ZEISS Microscopy
ZEISS Microscopy
Advanced Microfluidics Flow Analysis Made Easy
In this series "From Image to Results", explore various case studies explaining how to reach results from your demanding samples and acquired images in an efficient way. For each case study, we highlight different samples, imaging systems, and research questions.
In this case study, we demonstrate how to measure flow speed in µm/s and flow angle at specific positions within a microfluidic flow channel. Spatial information collected with ZEISS Airyscan can determine flow measurements that provide unique data related to microfluidics with ZEISS Dynamics Profiler. The flow speed and direction of active movement can be determined for defined spots across a microfluidics channel. A flow profile can be generated and laminar flow within the channel can be characterized. This will, for example, enable more accurate measurements related to the conditions for cells, organoids, or cultured tissues exposed to flow in microfluidic systems, including organ-on-a-chip set-ups.
Experimental Overview
Sample
|
Rhodamine 110 solution pumped through microfluidic flow cell (Fluidic138 chip from Chipshop, channel dimensions: 1000 µm width, 200 µm depth, 55.8 mm length) |
---|---|
Pump |
Fluigent Aria: Automated Sequential Injection System |
Imaging System |
ZEISS LSM 900 with Airyscan 2 (Axio Observer 7) and a 63x/1.2 W FCS objective |
Software |
ZEN 3.8 with ZEISS Dynamics Profiler |
Task |
Flow angle measurements and flow speed measurements in µm/s |
Introduction
Organ-on-a-chip systems are microfluidic cell culture systems with controlled conditions that are designed to replicate the complex environment and cell context of human organs in vitro (Leung et al., 2022; Figure 1). They have great potential to study organ function in vitro, establish disease modeling experiments, and offer insightful experimental designs for drug discovery and personalized medicine. Consequently, they are becoming increasingly popular. Various parameters can be controlled in such a system, including temperature and mechanical forces. Additionally, flow is an important aspect in such systems as it helps to mimic human physiological environments. The flow of media over cells removes waste and brings essential nutrients, or it might deliver drug substances. In addition, flow can be used to stimulate shear stress that can be critical for the behavior of endothelial cells or might influence stem cell differentiation (Campinho et al., 2020).
To create a system that most closely mimics the organ’s physiological environment, flow rates and pressure are monitored by microfluidic devices. Although the flow rate can be defined by microfluidics pumps very precisely in such systems, the actual flow in the system at a specific location, such as near specific cells of interest, remains unknown. Here, we use ZEISS Dynamics Profiler, which offers an experimental imaging approach, to measure speed and direction of flow for defined spots within a microfluidics channel and allows the comparison of different locations inside such a channel. This provides a flow profile of the chamber and detects heterogenous profiles created by the channel itself in the absence of cells. This application note demonstrates technical possibilities for flow rate measurements within microfluidic systems and could be expanded to include measurements proximal and distant from cells of interest, and further applied and tested in organ-on-a chip systems or other microfluidic-based experiments.
Material and Methods
Three sets of experiments were performed: 1) varying flow rates, 2) varying positions along the y-axis, and 3) varying positions along the z-axis. The Automated Sequential Injection System Aria by Fluigent was used to pump a rhodamine 110 solution through a microfluidic flow cell (1000 µm width, 200 µm depth, 55.8 mm length) (Figure 2). Three sets of ZEISS Dynamics Profiler measurements were performed with the ZEISS LSM 900 laser scanning confocal with Airyscan 2 detector using a 63x/1.2 W FCS objective. The reference image helped to position the spots and to orient within the sample (Figure 3).
The first data set was acquired to compare flow speed and flow angle at varying flow rates. For this, flow speed and flow angle were measured for two spots within the microfluidics channel in three consecutive measurements, with increasing flow rates each (40 µl/min, 160 µl/min, 320 µl/min). Spot positions inside the channel were not changed in-between measurements. The second data set was acquired (at 200 µl/min) to compare flow speed and angle at different positions across the channel along the y-axis (channel width). For this, eight spots were positioned; the stage moved twice by 50 µm to obtain the second and third set of eight measurement points. The “reuse Spot” option of ZEISS Dynamics Profiler facilitated spot positioning and ensured spacing between spots remained the same between the first, second, and third measurements. The third and last data set was acquired (at 230 µl/min) to compare flow speed and angle at different positions across the channel along the z-axis (channel height). For this, several rounds of 2 spot measurements were performed, starting at the bottom of the channel. In between the rounds, the stage was first moved up by 5 µm, and afterwards in 15 µm steps up to a total of 90 µm into the channel.
Flow Analysis
ZEISS Dynamics Profiler uses fluorescence correlation spectroscopy (FCS) methods in combination with the Airyscan area detector to provide new information about dynamic processes. For an introduction to FCS-based measurements with ZEISS Dynamics Profiler, please see
Spatial information collected with ZEISS Airyscan enables flow measurements that provide unique data related to microfluidics. Detector pairs are used for the cross-correlations along three different axes for the analysis. Each axis comprises nine bi-directional cross-correlations (Figure 4A). The cross-correlation curves and their respective fits along three different axes are calculated (Figure 4B). If there is flow along one of the axes, it results in a shift to the right of the correlation curve. In the example here, there is a shift in one of the cross-correlation curves of an axis-pair for 60°-240° and 120°-300° and there is no clear curve shift along the 0°-180° axis. This indicates that there is flow along two axes (60°-240° and 120°-300°), but no flow detected along one axis (0°-180). The results of all three axes determine the overall flow angle and flow speed in µm/s. In this example, the resulting flow angle calculated from the cross-correlations along the three axes is 86 ° (Figure 4C). The described flow analysis was performed within the ZEISS Dynamics Profiler environment in ZEN with easy access to flow visuals and tables with all metrics.
Results
1) Varying flow rate
The first experiment was setup to confirm that measured flow speed correlates with the actual flow rate applied by the microfluidic system. Flow speed and flow angle were measured for two spots within the microfluidics channel in three consecutive measurements with increasing flow rates. The cross-correlation curves and their fits (not shown) along three different axes were calculated (Figure 5A). The shift to the right of one of the correlation curves of one axis pair indicates flow along this axis (here 60°-240° and 120°-300°). There is no clear curve shift along the 0°-180° axis. These shifts become more obvious the higher the flow rate. Higher flow speeds result in steeper cross-correlation curves with an overall shift to the left. The resulting flow speeds of the two spots with the same flow rate show minimal speed differences (Figure 5C). Average flow speed at 40 µl/min is ~4000 µm/s. Quadrupled flow rate of 160 µl/min results in a measured flow speed of around 18,000 µm/s, which corresponds to 4.5x the previous flow speed. Doubling the flow rate once more to 320 µl/min resulted in a flow speed of around 40,000 µm/s, 2.2x the flow speed as before. As expected, there is a linear correlation between the flow rate and flow speed. The average flow angle from all measurements was 84.8°. The resulting flow angle of the two spots with the same flow rate show minimal differences (Figure 5D). In summary, varying the flow rate resulted in a linear change of flow speed but had no influence on the flow angle, as expected.
2) Varying positions along the y-axis
We next addressed how changing the spot position influences flow angle and flow speed. For this we changed the spot position along the y-axis (channel width) while keeping x and z constant. Flow rate was also set constant. Eight spots were defined per measurement group (Figure 6A) and the stage was shifted twice in y without further changing spot positions. The total of 24 spots covered 145 µm of the channel width, starting at the border. The measured flow speed ranged from about 1000 µm/s at the border to about 19,000 µm/s close to the middle of the channel (Figure 6B, 6C).
The flow speed confidence intervals were small for all spots. The further away from the border, the higher the measured flow speed for a spot. This corresponds to a laminar flow profile present in the channel, where the maximum speed is found in the middle and the lowest speed at the border of the channel. The median flow angle from all measurements was 82.8° (Figure 6D). The five spots closest to the border show the biggest angle difference compared to the average. These differences could be due to small turbulences that the liquid experiences at the channel border. The channel wall might also influence the imaging and thus the measurements. In summary, laminar flow like behavior could be detected with ZEISS Dynamics Profiler along the y-axis of the channel, with lower flow speed at the border and increasing speeds towards the middle.
3) Varying positions along the z-axis
Similar to the set-up along the y-axis, we also investigated how changing positions along the z-axis influences flow speed and angle. Two spots were defined per measurement group and the stage was shifted in z for seven consecutive measurements, while keeping x, y, and the flow rate constant (Figure 7A). The measurements covered 90 µm of the channel height, starting close to the bottom. The measured flow speed ranged from about 5,000 µm/s close to the bottom to 28,000 µm/s close to the middle of the channel (Figure 7B). The flow speed confidence intervals were small for all spots and very little variation was observed between the two spots per plane. The flow speed for the spots increased with increasing z-level into the channel. Some small differences in flow angle were observed (Figure 7C). In summary, laminar flow like behavior could also be detected with ZEISS Dynamics Profiler along the z-axis of the channel, with lower flow speed at the bottom and increasing speeds towards the middle.
Summary and Conclusion
With this experimental set-up, we demonstrate that flow speed and direction can be determined for defined spots in a volume of a microfluidics channel, generating a flow profile. A characterization of flow in 3D is thus possible. Flow speed increased from the border towards the middle and from the bottom towards the middle of the channel, which indicates laminar flow present within such a channel (Figure 7). This application showcases how ZEISS Dynamics Profiler can be used for flow cell characterizations. Such characterizations can be used to better study and document the conditions for cells in such microfluidics systems, such as organ-on-a-chip set-ups. This more technical approach will need further validation with cells in such a system. But measurements of that kind have great potential, first, to help optimize the microfluidic system used in an experiment and, second, provide new possibilities to answer biological questions concerning flow in such systems. In addition to flow, concentration, and diffusion of labeled proteins or drugs can also be easily measured with the help of ZEISS Dynamics Profiler.