C60-ToF SIMS imaging of frozen hydrated HeLa cells

Abstract

Sample preparation continues to be a major challenge for secondary ion mass spectrometry studies of biological materials. Maintaining the native hydrated state of the material is important for preserving both chemical and spatial information. Here, we discuss a method which combines a sample wash and dry protocol discussed by Berman et al^{1 (1)} followed by plunge freezing in liquid ethane for a frozen-hydrated analysis of mammalian cells (HeLa). This method allows for the removal of the growth media and maintains the hydrated state of the cells so that they can be prepared frozen-hydrated without the need for a freeze-fracture device. The cells, which were grown on silicon, have been successfully re-grown after the cleaning procedure, confirming that a significant portion of the cells remain undamaged during the wash and dry. Results from preliminary SIMS measurements show that is it possible to detect a large variety of bio-molecular signals, including intact lipids from the plasma membrane in the mass range of 700-900 Da from single cells, with little external water interference at the surface.

Figure 1

Optical image of a silicon shard 24 hours after a wash and dry procedure reported in the experimental section. Note the presence of new cells which have grown over the edge of the silicon, indicating cell viability after sample cleaning.

Figure 2

Mass spectral image of HeLa cells prepared frozen-hydrated for different chemical species as identified in the figure (total refers to a the total counts for the spectrum and HMR corresponds to high-mass range and is the cumulative image for ions between m/z 700–900. Analysis field of view is 298 µm × 298 µm with a spectral dose of 3.5 × 10¹¹ ions/cm².

Figure 3

Mass spectral image of HeLa cells prepared frozen-hydrated for different chemical species as identified in the figure (total refers to a the total counts for the spectrum and HMR corresponds to high-mass range and is the cumulative image for ions between m/z 700–900. Analysis field of view is 298 µm × 298 µm with a spectral dose of 3.5 × 10¹¹ ions/cm² following an etch dose of 2.0 × 10¹⁴ ions/cm².