Mounting of Escherichia coli spheroplasts for AFM imaging

Area of Science:

• Microbiology
• Biophysics
• Biochemistry

Background:

• The cytoplasmic membrane of Escherichia coli (E. coli) contains crucial transporters and recognition elements.
• In vivo investigation of this membrane using atomic force microscopy (AFM) necessitates cell wall removal and stable spheroplast immobilization.

Purpose of the Study:

• To establish a reliable method for immobilizing E. coli spheroplasts for in vivo AFM imaging.
• To enable repeated, long-term AFM analysis of the E. coli cytoplasmic membrane.

Main Methods:

• Preparation of E. coli spheroplasts by removing the cell wall.
• Immobilization of spheroplasts onto a mica substrate using warm gelatin.
• Imaging of immobilized spheroplasts using atomic force microscopy (AFM).
• Confirmation of spheroplast association with the gelatin layer via confocal fluorescence imaging.

Main Results:

• Warm gelatin effectively secures E. coli spheroplasts on a mica substrate.
• The prepared spheroplasts can be repeatedly imaged by AFM for several hours.
• Confocal fluorescence imaging verified the spheroplasts' association with the gelatin layer.

Conclusions:

• Gelatin immobilization provides a stable platform for in vivo AFM studies of the E. coli cytoplasmic membrane.
• The reordering of heated gelatin into a network underlies the effective entrapment and immobilization of spheroplasts.
• This technique facilitates detailed investigation of membrane components and their functions.