May 08, 2020
Imaging Mass Cytometer Tuning
- 1University of Zurich;
- 2University of Florida
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: Jeff.spraggins@vanderbilt.edu
Protocol Citation: Michelle Daniel, Marda Jorgensen 2020. Imaging Mass Cytometer Tuning . protocols.io https://dx.doi.org/10.17504/protocols.io.bf2gjqbw
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: May 05, 2020
Last Modified: October 02, 2020
Protocol Integer ID: 36648
Abstract
This SOP describes the tuning of an Imaging Mass Cytometer (IMC). The tuning process
encompasses parameters such as Interface alignment, gas flow rate optimizations, laser
power optimization and detector voltage optimization. All these factors aim at having a
machine that performs equally over time.
The machine needs to pass specifications listed in this SOP in order to be used for data
acquisition.
Machines do not need to be tuned daily but at least twice per week and upon every startup.
Additionally, the adjustment of the laser energy is described in this SOP, which
needs to be performed every 3 weeks.
Materials
MATERIALS
IMC (software 7.0 and higher)Fluidigm
3-metal tuning slideFluidigm
Before start
Ensure a sufficient supply and pressure of argon and helium gases prior to starting.
Start plasma and insert tuning slide
Start plasma and insert tuning slide
1. If the machine is running, check the “tuning log sheet.ods” and get the date of the last tuning. If
the last tuning was done more than two days ago proceed to step 3.3.
2. Check argon supply and pressure (5 bar). Make sure that one of the argon tanks has at least
25% which will last overnight. Get a new tank if required from the shop in the basement using the
PanGas pay card.
3. Check Helium pressure (2 bar). Check the Helium bottles in the cabinet. If the pressure drops
below 100 bar open the second bottle and close the one that was open. Move the empty bottle in
front of the room and hang a sign “Empty” on it. Inform Esther Danenberg or Stefanie Engler to
order a new bottle.
4. Open the CyTOF software on the computer and log in with your name and password.
5. Insert the tuning slide into the Hyperion.
6. Start plasma.
7. Wait for at least 30 minutes until you proceed with tuning.
Adjust Eref
Adjust Eref
1. Confirm that “Adjust E-ref” needs to be performed. If not, proceed to tuning.
2. Hit “Settings” under Manual Tune.
3. In the camera window move to an empty region next to an ablated region (right side).
4. Hit “Get Current Coordinate” in the Manual Tune Settings.
5. Copy the values of the Ablation Energy Range to the tuning log sheet.
6. Hit “Adjust Eref”.
7. Copy the new values of the Ablation Energy Range to the tuning log sheet.
Note
This process needs to be done every 2-3 weeks. Therein the laser energy is adjusted to
guarantee optimal ablation with respect to decreasing laser power output over time. Check the tuning “tuning log sheet.ods” on the IMC computer for the last time this was performed.
Tuning
Tuning
1. Hit “Auto Tune” under Instrument Control.
2. Hit “New” to create a new tuning template.
3. In the camera window move to an empty space next to previously ablated regions (right side).
4. Hit “Get Current Coordinate” in the Auto Tune window.
5. Under Parameters check “QC Report”, “HTI Current/Gases Calibration” and “Transient
Calibration”.
6. Hit “Run”.
7. If tuning does not pass repeat one more time with the same parameters.
8. If tuning does not pass again also check “Coarse XY Optimization” and “Fine XY Optimization”.
9. If tuning fails again contact Daniel Schulz or Esther Danenberg or Stefanie Engler.
Note
XY optimization does only need to be performed if the torch box of the machine has been opened
or if tuning continuously fails.
Check additional results if tuning passes
Check additional results if tuning passes
1. If tuning passes under Results check the Transient Cross Talk 1. This value should be below
0.05. If this is not the case the Hyperion and the Helios are not well aligned. Align tubing. If you
have not obtained training to align tubing contact Esther Danenberg, Stefanie Engler or Daniel
Schulz.
2. Check the detector voltage value and compare with previous tuning results. Large jumps (>50 V)
in detector voltage indicate abnormal deterioration of the detector potentially due to sample/gas
contaminations. Contact Daniel Schulz.
3. Copy all the parameters required in the tuning log sheet from the Results to the tuning log sheet
on the IMC computer.