Liquid Phase Electron Microscopy of Bacterial Ultrastructure

Abstract

Recent advances in liquid phase scanning transmission electron microscopy (LP-STEM) have enabled the study of dynamic biological processes at nanometer resolutions, paving the way for live-cell imaging using electron microscopy. However, this technique is often hampered by the inherent thickness of whole cell samples and damage from electron beam irradiation. These restrictions degrade image quality and resolution, impeding biological interpretation. Using graphene encapsulation, scanning transmission electron microscopy (STEM), and energy-dispersive X-ray (EDX) spectroscopy to mitigate these issues provides unprecedented levels of intracellular detail in aqueous specimens. This study demonstrates the potential of LP-STEM to examine and identify internal cellular structures in thick biological samples. Specifically, it highlights the use of LP-STEM to investigate the radiation resistant, gram-positive bacterium, Deinococcus radiodurans using various imaging techniques.

Keywords: deinococcus.radiodurans; energy dispersive x‐ray spectroscopy; graphene encapsulation; liquid phase; manganese uptake; radiation resistance; scanning transmission electron microscopy.

Figure 1

Schematic of graphene liquid cell (GLC) assembly and LM/EM analysis of encapsulated D.radiodurans. a) Freshly prepared single‐layer graphene grids placed in water on filter paper, with the second layer of graphene suspended above. b) After exchanging water with buffer (HEPES), the sample is added directly to the top of the graphene grids. c) The solution is removed slowly to ensure the graphene layer remains intact over the grids. d) Solution is completely removed, and grids allowed to air dry for GLC comprised of multiple blisters. e) Expanded view of the panel (d) of graphene blisters with encapsulated D.radiodurans.

Figure 2

D.radiodurans growth stages and membrane composition in graphene encapsulated environments. a) Bandpass (BP) filtered LP‐ADF‐STEM section of encapsulated late exponential phase (OD₆₀₀: 0.65) D.radiodurans cell envelope, with a density profile region of interest (ROI) highlighted as white dashed box (TCF: 37500 e⁻/nm²). b) Corresponding density profile of cell envelope components reveals a periplasmic space (Pp) between the inner and outer membrane, a peptidoglycan layer (PG) followed by an interstitial layer (IL) and a final high contrast layer (HCL). c i, ii) BP filtered LP‐ADF‐STEM images of encapsulated stationary phase D.radiodurans octads at various stages of division from the same sample (TCF: 93 e⁻/nm²). d–h): Expanded view of single cocci from octad ROIs showing septum progression stages, rotated to place the central septum in the bottom left of the ROIs. d) Characteristic septal aversions visible. e) Septa meeting side on, in the cell center. Asterisk (*): Small storage granules; Arrowheads: Phosphate storage granules. f) Two cocci separated by a completed septum. g) New septa begin to emerge perpendicular to the central septum. h) Later stage of tetrad formation. Scale bar: a: 100 nm; c i, ii: 1 µm, d–h: 250 nm.

Figure 3

Ultrastructural detail and elemental distribution in D.radiodurans using LP‐ STEM and EDX. a) BP filtered LP‐ADF‐STEM and b) LP‐BF‐STEM image of encapsulated bacteria internal structure (TCF: 11800 e⁻/nm²), Inset: Expanded PS of cytosolic (red) ROI, with spatial frequency at (18 nm⁻¹) marked (dashed red). c) EDX spectra from ROIs in (b). d) Gaussian filtered and contrast normalized phosphorous and sulfur EDX maps of (a,b) (TCF: 50300 e⁻/nm²). Dashed white lines: Outline of membrane and polyphosphates from (a). Scale bar for a, b, d: 250 nm; PS: (2 nm⁻¹).

Figure 4

Manganese accumulates in bacterial phosphate granules. Summed LP‐ADF‐STEM image of bacteria incubated in MnCl₂ (Average TCF: 2979 e⁻/nm²); White Arrow: Gaseous bubble. Top Row: Intracellular elements; Middle row: Cell membrane associated elements; Bottom Row: Polyphosphate associated elements. Scale bar: 500 nm.

Figure 5

Effect of different environmental stressors on D.radiodurans ultrastructure. a) LP‐ADF‐STEM image of vacuum desiccated bacteria in a torn graphene liquid cell (TCF: 93 e^‐/nm²); Dashed red line: Tear in graphene, White arrows: Examples of desiccation‐related membrane separation. b) LP‐ADF‐STEM image of encapsulated diad, grown in 10% LB media (TCF: 2334 e^‐/nm²); Dashed yellow line: Ring shaped membrane between individuals. Scale bar for a: 2 µm, b: 500 nm.