Injection workflows
ARYA provides two main injection workflows to suit different experimental needs: Constant Force Injection and Constant Z Position Injection. The Constant Force workflow keeps the same pressure on the sample by adjusting the probe height during injection, which works well for uneven surfaces or when gentle force control is important. The Constant Z Position workflow keeps the probe at a fixed height after touching the sample surface, providing consistent injection depth. Both workflows follow the same basic steps—Positioning, Injection, and Review—but handle the injection process differently, allowing researchers to choose the best approach for their specific samples and goals.
Constant Force Injection Workflow
In this setup, the probe
maintains a constant force setpoint while the injection takes place. The system
ensures that a consistent force is applied during the injection process. 
Overview of the Constant Force Injection workflow
The parameters to be defined for the workflow are related to the different phases of the injection. During the approach, the cantilever will only stop when a certain force is achieved (setpoint) with a set speed (approach speed) and will pierce the cell with the tip. The piercing is followed by the delivery; by applying pressure for a certain amount of time (injection time), the injection mix (IM) can be delivered into the cell. Afterwards, the cantilever will move out of the cell with a set speed (retraction speed). Idle pressure is applied during the approach and retraction phases.
- Setpoint: The setpoint is the deflection value, measured in newtons or volts, at which the system stops moving during approach. It represents the contact force applied to a surface (in this case, a cell). Always use Newtons for greater consistency across different probes and setups.
- Approach Speed: The speed at which the cantilever moves toward the surface.
- Pressure: The pressure in mbar applied to deliver the IM into the cell. It must be sufficient to overcome the cell’s internal resistance without causing the cell to burst.
- Injection Time: The duration for which pressure is applied. For this workflow, there is a 1-second delay before pressure begins.
- Retraction Speed: The speed at which the probe withdraws from the cell after injection. It is typically set low to minimize the risk of damaging the cell during retraction.
- Retraction Distance: The distance the cantilever retracts after injection. This should be far enough to avoid any contact with the cell when moving to the next target, reducing the risk of collisions or damage.
- Flush Configuration: Used to prevent clogging from cell debris and to maintain consistent pressure delivery. Sets up a high-pressure pulse between injections: the flush interval, position, pressure and duration can be defined.
💡 Tip: It’s recommended to flush the probe after each injection (interval of 1) for optimal performance.
To evaluate the results of an injection, the images, as well as the force curves of the injection [Link placeholder to section Injection/Postinjection assessment], must be taken into account.
The force curve for each injection can be evaluated in the Results history of the ARYA software. In a perfect injection, the Z position and the force should remain stable during the injection (as observed in figure below).
Force curve of a cell injection, showing a force overshoot at the start and stable force during the injection. In green, z position is indicated in mm. In yellow, force readout is shown. Important: exported force curves show force in Volts instead of Newtons.
Observation in brightfield and fluorescence can also provide an initial feedback about the injection success:
- Examine brightfield image to ensure that the cell is alive. Other than with a viability analysi, cell death can be indicated by:
- Cell partially detached
- Cell blebbing
- Missing cell
- Examine the fluorescent image to ensure that tracer is visible in the injected cells
Constant Position Injection Workflow
In this process, the system keeps a fixed Z-axis position, instead of a constant force as the previous one. After approaching the cell and prior to the injection, the cantilever retracts by a defined adjustment distance with a specific speed. During the injection, pressure is applied while the cantilever deflects freely, providing visual feedback of cell deformation via spectroscopy graphs.
Overview of the constant Z position injection workflows
This workflow has the same injection parameters as the previous one. The following ones are specific for the constant Z workflow:
- Adjustment Distance: After reaching the setpoint, the probe retracts by this specified distance. This retraction creates space for the injection phase and is defined as a relative movement upward from the setpoint position.
- Adjustment Speed: The speed at which the probe performs the retraction to the adjustment distance.
Which workflow to choose?
Selecting the appropriate injection workflow depends on the experimental goals and the behavior of the target cell culture: there’s no one-size-fits-all solution.
The Constant Z Position Injection workflow is generally recommended as a starting point, as it allows for higher injection volumes at lower pressure ranges and offers greater control over the injection process. However, switching to the Constant Force Injection workflow may be beneficial if yields are suboptimal with Constant Z.
💡 Tip: Begin with Constant Z Position Injection and transition to Constant Force only if needed based on yield and cell response.
Injection optimization
The injection calibration workflow is the dedicated workflow in ARYA software for determining optimal injection parameters for a given cell line and IM. To run the injection parameters optimization:
- After running a full Preparation Advanced workflow, open the optimization workflow in the right tool bar of the ARYA software by going to Settings > Workflows > Injection Calibration
- Under “Injection type”, select the workflow chosen (Constant Z injection or Constant Force injection)
The full parameter optimization only needs to be run when using a new cell line or IM. When the same cell line and IM are reused, only the pressure needs to be swept, unless there are large difficulties in the injection process.
When starting with a new cell line and IM:
- Select the nucleus from 30 cells
- 💡 Tip: To avoid any disturbances to the target cell culture, we recommend using a separate well for Injection optimization.
- Configure
the initial sweep settings. The parameters listed here are identical to those
used in the standard Injection workflow. During calibration, these parameters
can be tested by incrementally increasing their values (Δ setting) within a
defined interval (repetition count: how many points or cells are included).
Parameter
Constant Z injection
Constant F injection
Setpoint
700 nN
700 nN
Approach Speed
50 um/s
50 um/s
Adjustment Distance
1 um
-
Adjustment Speed
1 um/s
-
Pressure
50 mbar
50 mbar
Injection Time
2 s
2 s
Retraction Speed
5 um/s
5 um/s
Retraction Distance
20 um
20 um
Repetition Count
30
30
Δ Setpoint
0
0
Δ Approach Speed
0
0
Δ Adjustment Distance
0
0
ΔAdjustment Speed
0
0
Δ Pressure
0
0
Δ Injection Time
0
0
Flush Configuration
- Interval
- Flush Position
- Retraction Distance
- Pressure
- Flush Duration
1
Retract by distance
150 um
500 mbar
2 s
1
Retract by distance
150 um
500 mbar
2 s - This will inject 30 cells with constant settings
- Check the cells during and after injection:
- Record if a cell bursts during injection
- Cells should show a slightly enlarged nucleus
- With fluorescent imaging presets, cells should show the fluorescent tracer contained in the IM.
In order to evaluate the results of a parameters sweep, take two images of the injected cells (in bright field and in fluorescence preset for the specific injection mix).
When exporting the images, include annotations on the image. This will label the injection points with inj_x, where x is the group number.
Brightfield and fluorescent images of injected cells following an injection parameter sweep.
The next parameter sweeps depend on the result of the initial sweep.
- A result of more than 50% of cells expressing fluorescence and remaining viable is considered a positive outcome. For some cell/IM combination, success rates can be as high as 70%.
- During parameter evaluation, verify flow rates at 0, 100, 200 and 300 mbar using the fluorescent preset, to ensure that the probe has not blocked.
The force curves also
provide valuable information about the injection (see previous sections).
Injection Optimization examples
If the cells explode (more than 40% of cells burst during injection), run the following sweeps one by one. Evaluate the results of the sweep as described above, and only continue to the next sweep if injection success is not satisfactory enough:
Pressure Sweep:
- Select 50 cells
- Apply the same settings as in the initial sweep above except for:
- Pressure → 10 mbar
- Repetition Count → 10
- ΔPressure → 10 mbar
- This will inject 5 groups of 10 cells with pressures 10, 20, 30, 40 and 50 mbar
- Continue with the lowest pressure where:
- Cells are visibly enlarged after injection
- Cells express fluorescence
Injection Time Sweep:
- Select 40 cells
- Apply the same settings as in the initial sweep above except for:
- Injection Time → 0.5 s
- Repetition Count → 10
- ΔInjection Time → 0.5 s
- This will inject 4 groups of 10 cells with injection times 0.5, 1, 1.5 and 2 s
- Continue with the injection time where cells are still successfully injected but the cells explosions are lowered
(Only for Constant Z workflow) Adjustment Distance Sweep:
- Select 50 cells
- Apply the same settings as in the initial sweep above except for:
- Adjustment Distance → 1 µm
- Pressure → As chosen in the pressure sweep
- Repetition Count → 10
- Δ Adjustment Distance→ 0.5 µm
- This will inject 5 groups of 10 cells with adjustment distances 1, 1.5, 2, 2.5 and 3 µm
- Continue with the adjustment distance with the lowest number of cells exploding
Examples of injection parameters for multiple cell lines
The parameter values can be used as a starting point for injection workflows, and are meant to be adjusted for each cell line and injected solution.
Cell line | Setpoint (nN) | Approach Speed (um/s) | Retraction Speed (um/s) | Pressure (mbar) | Injection time (s) | Retraction Distance (um) |
CHO | 1000 | 50 | 5 | 30 | 3 | 20 |
HeLa | 1000 | 50 | 5 | 30 | 10 | 20 |
HEK | 1000 | 50 | 5 | 30 | 10 | 20 |
C2C12 | 800 | 50-70 | 5 | 80-150 | 2-3 | 20 |
MCF7 | 700 | 60 | 5 | 100-150 | 2 | 20 |