Subcellular chemical mapping using correlated cryogenic electron and mass spectrometry imaging

Area of Science:

• Biophysics
• Chemical Biology
• Microscopy

Background:

• Electron cryomicroscopy (cryo-EM) provides high-resolution imaging of biological samples but struggles with chemical identification.
• Accurate chemical identification of structures within cryo-EM is crucial for understanding cellular processes.

Purpose of the Study:

• To develop and validate an integrated spatiochemical analysis workflow combining cryo-EM and focused ion beam secondary ion mass spectrometry (FIB-SIMS).
• To enable subcellular localization and chemical identification of molecules in untagged biological specimens.
• To investigate the cellular uptake and storage mechanisms of the chemical pollutant bisphenol-AF in environmental bacteria.

Main Methods:

• Correlative workflow integrating cryo-electron microscopy (cryo-EM) with focused ion beam secondary ion mass spectrometry (FIB-SIMS).
• Application to untagged bacterial and eukaryotic specimens, compatible with cryogenic light microscopy.
• Analysis of bisphenol-AF uptake in environmental bacteria using the integrated cryo-EM-FIB-SIMS approach.

Main Results:

• The cryo-EM-FIB-SIMS workflow successfully achieved subcellular localization of molecules within bacterial cells.
• The method demonstrated compatibility with cryogenic light microscopy and FIB-milled eukaryotic samples.
• Environmental bacteria were shown to store bisphenol-AF within cytosolic phase-separated aggregates.
• These stored chemicals were not removed by bacterial efflux machinery, despite its upregulation.

Conclusions:

• Cryo-EM-FIB-SIMS is an effective method for mapping elemental and molecular signatures in near-native biological samples.
• The study reveals a novel mechanism of pollutant sequestration in bacteria via cytosolic aggregates.
• This integrated approach provides significant biological insights into chemical interactions within cells.