Label-free sub-micrometer 3D imaging of ciprofloxacin in native-state biofilms with cryo-time-of-flight secondary ion mass spectrometry

Abstract

High spatial resolution mass spectrometry imaging has been identified as a key technology needed to improve understanding of the chemical components that influence antibiotic resistance within biofilms, which are communities of micro-organisms that grow attached to a surface. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) offers the unique ability for label-free 3D imaging of organic molecules with sub-micrometer spatial resolution and high sensitivity. Several studies of biofilms have been done with the help of ToF-SIMS, but none of those studies have shown 3D imaging of antibiotics in native-state hydrated biofilms with cell-level resolution. Because ToF-SIMS measurements must be performed in a high-vacuum environment, cryogenic preparation and analysis are necessary to preserve the native biofilm structure and antibiotic spatial distribution during ToF-SIMS measurements. In this study, we have investigated the penetration of the antibiotic ciprofloxacin into Bacillus subtilis biofilms using sub-micrometer resolution 3D imaging cryo-ToF-SIMS. B. subtilis biofilms were exposed to physiologically relevant levels of ciprofloxacin. The treated biofilms were then plunge-frozen in liquid propane and analyzed with ToF-SIMS under cryogenic conditions. Multivariate analysis techniques, including multivariate curve resolution (MCR) and inverse maximum signal factor (iMSF) denoising, were used to aid analysis of the data and facilitate high spatial resolution 3D imaging of the biofilm, providing individually resolved cells and spatially resolved ciprofloxacin intensity at "real world" concentrations.

Keywords: 3D imaging; Antibiotic; Biofilm; Cryogenic analysis; Multivariate analysis; ToF–SIMS.

Fig. 1

Selected ion signals from ToF–SIMS depth profile through a B. subtilis biofilm exposed for 5 min to 50 µg/ml of ciprofloxacin. The cell-free frozen aqueous layer is highlighted in blue, and the biofilm layer is highlighted in orange

Fig. 2

3D ToF–SIMS image of a frozen hydrated B. subtilis biofilm prior to exposure to ciprofloxacin. a “Live” (red) and “inactive” (green) cells. b Background signal at m/z 332.14 where the ciprofloxacin (M + H)⁺ signal would appear

Fig. 3

3D ToF–SIMS image of a frozen hydrated B. subtilis biofilm after 5 min exposure to 1000 µg/ml ciprofloxacin. a “Live” (red) and “inactive” (green) cells. b Raw ciprofloxacin (M + H)⁺ signal (m/z 332.14) (8 voxel binning). c iMSF denoised ciprofloxacin (M + H)⁺ signal showing preferential binding of ciprofloxacin to the “live” cells

Fig. 4

3D ToF–SIMS image of a frozen hydrated B. subtilis biofilm after 5 min exposure to 100 µg/ml ciprofloxacin. a “Live” (red) and “inactive” (green) cells. b iMSF denoised ciprofloxacin (M + H)⁺ signal showing preferential binding of ciprofloxacin to the “live” cells

Fig. 5

3D ToF–SIMS image of a frozen hydrated B. subtilis biofilm after 5 min exposure to 50 µg/ml ciprofloxacin. a “Live” (red) and “inactive” (green) cells. b iMSF denoised ciprofloxacin (M + H)⁺ signal showing preferential binding of ciprofloxacin to the “live” cells

Fig. 6

3D ToF–SIMS image of a frozen hydrated B. subtilis biofilm after 5-min exposure to 10 µg/ml ciprofloxacin. a “Live” (red) and “inactive” (green) cells. b iMSF denoised ciprofloxacin (M + H)⁺ signal showing preferential binding of ciprofloxacin to the “live” cells