1) Calibrate Orbitrap MS
a. Use the 3D OrbiSIMS control software, SurfaceLab 6.7, to set up gas cluster ion beam (GCIB) to provide a pulsed beam of Ar3000^+ clusters with an energy of 20 keV and a time-averaged current above 20 pA.
b. Set the He gas pressure in the secondary ion transfer optics to the “High” setting for optimal transmission of small inorganic cluster ions to the Q Exactive™ HF.
c. Navigate to the Ag sample and adjust working distance (sample-to-extractor) and target potential scheme to optimise secondary ion transmission to the Q Exactive™ HF. For a standard instrument configuration these are 1.5 mm and 57 V, respectively.
d. Use the Q Exactive Tune software to calibrate mass spectrometer based on positive and then negative ion spectra obtained from the Ag film using the argon GCIB. Table 1 lists recommended secondary Ag cluster ions for the calibration.
Table 1: Positive and negative secondary ions used for Orbitrap MS calibration.See figure in Figures section.
2) Mount tissue section and load sample
a. Section tissue in the cryo-microtome with 10 μm thickness at -22 °C.
b. Thaw mount section onto the conductive side of a ITO coated conductive microscope slide for OrbiSIMS analysis.
c. Optionally cryo-microtome an adjacent tissue section for subsequent H & E staining and imaging (protocol not given here).
d. If OrbiSIMS analysis is not done immediately after sectioning, store the microscope slide in a suitable container at -80 °C until analysis. Care should be taken to avoid condensation when taking the microscope slides out of the freezer, e.g. by vacuum packing them before freezing, and thawing under vacuum.
e. Introduce the slide with the dried tissue section into the hybrid instrument’s vacuum system.
3) Acquire mass spectrum (optional)
a. Set up GCIB to provide a 20 keV Ar3000+ beam with a current greater than 1 pA. A cluster size of 3000 is the minimum recommended. Below this, increased fragmentation of molecules occurs. Larger clusters can be used but with lower beam current and longer acquisition times.
b. Measure and record the beam current, this is needed to calculate the dose. It is good practice to also measure the beam current at the end of the experiment.
c. Set up electron gun to flood sample surface with > 10 μA low energy (20 eV) electrons for charge compensation between GCIB ion pulses.
d. Set He pressure in secondary ion transfer optics to the “Low” setting for optimal transmission of organic secondary ions to the Q Exactive™ HF.
e. On tissue section, adjust target potential pulsing scheme to optimise secondary ion transmission to Q Exactive™ HF mass analyser for spectrum acquisition conditions, using a random raster pattern for the GCIB.
f. Use in-instrument video camera system to find region of interest on tissue section. Optionally, a secondary ion image of a larger area of the sample may be obtained using a Bi3+ beam and the ToF MS to help identify regions of interest for imaging with the GCIB and the Q Exactive™ HF. Note that doing this before the Orbitrap acquisition can result in damage. A low ion dose is needed < 1 x 1011 ions / cm2.
g. Start spectrum acquisition using SurfaceLab 6.7 with a set mass resolving power of 240,000, using a random raster pattern for the GCIB.
4) Acquire image
a. Set up 20 keV Ar3000+ focused pulsed beam with a spot size smaller than 2 μm (1.5 μm can be achieved) and a current between 1 pA and 30 pA.
b. Set up electron gun to flood the sample surface with > 10 μA low energy (20 eV) electrons for charge compensation between gas cluster ion pulses.
c. Set He pressure in secondary ion transfer optics to the “Low” setting for optimal transmission of organic secondary ions to the Q Exactive™ HF.
d. On tissue section, adjust target potential pulsing scheme to optimise secondary ion transmission to Q Exactive™ HF mass analyser for imaging conditions, using a sawtooth raster pattern.
e. Use in-instrument video camera system to find region of interest on tissue section.
f. Start image acquisition using SurfaceLab 6.7 with a set mass resolving power of 240,000 and an image resolution for which the pixel-to-pixel distance corresponds to the GCIB spot size, e.g. an image resolution of 150 × 150 pixels for a field of view of 250 μm × 250 μm. Use a saw tooth raster pattern for the GCIB.
5) Obtain MS/MS spectrum for secondary ions of interest (optional)
a. Inspect mass spectra obtained in steps 3 and 4 in SurfaceLab 6.7 and identify mass peaks of interest.
b. Set up the instrument for spectrum or image acquisition following step 3 or step 4.
c. Start spectrum acquisition from region of interest using SurfaceLab 6.7, selecting the option for tandem mass spectrometry using the higher-energy collision-induced dissociation cell in the Q Exactive™ HF. The normalised collision energy needs adapting to the analyte. Useful normalised collision energies are typically in the range from 15 to 70 eV.
6) Interpret data and generate biomolecule distribution images
a. Inspect mass spectra in SurfaceLab 6.7 and review potential putative secondary ion assignments for peaks of interest.
b. Select peaks for image generation, ensuring that peak integration windows encompass the full width of the mass spectral peaks.
c. Adjust secondary ion intensity-to-colour transformation functions for individual images to highlight relevant features of the data.
d. Where required, make composite multi-colour images, e.g. RGB, based on individual secondary ion images or summed images.