Principles of Cell Sorting and Tiny Flow Cytometers

Honey I Shrunk the Kids, Walt Disney Pictures.
Many of our readers should be familiar with the larger cell sorters that may be available for use at your local flow core. Some of these instruments are so big and expensive that most of us will not have even been allowed to touch one, and instead are required to pay a core facility staff to run our samples for us. Though FACS (fluorescence-activated cell sorting) is a powerful system that will not be going away any time soon, there are important limitations to many common cell sorting instruments, including:
 
1. They are expensive (both to purchase and to maintain).
2. They take up a huge amount of space (footprint).
3. For biohazardous samples, they generate large amounts of biohazardous waste.

Though there are many instruments available today that get around some of these limitations, there is still room for innovation. A collaborative group is working to do just that, and recently published some progress into their endeavor. But first, let's talk about how FACS works.

Futurama, Fox.
In most FACS instruments, cells pass through a fluidic system that hydrodynamically focus the cells so that the cells pass through an interrogation point one cell at a time. Think of this as a giant funnel that is eventually small enough that only one cell has enough space to pass through, creating a single file of cells to pass through the interrogation point. At the interrogation point, the stream passes through lasers of different wavelengths. The laser light may be meant to be scattered, as in forward and side scatter parameters, or it may be meant to excite a fluorophore on or in the cell, which then emits light at a different wavelength. The light is then filtered and collected by a detector which quantifies the intensity of the light into the familiar histograms or dot plots that you use for analysis. This is the basis behind all of the flow cytometers that can be used for immunophenotyping.

So how does a cell sorter separate certain cells into different tubes? Well, just after the point of interrogation, these FACS instruments have a nozzle with a vibrating mechanism that separates cells into individual droplets of water. The information collected from the detectors is used to assign a charge to each individual droplet, and as it passes through an electrostatic deflection system, the charge will gravitate towards the opposite charged pole, falling into a container of similarly sorted cells. For example, you may want to sort out cells that are above a certain size. You can direct the instrument to assign cells a positive charge based on forward scatter above a certain threshold, and these cells would be attracted to the negative pole of the electric field, while all other cells are assigned a negative charge and fall to the oppositely charged side. There are many different flow cytometers on the market today and some operate under different principles. However, the goal of identifying a cell based on a specific parameter (like size or fluorescence), and separating it from other cells, is the fundamental principle of these instruments.

Wikimedia Commons, courtesy of CFCF.
Exactly how is this tried-and-true technique being improved? Teams consisting of investigators from MIT, Penn State, Carnegie Mellon, and the NIH have been working on a cell sorting instrument that uses a different technique, acoustic cell sorting, which uses sound waves instead of scattered light to differentiate between cells. Sound waves are generated on two sides of a single channel, and the points at which the opposite waves meet create waves in a fixed position. These waves are called pressure nodes, and cells respond to bumping into a pressure node by getting pushed at a distance proportional to their size. Thus, cells can be sorted on the basis of size by some important fine tuning of the waves.

Via Media College.
The prototype for this type of cell sorter was originally presented in 2014, and at the time, the device could only sort 1-2 µL of a cell suspension per minute. Meaning, 100 hours to sort 100 µL of sample. Obviously, this was a huge limitation of the technology, though the team stressed that the primary advantage, and the rationale for developing the technology, was to minimize the amount of mechanical and chemical stress placed on a cell, allowing it to be minimally processed for downstream applications. You see, instead of a relatively harsh vibrations that may cause cell shearing, the pressure nodes are much gentler and cells are essentially rolling along without encountering any mechanical force. Additionally, the cells do not require fluorescent tags and thus antibody binding to an important receptor is not required. You’ll find that certain flow cytometers are already on the market that may use acoustic cell sorting in combination with the typical optical system.
In a paper recently published in Lab on a Chip by a group at Penn State (in collaboration with the NIH), they not only increased the speed of sorting to over 3,300 events per second (with a potential scalability to 13,000 events per second), but this acoustic cell sorter is also built upon lab-on-a-chip technology and is no larger than a cell phone. Incorporating FACS into the acoustic system by adding optics and optical detectors, the group estimates the sorter will still be no larger than a textbook (a far cry from the huge tabletop cell sorters available today). It remains to be seen what kinds of limitations this instrument will have or how practical it will be for typical use, but it seems like the acoustic FACS instrument may be a safer, smaller, and less expensive improvement on today's current sorters, at least in due time. Heard of any other exciting new technologies? Contact tech@biolegend.com and tell us about it!

Zoolander, Paramount Pictures.
References:
  1. Acoustic Cell Sorting
  2. A high-throughput acoustic cell sorter, Lab on a Chip
  3. Cell Sorter Shrinks to the Size of a Cell Phone
Contributed by Ed Chen, PhD.
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