Nov 03, 2022
Cryogenic (H2O)n-GCIB-SIMS imaging
- 1University of Pittsburgh
Protocol Citation: Hua Tian 2022. Cryogenic (H2O)n-GCIB-SIMS imaging . protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbyynovpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 03, 2022
Last Modified: November 03, 2022
Protocol Integer ID: 72233
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).
Materials
HPLC plus water, Sigma
Liver tissue section (Cryomicrotomed at the thickness of 10 microns)
Liquid nitrogen
Instrumentation: a buncher-ToF SIMS instrument, J105 3D Chemical Imager (Ionoptika, Southampton, UK. Abbv. J105)
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.