Cryogenic (H2O)n-GCIB-SIMS imaging
Hua Tian
Abstract
The protocol describes the imaging of frozen-hydrated biological sample using high resolution mass spectrometry imaging, water gas cluster ion beam secondary ion mass spectrometry (H2O)n-GCIB-SIMS).
Steps
Both (H2O)n-GCIB and C60-SIMS were performed on a buncher-ToF instrument, J105 3D Chemical Imager (Ionoptika, Southampton, UK. Abbv. J105). The water cluster ion beam is pulsed through a pulser in the gun column, where the distance to the sample surface is 0.533 m. Beam tuning was assisted with an oscilloscope (Tektronix TDS 2024, USA) with detection by a secondary electron detector (SED). The singly-charged (H2O)n cluster size at beam energy of 70 kV with a time of flight (ToF) of 103 µs was calculated using the ToF equation as n = 30,900 (Figure S15). The SED offset was 8 µs. Beam focus was measured by scanning a 1000 mesh grid (Agar Scientific, Essex, UK). The average beam spot sizes were calculated using 20/80 percent of maximum intensities and were 1.60±0.01 µm and 1.16±0.45 µm for 70 keV (H2O)30k+ and 40 kV C60+, respectively (Figure S14). The beam dither was adapted to match the image pixel size. The mass resolution m/Δm was 6875 around m/z 100, and 10,000~12,000 up to m/z 2000. The live readout of mass resolution was from the software, Ionoptika SIMS Mainframe during the data acquisition.
The gold coated Si wafer with the frozen-hydrated mouse/human liver tissue section was plunged into liquid nitrogen and inserted to the pre-chilled cold sample stage in J105 instrument and kept at 100 K during GCIB-SIMS imaging. This cryogenic sample handling preserved the frozen-hydrated state thus maintaining the chemical gradients in the tissue section.
Guided by the anatomical features on the semi-serial H&E stained section, an area of interest was selected for SIMS imaging in negative ion mode using a 70 keV (H2O)30k+beam. The acquisition was in negative ion mode with 256 × 256 pixels using a 2 × 2 tiled image mode for mouse liver tissue sections, or 768×768 pixels using a 3 × 3 tiled image mode for human liver tissue sections. Each tile covers 400 × 400 µm2 (3.1 µm per pixel) for each section. With 1 pA of beam current and 296 shots per pixel, the ion doses were 3.01×1012 ions/cm2 each tile.
