encountered when performing LC-TEM and is particularly problematic for large samples such as cells which are typically a minimum of 300-400 nm in thickness and can reach 1000 nm and greater depending on the cell type.…”