The laser detection system is a fundamental component of the FluidFM OMNIUM, essential for monitoring cantilever deflection and ensuring precise force control. Achieving optimal signal alignment and intensity is critical for accurate data acquisition. This guide outlines the principles of laser signal management and provides troubleshooting steps for common issues, including signal saturation, weak reflection, and baseline drift. Whether you are diagnosing specific error messages in ARYA or seeking to maximize instrument sensitivity, this article details the necessary procedures to ensure reliable system performance.
Laser detection in the FluidFM OMNIUM
Fluid force microscopy (FluidFM) is a fusion of atomic force microscopy (AFM) with integrated microfluidics (Meister et al., Nano Lett., 2009). FluidFM combines the accurate force-controlled positioning of AFM with the versatility of fluidics. The laser's main function in AFM is detecting cantilever deflection. A laser beam (Figure 1, A) is focused on the back of the cantilever (B), and its reflection (C) is monitored by a position-sensitive photodetector (PSPD) (D). PSPD consists of two photodiodes (Figure 1D, A and B). Changes in the cantilever's position cause the laser spot to move on the detector (B-D), which is then translated into a deflection signal (E). Angular displacement of the cantilever results in one photodiode collecting more light than the other photodiode, producing an output signal (the difference between the photodiode signals normalized by their sum), which is proportional to the deflection of the cantilever.
Figure 1. Laser detection system in FluidFM.
The laser must be precisely aligned to the center of the PSPD, as well as centred on the reflective layer of the cantilever. It is important that the laser is positioned close to the cantilever tip to have the best sensitivity for controlled cantilever movements (Figure 2). Placing the laser near the tip end of the cantilever uses the lever arm effect, where a given force causes a larger angular deflection at the tip compared to the base. The farther the laser is from the pivot point (the fixed end of the cantilever), the greater the angular change for a given displacement of the tip. This results in a larger deflection signal being detected by the photodetector. The deflection sensitivity of the detectors must be calibrated in terms of how many nanometers of motion correspond to a unit of voltage measured on the detector (“Measure Sensitivity” step). The best position of the laser is around the third row of pillars from the tip of the cantilever (Figure 2, after alignment). Misalignment can lead to flawed deflection signals.
Figure 2. Laser position before and after alignment.
For best force control, the signal needs to be maximized. Maximal signal is achieved when the two photodiodes within the FluidFM Omnium receive an equal amount of laser signal and are within 30% and 90% of their range. For that, a mirror inside the head (Figure 1, C) must be brought into the best position. During the ‘Maximize Signal – Center automatically’ step, the mirror, located inside the FluidFM head, will move causing the laser reflection signal to go from one side to the other on the PSPD (Figure 3a). The intensity signal collected from both sides of the two photodetectors is plotted (Figure 3b). The intensity plot will give one side of the photodetector as indicated in the image in blue, and the other side as shown in red. The best position is where the two signals intersect.
Figure 3. a) Drawing of the laser reflection signal movement (blue stands for one photodetector, red another one). b) Laser reflection signal plot after sweeping the signal over the two photodetectors.
However, laser signal plots are not always perfect, and special attention should be paid to the intensity and the shape of the signal (Figure 4).
Figure 4. Examples of laser signals.
Positioning of the Laser and ARYA feedback
As mentioned earlier, for the best sensitivity of the system, position the laser on the top of the cantilever, around the third row of pillars. However, during the bending of the cantilever, a laser that is positioned too high can ‘fall off’ the reflective area of the cantilever, and the PSPD will not detect any signal. This is mostly seen in experiments that are run in the air, because there are strong electrostatic forces that bend the cantilever strongly towards the surface. Electrostatic forces get stronger closer to the surface, therefore the bending is more visible when cantilever is close to the surface.
Below are example images on how the laser signal position affects the maximize signal plot (Figure 5).
Figure 5. Maximize signal outcomes based on laser position in air. Green arrows show laser position.
A high signal strength implies that the photodetector can detect minor changes in the cantilever's position with high accuracy, enhancing the sensitivity of the AFM system. A well-optimized laser alignment, as shown by a maximized signal plot, ensures a high signal-to-noise (SNR) ratio. High SNR means that the system can distinguish small deflections of the cantilever from noise, improving the detection of weak interactions between the cells and the cantilever.
The laser signal can be monitored in the ARYA software via the status bar, found at the bottom of the screen (Figure 6a) or oscilloscope tool ( 
, Figure 6b). The laser signal on the status bar is displayed as the output signal (the difference between the photodiode signals normalized by their sum), as well as the signal coming from both photodetectors (Figure 6a).
The oscilloscope icon toggles an animated graph which displays the deflection and the pressure of the last few seconds.
Figure 6. a) Laser signal via the status bar in ARYA, b) oscilloscope tool in ARYA.
Right next to the graph, you can see the current values of the deflection and the pressure. The scaling of the rulers on either side can be adjusted. Per default, it will automatically scale to show all recent values. Note that the deflection signal always shows uncorrected values. Therefore, one will see a corresponding shift of the signal when applying a relative setpoint but the signal itself will not necessarily correspond to that setpoint.
The initial laser and mirror alignments are carried out in air, however the majority of the FluidFM applications take place in liquid. Due to refractive index differences (liquid > air), the laser beam endures significant refraction and distortion of the beam in liquid. It is important to keep in mind that mirror alignment needs to be repeated when entering a different medium.
Troubleshooting Laser Signal
The status bar can also display warnings on the laser signal, usually indicating a maxed-out sensor (1024k/1024k) or no signal (0/0) , shown in red.
When the laser signal is too strong, the photodetector can become saturated, so it reaches its maximum output limit and cannot accurately respond to further increases in signal intensity. Saturation causes a loss of linearity in the detector’s response, making it impossible to accurately measure small deflections of the cantilever. This leads to incorrect force measurements. To spot this issue, pay attention to the detector’s output values (Figure 7a, red rectangle). In Figure 7a, before maximizing the signal, detector values are already close to reaching saturation. After maximizing the signal, ARYA reports back no signal (Figure 7b). The maximize signal plot also shows saturation, as the intercept of the signal reaches the top of the plot.
Figure 7. Laser signal strength a) before maximizing the signal, b) after maximizing the signal.
Saturation is always achieved by having too much laser light reaching the photodetector. If the issue is caused by laser power, users probably experience this issue regularly and it is independent of the used probe type. If the issue is caused by the reflective layer of the probe, the users experience this issue only when using a specific type of probe from the same wafer.
To mitigate this issue, reduce laser signal reflecting from the cantilever by realigning the laser on a different spot. The laser can slightly ‘fall off’ the cantilever (left-right direction) or move closer to the base. Verify the result by maximizing the signal again. In case of repeated issues, contact our support (support@cytosurge.com ). Please report the frequency of the issue, dependencies of the used probes (write down also wafer serial number found on the package) and attach videos and pictures of the maximized signal plots.
A weak laser reflection signal means the photodetector isn't receiving enough light from the cantilever, making it difficult to accurately detect the cantilever's deflections. A bad signal (Figure 4) can be retrieved during the ‘Maximize Signal’ workflow step. The status bar will display (0,0) and it is likely caused by disrupted laser path, laser beam not being placed on a cantilever, faulty gold coating or wrongly angled cantilever.
The laser path can be disrupted by dirt or other contaminants landing on the laser path (Figure 8a, b). Contaminants can lie on the surface of the prism because of insufficient cleaning. Therefore, maintain the OMNIUM head regularly to ensure the laser path is free from any contaminants (dried salt/dye residues), dust and debris. If any contaminants are present on the prism, clean the head with 2% SDS solution in water using optics-compatible cleaning swabs, MilliQ water and isopropanol.
Figure 8. a) Prism contaminated by liquid droplets and b) the corresponding laser signal from a probe.
If a poor laser signal happens after changing the medium, check the position of the laser on the cantilever and for the occurrence of an air bubble. It is likely the laser has shifted off the cantilever and the problem can be eased by repositioning it back on the cantilever. Additionally, a wrongly positioned probe in the drying step of the Extraction workflow can induce its physical movement, which will likely result in poor or no laser signal, because the laser is not reflected from the cantilever anymore. By realigning the laser, this issue can be solved.
Lastly, when a dry probe is immersed in liquid, an air bubble can sometimes become trapped on its surface. This bubble often interferes with the laser signal, leading to a "no signal" or a maxed-out signal on the status bar. To resolve this issue, try re-entering the well several times to dislodge the bubble. If this doesn’t work, altering the surface tension may be necessary to burst the bubble. To do this, first immerse the probe in MilliQ water, then in a 70% ethanol (EtOH) solution, and finally return it to MilliQ water. Because ethanol can fix biomolecules, it is important to wash the cantilever with MilliQ water before entering the well and to minimize the immersion time in ethanol to about 1 second. Be careful not to aspirate any ethanol into the probe or carry it over to the cells. Keep in mind that air bubbles can form anytime a dry probe is immersed in liquid, which is particularly important to consider during the Extraction workflow.
Poor laser signal is not only caused by user-related issues, but manufacturing problems. Sometimes, the reflective layer of the cantilever might be absent (Figure 9a), contaminated, damaged or detaching from the cantilever (Figure 9b), and therefore laser signal is insufficient. To improve laser signal strength, make sure to use high-quality, new cantilevers with reflective coatings. If unused cantilevers produce poor laser signal and you have troubleshooted all potential issues mentioned above, contact our support team (support@cytosurge.com). Please report the frequency of the issue, dependencies of the used probes (write down also wafer serial number found on the package) and attach videos and pictures of the maximized signal plots.
Figure 9. Probe missing a reflective layer a) completely and b) partially (peeling off).
Laser drifts
Laser drift is a common issue that can affect the accuracy and reliability of AFM measurements. Laser drift can be broadly categorized into environmental, instrumental, and operational factors. The main source of drifts in FluidFM OMNIUM systems is due to temperature fluctuations. Liquid environments can be more susceptible to temperature changes, affecting viscosity and refraction properties, therefore, for sensitive measurements, it is good to run them in temperature-controlled systems.
- Thermal Expansion: Changes in temperature can cause thermal expansion or contraction of the AFM components, including the cantilever, sample stage, and laser optics. This can shift the position of the laser beam. It is often detected in one-directional shift in the output signal. Thermal drifts reduce over time as the temperature stabilizes.
Figure 10. Thermal expansion happening over time.
- Refractive Index Changes: Temperature variations can alter the refractive index of the air or any medium through which the laser passes, causing the beam to deviate
- Rapid changes in pressure within the FluidFM system can induce vibrations or mechanical shifts in the cantilever or surrounding components. These vibrations can affect the stability and position of the laser spot on the photodetector. Figure 11 shows shift in deflection signal (9 mV to 42 mV) after switching the pressure from 10 to 100 mbar.
Figure 11. Oscilloscope view of pressure (yellow) and deflection signal (orange line) after changing the pressure from 10 to 100 mbar.
- Crosstalk fluorescent light attenuator: Thermal drift can also occur when using fluorescent light with an open attenuator (50-100%). Figure 12 shows a shift in laser signal (orange) when closing the attenuator from 100% to 50%. The expected range for the shift is around 50 mV compared to brightfield conditions or 1.5% attenuator.
Figure 12. Oscilloscope view of deflection signal (orange line) after partially closing the attenuator (from 100% to 50%).
Software errors related to laser signal
- NO_PEAK
- Runtime errors: (Cannot convert x mV, signal too high)
- E_SQUIGGLE_ERR
The system was not able to find a suitable position to direct the laser reflection to the detector. This usually means no signal, or the maximum signal was detected. To overcome this problem, try to re-position the laser in the X-Y direction (Step 2 “Align Laser” in Preparation Advanced) on the cantilever and re-sweep the signal (Step 3 “Maximize Signal” in Preparation Advanced).
This error appears when the laser signal is too close to saturation and the requested setpoint would reach it. To address this issue, try moving the laser slightly in the X and Y directions, further away from the tip of the cantilever, or re-maximize the signal in the media you primarily work in. Additionally, using a lower setpoint that avoids reaching saturation during the approach can help mitigate this problem. We are aware of this issue in the 'Extraction' workflow and are currently working on a software solution.
During initialization of the laser or mirror positioner an error occurred. Retry the laser alignment and maximization of the signal. Contact our support (support@cytosurge.com) when encountering this error, as the support team can re-configure the system to mitigate this issue.