The fluidics system of a flow cytometer is responsible for transporting sample from the sample tube to the flow cell. Once through the flow cell (and past the laser and detector), the sample is transported to waste.

## Importance of focusing the sample stream (hydrodynamic focusing)

Prior to the sample entering the flow cell (also called flow chamber), the cells or particles are moving in a random and disorganized manner within the fluid suspension (Figure 1A). If you map the distribution of same-sized cells in terms of their movement in a cylindrical column of liquid, it would look like a hyperbola (Figure 1B) in that some of the cells would be moving faster than normal and some will be moving slower than normal. Additionally the cells are not guaranteed to be moving in a single file at any given time. The end results of these two phenomena are that (1) cells traveling at different speeds introduce variability in the data and (2) cells that are in too close proximity to each other result in coincident events (two cells being analyzed at the same time instead of separately)being collected (Figure 2A). Accurate data rely on a fluidics system that ensures that all of the particles within the sample are focused as single cells and that they are moving at the same speed before they reach the interrogation point.

Figure 1. Schematic of particle movement. (A) Individual particles in an unfocused system travel in a chaotic way. (B) If you plot the general direction of those same particles now traveling in liquid in a cylindrical tube they appear to have an overall hyperbolic velocity (some particles will be moving faster and some particles will be moving slower).

The flow cytometer fluidics system addresses these problems by utilizing hydrodynamic focusing. In other words, the system uses liquid (hydro) to force the cells to travel at the same speed and to travel as single cells. In hydrodynamic focusing (Figure 2B), a faster-moving sheath fluid (which is simply a saline solution)is used to force the sample into a smaller core stream (also called hydrodynamic core) so that all particles are travelling along the same axis at approximately the same velocity. The core stream is maintained by ensuring laminar flow, which is characterized by layers moving parallel to each other in a stream without mixing. Once focused into a tighter core stream, the sample will travel through the interrogation point before either going to waste (analysis instruments) or being sorted (cell sorters).

Figure 2. Hydrodynamic focusing.(A) Before hydrodynamic focusing is applied, cells are unfocused, traveling at different speeds and very close to one another when they reach the interrogation point. This can result in multiple cells arriving at the interrogation point at the same time, leading to detection of multiple events at the same time (also called coincident events). (B) Addition of the sheath fluid focuses the cells into the core stream causing the cells to travel at the same speed and arrive at the interrogation point as cells in a single file line. Collection of coincident events is minimized.

## Impact of changing the pressure

The pressure of the sheath fluid sets the speed of the system, so if you want to change the event rate—that is, the number of cells or particles passing the interrogation point in a given period—you will have to change the differential pressure between the sample and sheath fluid. It can be tempting to increase the pressure, which increases the event rate so that you can get your data faster, but if you do, there may be multiple consequences. As demonstrated in Figure 3A, the low differential pressure allows the cells to move past the interrogation point one at a time. Increasing the pressure (Figure 3B) causes the core stream to widen, allowing more cells to pass through the flow cell. This may mean, however, that two or more cells can be in the interrogation point at the same time. These coincident events generate data anomalies including increased variability, which can be seen in Figure 3C. A plot of forward scatter versus time where the pressure is increased at different time points shows the increased data spread as more events are captured at the same time.

Figure 3. Impact of increasing pressure on flow cytometry data.(A) At low pressure, the cells travel through the interrogation point one at a time. (B) Increasing the pressure increases the width of the core stream and the rate of the cells flowing past the interrogation point. This causes more than one cell to pass by the laser at a given time which results in collection of coincident events. (C) In this example, particles were run first at low pressure, but then the pressure was increased to medium and finally to high pressure. Note that the number of forward scatter events increased as the pressure increased, indicating more cells were collected at the higher pressure. The spread of the data also increased, which indicates more variability in signal collection at the higher pressure.

Your best bet for generating the most accurate data is to run the cells at a low concentration and as s l o w l y as possible. A slow rate will maintain a tight core stream, which will minimize coincident events and data spread and provide the optimal precision of signal. This practice is especially important when performing rare-event analysis and DNA content cell cycle analysis, or particularly sensitive measurements. If the experiment is aimed at getting at a quick yes or no answer, such as seeing if a transfection or some enrichment worked, and you are not worried about coincident events or the spread of the data, you can run the instrument faster. In addition, if you want to compare results from several samples, ensure that you run them all at the same flow rate. Don’t change flow rates between tubes, as the difference in data spread may lead to incorrect conclusions about the data.

## Acoustic-assisted hydrodynamic focusing

Acoustic-assisted hydrodynamic focusing is a recent development in the field of cytometry that minimizes the impact of higher pressure and higher flow rates on the ability to collect single cell data. With this technology, the cells are focused using a combination of hydrodynamic forces and a device that produces sound waves to align the cells. This allows the cells to remain confined to a narrow region during increased flow rates, increasing data fidelity and decreasing the amount of data spread associated with increased flow rates in traditional cytometers (Figure 4).

There is only one commercially available line of flow cytometers that use acoustic-assisted hydrodynamic focusing at this time (the Invitrogen Attune CytPix and the Attune NxT Flow Cytometers). At low flow rates (10–20 µL/min) the Attune Flow Cytometers use hydrodynamic focusing to align the cells, just as a traditional flow cytometer does. However, at higher flow rates (100–1,000 µL/min) the instruments use sound waves to focus the cells into a single cell flow (Figure 4). Acoustic focusing is doubly useful for cellular imaging on the Attune CytPix instrument, helping to position the cells precisely so that a sharp image is obtained. The videos below show how the cells are aligned into a single cell flow by the application of the sound waves (Figure 5).

Figure 4. Comparison of traditional and acoustic-assisted hydrodynamic focusing at higher flow rates. (A) With traditional hydrodynamic focusing, increasing the flow rate (and pressure) allows more than one cell at a time to pass through the interrogation point. (B) In acoustic-assisted hydrodynamic focusing, the sound waves force the cells to remain aligned and pass through the interrogation point one at a time.

### A

Figure 5. Principle of acoustic-assisted hydrodynamic focusing. (A) This video demonstrates the event alignment that occurs when acoustic focusing is applied. (B) View of the end of the capillary with cells run at 500 µL/min.

## Differential pressure systems versus peristaltic pump systems

Flow cytometry fluidics systems typically follow one of two designs. One kind involves generating pressure using a pump and regulator system (sometimes called a differential pressure system). A typical layout of a differential pressure system is shown in Figure 6. The differential pressure is set with a variable regulator, which applies pressure to the sample tube, pushing the fluids into the flow cytometer. For a variety of reasons, the volume delivery control on these instruments is not absolute. In other words, the volume of sample injected will be consistent each time within the same instrument, but you will not know the exact or absolute sample volume that was injected. This is not a problem unless you want to determine the absolute concentration of the sample you are analyzing since

Cell concentration = ((number of cells)/(volume of the sample))

If you’d like to count cells that have been phenotypically defined by your flow experiment, you’d have to add a defined number of counting particles (called absolute counting beads) to the sample in order to be able to calculate this information.

Figure 6. Differential pressure based fluidic system. With two controls (sheath pressure and sample pressure) the flow rate of the sample can be adjusted.

Several new cytometers integrate the second system that uses peristaltic and/or syringe pumps to deliver the sample into the instrument. A typical layout of this type of instrument is shown in Figure 7. Since the control of these pumps is more precise than the fluid delivery system in differential pressure flow cytometers, they can be reliably used to generate absolute cell counts from which the concentration of a particular cell type in your sample can be derived.

Figure 7. Schematic of a peristaltic, syringe-pump fluidic system.

## Summary

The fluidics system is the lifeblood of the flow cytometer. It is responsible for aligning the cells in single file in the core stream, and passing them through the interrogation point for data collection. Without consistent sample introduction and correct cell alignment, the data spread will be large and your confidence in the data will be reduced. Flow cytometers use hydrodynamic focusing or acoustic-assisted hydrodynamic focusing to control the flow of the cells through the interrogation point, allowing more precise data collection.

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