This guide provides step-by-step instructions for preparing the FluidFM system before conducting experiments. Completing this workflow ensures proper calibration for precise force measurements and optimal experimental results.
In the ARYA software interface, locate the right-hand menu panel. Under 'Preparation' workflows, select 'Preparation Advanced.'
Preparation Advanced workflow under the Preparation Workflows section in ARYA.
Choose Probe
Grip the FluidFM probe with the FluidFM head.
1.Check the bottom status bar to verify whether a probe is already mounted. If a probe is mounted, select a dropping location from the 'Drop' menu before proceeding.
Figure 1. Status bar in ARYA, showing the absence of a probe.
Figure 2. Choose probe step, showing locations for dropping and picking the probe.
2. Select the new probe location from the ‘Pick’ menu.
3. Focus on the probe using the 10× objective lens.
- Note: The 20× objective lens cannot achieve proper focus during probe setup. Use the 10× objective.
4. Using the mouse, drag the field of view to position the probe aperture directly beneath the crosshair marker.
Figure 3. Adjust the gripping position of the FluidFM probe.
5. The system will pick the probe and perform an air tightness test at 20 mbar pressure.
- If the air tightness test fails, an error message will appear. Verify the probe is properly seated and retry.
Laser Alignment
Adjust the laser position on the cantilever's reflective surface to ensure accurate force measurements.
1. Click ‘Align’.
Figure 4. Align laser step.
2. Use arrow controls to position the laser on the probe reflective layer.
Figure 5. Position the laser on top of the probe reflectie layer.
3. For optimal signal readout, position the laser between the 3rd and the 4th cantilever pillars.
Learn more about the laser signal and troubleshooting
Maximize Signal (air)
The mirror inside the FluidFM Head moves to optimize laser reflection on the photodetector. This process balances the laser signal between two photodiodes. The intensity signals from both photodetectors are plotted. Signal maximization is achieved when both photodiodes receive equal laser intensity and each operates between 30% and 90% of its detection range. The best position is where the two signals intersect.
Signal maximization must be performed twice:
- In air.
- In experimental medium (liquid), done later in the workflow.
1.Select ‘Center automatically’.
Figure 6. Choose probe step, showing locations for dropping and picking the probe.
2. Click 'Ok' to proceed.
Figure 7. Plot of the intensity signals from both photodetectors.
Learn more about the laser signal and troubleshooting
Configure Probe
Select one of the following configuration options:
- Scan the QR code on the FluidFM probe blister pack using the barcode scanner.
- Select the type of cantilever mounted (FluidFM Micropipette, FluidFM Nanopipette, FluidFM Nanosyringe).
- Enter the serial number of the cantilever.
The system displays probe specifications and aperture image:
Figure 8. FluidFM probe specifications when the QR code is scanned.
Measure Spring Constant
The spring constant (k) quantifies cantilever stiffness by relating applied force to resulting displacement. It is calculated using Hooke's law:
Where:
= Force on the cantilever (nN)
= Spring Constant (N/m)
= Cantilever deflection (nm)
In atomic force microscopy (AFM), the spring constant is determined using the Sader method, which analyzes the thermal resonance spectrum peak. The nominal value of the spring constant for a cantilever is printed on the FluidFM probe blister pack (configured in Step "Configure Probe"). Although an empirical value is measured for each wafer during quality control, the specific cantilever value must be determined during the Preparation Advanced workflow.
In practice, the probe contacts the surface at an angle through its pyramidal tip, located a few micrometers from the cantilever edge. The force required to deflect the cantilever increases with distance from the cantilever edge. This geometric effect requires correction of the Sader method calculation, resulting in the effective Spring Constant (keff). For typical FluidFM probes, the total correction factor is as follows:
FluidFM Probe type | keff/kmeas |
FluidFM Nanopipettes and Nanosyringes | 1.229 |
FluidFM Micropipette | 1.155 |
1.Adjust the 'Ambient Temperature' setting if necessary.
2. Retain the default probe dimensions.
3. Click ‘Measure’.
4. After measuring, the following output is shown:
Figure 9. Results of the Spring Constant measurement.
- The thermal resonance spectrum plot displays:
- A: Beginning of the fitting rang.
- B: Full width at half maximum (FWHM) lower boundary.
- C: Peak.
- D: Full width at half maximum (FWHM) upper boundary.
- E: End of the fitting range
- Quality Factor: Ratio of stored energy to energy dissipated per oscillation cycle. Higher values indicate lower energy loss and more stable measurements.
- Resonance frequency: The frequency at which the AFM control loop oscillates the probe during tapping mode operation.
- Measured Spring Constant value (calculated through the Sader method).
- If the probe type has been configured, the effective spring constant value is also displayed.
Fill Probe
Fill the FluidFM probe microchannel with solution from the reservoir (see Probe filling section). The software will display a differential image of the cantilever to detect when the cantilever is filled.
1.Select an area that includes the visible cantilever. This enables differential imaging to detect when filling is complete.
Figure 10. Area selection to obtain the differential image of the cantilever.
2. Set an initial pressure using the Fill pressure bar.
Figure 11. Fill probe step, with the pressure bars for Fill and Idle conditions.
3. Press 'Fill'.
4. Gradually increase pressure in 100 mbar increments.
5. Monitor the cantilever channel until liquid becomes visible (typical filling pressure: 200–400 mbar).
6. Once filling is complete, switch the pressure control to 'Idle.'
Higher pressures may be required depending on solution viscosity. Filling time ranges from seconds to minutes depending on solution properties and probe characteristics.
Note: Some liquids (e.g., trypsin) evaporate rapidly. Immediately move the filled cantilever into liquid medium.
Go to sample
Once the probe is filled, move it to the experimental plate.
In the Navigation Tool, select the target well on the experimental plate. Maintain positive pressure in the cantilever during movement.
Maximize Signal (liquid)
Repeat the signal maximization procedure with the probe immersed in experimental medium. Follow the same steps as Section "Maximize Signal (air)".
Measure Sensitivity
Sensitivity converts voltage readings from the photodetector to cantilever deflection in nanometers, enabling force measurements in nanonewtons. Sensitivity depends on cantilever properties and laser positioning. It allows direct force measurement in nanonewtons, accounting for variations in cantilever stiffness.
Where:
=
Cantilever deflection (nm)
=
Voltage (mV)
=
Sensitivity (nm/mV)
During sensitivity measurement, the system moves the probe to the dish bottom and performs a series of indentations.
Figure 12.
Parameters for sensitivity measurement.
Recommended measurement settings:
- Initial setpoint: 100 mV
- Number of measurements: 5
Verify the following conditions:
- The cantilever is positioned over a hard surface (i.e., plastic or glass).
- No cells or debris are present under the probe.
After the measurement, inspect the results in the Results Viewer:
- Verify the probe contacted the dish base, not floating debris.
- If necessary, repeat the measurement.
Figure 13. Example of a sensitivity measurement.
Note: Recalculate sensitivity after laser realignment, probe exchange, or transfer to different medium.
Align Probe
Define the probe aperture position in the software to ensure accurate targeting of selected points. This alignment ensures precise targeting. This step can be performed at any time using the dedicated workflow in the Preparation section.
- Position the crosshairs over the probe aperture.
- For optimal targeting accuracy, ensure the probe is at the same focal height as the cells.
- Click ‘OK’.
Note: Realign the probe after changing the objective lens, medium, or well.
Calibrate Focus Level
Adjust focus settings to match current working conditions. Although the system is pre-calibrated, changes in probe position or optical conditions may affect focus quality.
- Switch to the highest magnification objective that can reach the probe at its current position.
- Manually bring the probe into sharp focus.
- Click ‘OK’ to save the calibrated position.