They way that droplets are charged is one of the more counterintuitive and complex aspects of cell sorting.

My own experience as a past fledgling cytometrist certainly echoes this assertion. I distinctly remember my struggle to understand how exactly all of the sorting components coalesced to accomplish the instrument’s tasks, but when I finally did, I gained a much deeper understanding of cell sorting as a holistic process rather than a vague sum of its parts.

It’s my hope here to be able to facilitate a more accurate conception of the process, grounded in a reality of physics and mechanics rather than one shrouded in magic.

Towards this end, the most crucial point is that the technique of electrostatic cell sorting, which is the most widely employed type of sorting, is fundamentally built upon droplet charging.

How Flow Cytometry Electrostatic Cell Sorting Works

Electrostatic cell sorting is a complicated process that continues to be improved.

For most electrostatic cell sorters, a stream of sheath fluid that contains a mixture of particles is interrogated by a laser and the resulting optical signal is collected and processed by optical and signal processing systems that are nearly identical to those on top analytical flow cytometers.

The fluidics are most often comprised of a sheath fluid, the first component, that runs through the system in laminar flow. The movement of this sheath can be achieved by several mechanisms, the most common method using pressure provided by pumps. The second component of the fluidics is the sample injection port. This is where the sample is pushed through to be introduced to the sheath fluid.

Based on the principles of hydrodynamic focusing, these cells become strung out, single file, in the direction of the flow, where they will pass the interrogation point. The final main component of the fluidics is the flow cell, which is where everything comes together.

Anything can be sorted as long as it’s big enough to be detected by the instrument (this depends on the instrument, but >0.5 μm is reasonable for many instruments) and small enough to fit through the nozzle tip at the end of the flow cell without clogging it or causing side stream instability (usually < 1/5 the size of the nozzle tip).

After interrogation, the stream is partitioned into identically sized droplets that are generated at an extremely fast, stable, and predictable rate.

These droplets are formed by the application of an acoustic wave, through a piezoelectric device, in the flow cell of the instrument. When a target particle is detected, a charge is applied to the droplet that contains this target particle.

All of the droplets, charged or uncharged, then pass through an electrical field that is generated by the deflection plates.

Charged particles are pulled towards the plates, away from the uncharged droplets, and fall into collection tubes, forming side streams, while uncharged droplets pass into the waste (see below figure).

How Droplets Are Charged And How A Drop Delay Is Calculated

The first key to understanding the application of the charge is that it occurs in the flow cell and is applied to the entire connected stream rather than to droplets individually.

This may seem counterintuitive—the point at which a target droplet is charged (at the break-off) is significantly downstream of where the charge is applied (at the nozzle).

However, the charging must occur at a point in the fluidic path where the stream can interact with a wire without being disturbed. The only place that this can occur is in the flow cell.

The second key to understanding the application of the charge is the calculation of the drop delay. The drop delay value, which is invariably the most critical sorting parameter, is defined as the distance in time between the laser interrogation point and the droplet break-off point.

The droplet break-off is the stable and predictable point on the stream where it begins to be partitioned into droplets. Above the break-off, the stream is continuous and connected; below it, the stream is disjunct as discrete droplets.

The drop delay determines how long the system must wait before it applies a charge once a target particle is detected.

Interestingly, the drop delay is not reported in units of seconds but rather, as units of droplet periods, which can be calculated as 1/droplet frequency. For example, a drop delay of 34.67 means that the sorter must wait 34.67 droplet cycles before it applied the charge.

The timing involved is determined by the duration that the particle spends traveling from the laser interrogation point to the droplet break-off. Calculating this timing is critical for getting adequate values of sort recovery, which is the best measure of cell sorting performance.

What Is A Flow Cytometry Cell Sorting Decision?

Once a target particle is detected at the laser, the drop delay countdown timer begins.

The particle leaves the laser interrogation point and begins traveling down the stream, approaching the break-off. Once the particle enters the break-off, a potential is applied to the continuous stream from the wire in the nozzle.

The entire connected stream is held at this potential for the duration that the target particle spends in the break-off, which is exactly one droplet period. At the exact instant that the particle leaves the break-off period and is encapsulated into a droplet, the stream is brought back to its near-zero potential.

As a result, the droplet containing the target cell has retained the potential that was just applied, but the rest of the stream is now uncharged. This process is repeated every time that a target particle is detected at the laser, whether it’s a green laser, yellow laser, violet laser, UV laser, or other flow cytometry laser, and a sort decision is made.

The story doesn’t end here. Isolated charge on broken-off droplets exerts electrostatic force on the ions (Na+ or Cl–) in the now-neutralized stream.

The appropriate ions then migrate down the stream towards the break-off and oppositely charged droplet, resulting in slight charge isolation on other droplets as they break off. To compensate for this, the instrument must apply a small amount of neutralizing charge to these droplets to ensure only target droplets are charged.

This process is termed in various ways depending on the instrument, including “Defanning” and “2nd, 3rd, and 4th drop.”

How Two-Way Cell Sorting Experiments Work

Two-way sorts can be performed by charging droplets at positive and negative potentials.

Droplets charged at positive potentials will be attracted to the deflection plate with negative potential and droplets charged at negative potential will be attracted to the deflection plate with positive potential.

Four or six-way sorts can be performed by modulating the amount of positive or negative charge on droplets. Droplets with large magnitudes will be attracted closer to the plates than droplets with lower magnitudes, forming two (or three) side streams on either side of the center stream.

An analogy may be helpful to understand this better. Imagine a very large department store with only one set of revolving doors that customers must use to enter. This portal between the outside world and inside of the store is the break-off. There is a very big promotion going on, and the customers are politely lined up outside, single-file, waiting to enter the store.

In order to ensure an orderly influx of the large number of customers who have shown up for the promotion, each customer is told to either go left or right (or given no instructions) once they enter the store so that they can interact with sales staff who are not already helping other customers.

However, they are only given these instructions as they pass through the revolving doors. As the customers enter the revolving door, they are told, “Once inside, please go to the left,” “Once inside, please go right,” or simply silence.

Those who are given instructions diverge to the left and right of the doors, and those who are not continue forward, resulting in an orderly stream of customers in three different directions inside the store.

Like these customers, droplets receive their instructions as they pass through the break-off, but are still attached to the stream, and can then act on these instructions (with the help of the deflection plates) once they are liberated as individual droplets.

By understanding how electrostatic cell sorting works, in particular how droplets are charged and how drop delays are calculated, you will be in a better position to set up a successful flow cytometry cell sorting experiment. Running a successful sorting experiment will help you achieve high sort recovery values, which will allow for accurate analysis of your cells as well as more cells to work with for your downstream experiments.

To learn more about electrostatic cell sorting, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.