Non-contact wettability assessment system
We developed a non-contact wettability assessment system (Fig. 2a) based on our previous study [19] and related patent [20]. The system is equipped with a particle filter (Fig. 2a), an XYZ-three-axis motorized stage (Fig. 2b), and image processing instrumentation to allow detection of the liquid squeezed area automatically. The particle filter is readily available commercially as an item for clean rooms. Because various types of motorized stages are provided by several companies, we selected an appropriate type for the system from the viewpoints of position accuracy, size of the system and samples to be analyzed. The main system is W400 × L300 × H250 mm, and it can be installed on a standard clean bench. We also use a PC for system control and a compressed-gas source, such as an air compressor and a pressurized gas cylinder.
Assessment of particle contamination
First, we counted the particles in air flow from an oil-free air compressor (Fig. 3a) or non-contact wettability assessment system (Fig. 3b). The air flow from the compressor contained a large number of micro-particles: 1.8 × 104, 1.0 × 104, and 7.2 × 103 particles in a cubic foot with diameters of 0.3, 0.5, and 1.0 μm, respectively (Fig. 3c). These values were still smaller than that in a typical laboratory room, which reaches particle numbers of 105 to 106 particles (with a diameter of 0.3 μm) in a cubic foot. By installing the particle filter before the solenoid valve, the particles in the air-jet flow from the air nozzle were captured and removed (Fig. 3d). Therefore, filtration of the compressed air was quite useful for the risk reduction of particle contamination and necessary to achieve class 5 level in the ISO 14644-1 cleanroom standards, which should be met to perform therapeutic cell culturing.
The microorganism test was also performed with agar culture plates (Fig. 3e). A colony was observed on the Sabouraud agar plate, which was exposed to the air directly from the air compressor and incubated. On the other hand, no colonies were observed on both trypticase soy and Sabouraud agar plates in the case of air-jet application from the air nozzle. Because trypticase soy and Sabouraud agar plates are suitable for cultivation of bacteria and fungi, respectively, the colony on the Sabouraud agar plate exposed to compressed air might consist of fungi derived from inside the air compressor. The result indicated that the air-jet from the non-contact wettability assessment system never contained bacteria and fungi. Therefore, the particles including microorganisms derived from the air compressor were effectively suppressed by insertion of the particle filter into the air supply line.
Advantages obtained by using robotic technology and image processing instrumentation
The main manual operation in the non-contact wettability assessment is the adjustment of measurement position, because the operator wants to make assessments on various points of the sample surface. The three-axis motorized stage enables system users to avoid the manual handling of target samples. During continuous movement of the three-axis electric stage for 1Â min, no particles were detected. Although mechanical movement elements such as screws, bearings, and motors could be a particle source, the components of this system and the movement condition never generated particles.
To avoid the need for human operators to approach the measurement area, a digital camera and image processing instrumentation are included in the system. Obviously, these elements are not particle source, and the problem of contaminating particles derived from human operators was well eliminated. Furthermore, remote observations were implemented using software for the image instrumentation. This software detects the liquid squeezed area and measures its diameter. By automating this work, the time needed for analysis is shortened. In our previous study [19], human operators had to analyze huge numbers of recorded images to measure the diameter of the liquid squeezed area. But the simple algorithms of the image processing (Fig. 4a), allow the liquid squeezed area to be detected from just one monitored image (Fig. 4b–d; Additional file 3: Movie 1).
The performance of the image processing instrumentation was evaluated using circle patterns with known diameter values that had been printed on a sheet of paper (Fig. 4e–g). The diameter values of the actual and detected circles coincided within the worst coefficient of variation being 0.05% (=SD/mean) (Fig. 4h).
Comparison with contact angle method
To confirm the usability of our developed system, we compared the two indexes, liquid-squeezed diameter during air-jet application and the liquid recovery time after ceasing the air-jet application, with the conventional contact angle measurement on the same surfaces (Fig. 5). To obtain surfaces with various wettability values, polystyrene petri dishes were treated with atmospheric pressure nitrogen-plasma for 0, 3, 6, 12, 18, 30, and 60 s. With the extension of treatment time, the contact angle was decreased, indicating that the wettability was improved (Fig. 5a). Both liquid-squeezed diameter and liquid recovery time were also measured on these surfaces with the developed system and the relationship to the contact angle was investigated. The line expressing the relationship between the liquid-squeezed diameter and contact angle had a positive slope (Fig. 5b) with a small squeezed diameter at a small contact angle and a large diameter at a large angle, as observed earlier in Fig. 1c–f. Interestingly, the increase of the liquid-squeezed diameter had two different slopes with the turning point being around 72° for the contact angle; that is, the slope for the contact angle below 72° (Fig. 5c) was quite a bit gentler than that over 72° (Fig. 5d). In each region (Fig. 5c, d), the relationship seemed to be almost linear and the linear approximation lines well fit the data points with a coefficient of determination R
2 over 0.9. Unfortunately, we could not identify the mechanism causing the slope change; however, we could confirm assessment usability between the liquid-squeezed diameter and contact angle, which is the gold standard among wettability indexes, but contact angle measurement cannot be applied to cell surfaces.
Through this experiment, we investigated the relationship between the liquid recovery dynamics after ceasing the air-jet application and the contact angle (Fig. 5e). In the case of the surface with a contact angle over 70.7°, the liquid never recovered the surface and the liquid squeezed area caused by the air-jet application remained. On the other hand, the surface with a contact angle below 65.6° was always recovered by the liquid. Furthermore, whenever the liquid recovery occurred, the liquid recovery time increased with the contact angle. And this result suggested that the liquid recovery time could be used as an index of wettability, especially for high wettability surfaces with small values of the contact angle. Supposing the relationship was modelled as linear, we saw the approximation line provided a good estimation of contact angle from the liquid recovery time with the coefficient of determination R
2 over 0.8.
The coefficient of variation (CV), which was defined as the standard deviation per mean value, of each wettability index was calculated (Fig. 5f). On a low wettability surface with plasma treatment time from 0 to 12 s, the contact angle was the best index among the three indexes. On the other hand, the CV value of liquid-squeezed diameter was the best index for a higher wettability surface with 18-s or more treatment time. Therefore, the air-jet based liquid squeezing method implemented in our non-contact wettability assessment system would be the most suitable for the in-liquid wettability assessment of high wettability surfaces. The liquid recovery time had 4–30 times larger CV values compared with those of contact angle and liquid-squeezed diameter. Because the liquid recovery is a quite fast behavior, the measurement system with more frequent sampling, which exceeds the current system with a normal video rate of 30 Hz, is required for accurate evaluation.
We found the contact angle measured in the gas phase was strongly dependent on ambient humidity and the trend for the dependency varied with the surface materials (Additional file 1: Figure S1). Therefore, the ambient humidity must be controlled for accurate measurement of contact angle. On the other hand, this non-contact wettability assessment system is free from the effect of ambient humidity, because it is an in-liquid method, although the proposed method cannot be applicable to the wettability assessment in dry condition.
Influence on cultivated cells during wettability assessment
To investigate physical and biochemical influences during wettability assessment, we cultivated mouse skeletal myoblast cells on cell culture dishes (Fig. 6a).
First, mechanical strength was assessed by observing peeling off of cultivated cells from the dish during air-jet application. However, cells were never peeled from the dish and the cellular shape was seemingly never changed before and after the air-jet application of 10 kPa pressure (Fig. 6b, c). When a gentler air-jet is required to reduce the pressure on the surface, not only the regulation of gas pressure but also the nozzle height from the surface is effective. From the spatial distribution of pressure (Additional file 2: Figure S2), the maximum pressure, which was caused at the point just under the nozzle center, can be suppressed by controlling the nozzle height.
Next, to investigate biochemical influences, glycometabolism and damage to the cytomembrane were assessed for the 10 kPa air-jet application, which never caused the peeling off of cells. The condition without the air-jet application was defined as the control. Generally, cells consume glucose and convert it into lactate in glycometabolism. Therefore, the concentration changes of both glucose and lactate in culture media were quantified during a 1-day culturing after the air-jet application (Fig. 6d, e). As a result, we saw no differences in the glucose consumptions and lactate productions between the control and air-jet application conditions. The released concentrations of lactate dehydrogenase (LDH) were also quantified to assess damage to the cytomembrane, because LDH is normally located in the cytoplasm and released through a damaged cytomembrane (Fig. 6f). The LDH release from cells showed no significant difference between the control and air-jet application conditions. Therefore, these results suggested that there were no influences on the cells during the wettability assessment in comparison with the control condition.
Wettability assessment
Here, we assessed the wettability of cultivated mouse skeletal myoblasts with air-jet based liquid squeezing. The behavior of the liquid squeezing and liquid recovery could be monitored by the developed system (Fig. 6g; Additional file 2) and we analyzed the image data to obtain the time-courses of squeezed diameter from the image instrumentation (Fig. 6h). The time-courses of three individual measurements were in almost complete agreement. As compared with the squeezed diameter using the normal polystyrene dish during air-jet application (Fig. 4b; Additional file 3: Movie 1), the diameter of the squeezed culture medium area was almost the same as that when using the dish. On the other hand, although the squeezed area remained in the case of the polystyrene dish after the air-jet cut-off, the culture medium fully recovered the cell culture surface (Additional file 3: Movie 1, Additional file 4: Movie 2). Therefore, these results suggested that hysteresis between liquid-squeezing and rewetting was also important information about surface wetting.