Phosphorus detection in vitrified bacteria by cryo-STEM annular dark-field analysis

Area of Science:

• Microscopy
• Cell Biology
• Biochemistry

Background:

• Bacterial cells contain dense granules, including polyphosphate bodies (PPBs), which store inorganic phosphate.
• Traditional methods for identifying PPBs require cell drying or chemical fixation, necessitating high electron doses incompatible with low-dose cryo-imaging.
• A need exists for methods to identify PPBs in intact, hydrated bacterial specimens without damaging them.

Purpose of the Study:

• To establish a low-dose electron microscopy technique for mapping phosphorus-containing granules in hydrated, vitrified bacteria.
• To demonstrate that Scanning Transmission Electron Microscopy (STEM) can identify polyphosphate bodies (PPBs) without chemical fixation or drying.

Main Methods:

• Utilized Scanning Transmission Electron Microscopy (STEM) on fully hydrated, intact, vitrified bacterial specimens.
• Leveraged the quantitative sensitivity of electron scattering to atomic number for elemental mapping, distinguishing phosphorus from lighter elements (C, N, O).
• Employed scattering angle resolution, analogous to Z-contrast in materials science, for phosphorus identification.

Main Results:

• Successfully mapped phosphorus-containing dense granules in hydrated bacteria using STEM.
• Demonstrated that phosphorus can be distinguished from carbon, nitrogen, and oxygen based on electron scattering properties.
• Showed that this method requires a low electron dose, comparable to standard imaging.

Conclusions:

• STEM imaging of hydrated, vitrified bacteria offers a simple and effective method for mapping phosphorus-containing granules like PPBs.
• This technique avoids damaging sample preparation steps, preserving cellular structure.
• The approach is broadly applicable for mapping other known heavy elements in cryopreserved biological specimens.