Picture this: there are 500 numbered balls in front of you. If you were asked to pick out the even-numbered balls in one second, how would you do so? To further complicate this, what if the balls were only 0.01mm in size, which is smaller than the width of a hair? How would you even begin to identify and sort these balls? Sounds like a trick question, doesn’t it? The reality is, biologists have to deal with the same problem all the time. Only they’re not handling balls, they’re handling cells. Each of our organs are composed of many different types of cells. What if we are only interested in studying one type? The first step is to dissociate an organ into individual cells and “pick” the cells you like. The method to solve this problem was invented in the late 1960s [1], when biologists came up with an elegant solution to this riddle: flow cytometry.

Before we go into the mechanism of flow cytometry, we first have to understand the targets in question, namely the cells. Similar to how different people have unique identifying features, different cell types also have unique characteristics. The simplest identifying features of a cell are its relative size and complexity: some cells are larger than others and some cells have higher internal complexity (that is, having more cellular components like organelles or granules). Other than cell size and complexity, we can also identify cells by proteins that are preferentially produced by the cells. These proteins can either be positioned on the cell surface or sometimes contained within the cell’s cytoplasm, like a transgenic fluorescent marker. With this in mind, as long as we are able to identify these features, we should be able to identify the cell type. For example, we can distinguish the two major types of mature T cells, helper T cells and killer T cells (a.k.a. CD4+ cells and CD8+ cells respectively), by figuring out which surface protein, CD4 or CD8, is expressed on the T cell (footnote 1).

The next question, of course, is how can we identify these cellular features? Size and complexity can be measured with lasers (more on that later), but unique protein markers are harder to identify. The solution to this comes from antibodies, which are proteins that can bind specifically to certain molecules. These antibodies can be selected such that they only bind proteins that are specific to certain cell types. These engineered antibodies are then joined (or conjugated) with unique fluorophores, which are chemical compounds that re-emits fluorescent signals at a longer wavelength upon excitation by a laser. Scientists can thus “label” the cells using these fluorophore-conjugated antibodies before the application of flow cytometry to identify the cell type. Using the example of mature T cells again, an engineered antibody-fluorophore conjugate, which can bind CD8 proteins on cell surfaces, will be able to stain CD8+ cells (killer T cells). Upon laser excitation, the fluorophore will give a unique fluorescent signal, differentiating CD8+ cells from other cell types.

As discussed, scientists are able to identify specific cell types thanks to unique markers on their cell surface or inside their cytoplasm. With this background knowledge in mind, we can now get into the procedure of flow cytometry. First, the biological sample in the form of a single-cell suspension (i.e. free-floating single cells in liquid medium; connected cells in a tissue need to be dissociated first) is loaded into the flow cytometer. The suspension is then focused into a single stream of liquid, like how water comes out of a tap in a single stream, whereupon cells will be arranged into a single file before passing through an array of lasers one by one. After that, an accompanying array of sensors will detect any signals that are subsequently generated. The photons in the laser beam will be able to pass through the liquid, in which the cells are suspended in, unobstructed but photons that encounter cells in the stream will be forced to divert from their original trajectory. Such divergence of light is known as scattering. Forward scatter, which measures the amount of light diffracted slightly due to contact with the cell membrane, can be used to give a measurement of the cell’s relative size; the larger the cell, the more forward scatter there is [2, 3]. On the other hand, side scatter, which measures the amount of the light reflected at a greater angle upon contact with internal organelles or granules inside the cell, can give a measurement of the cell’s relative internal complexity; the more objects there are inside the cell, the more side scatter there is [2, 3]. In addition, the lasers serve to excite any fluorophores conjugated to antibodies, allowing the labeled cells to be detected. With that, cells can be identified efficiently.

Besides identification, an extended function of flow cytometry is cell sorting. This is enabled by vibrating the stream of cells in the cytometer, causing the stream to break off into droplets containing mostly single cells. Then, the cell-containing droplets are each given different electrical charges according to their characteristics we tested before, such as the strength of fluorescence. The droplets with different charges will be deflected and sorted into different receptacles in the fluorescence-activated cell sorting (FACS) machine, so selected populations of cells, for example, successfully transformed cells that express the fluorescent marker, or tumor cells or white blood cells (including B cells and T cells) that express a specific surface protein marker [4], can be retained for analysis or further experiments.

Before the invention of flow cytometry, the accurate identification of specific cells from a diverse pool was almost impossible, let alone its isolation. Now, flow cytometry has become a common, yet indispensable technology in basic and clinical laboratories.

1 The HIV virus attacks CD4+ T cells.

References:

[1] Herzenberg, L. A., Parks, D., Sahaf, B., Perez, O., Roederer, M., & Herzenberg, L. A. (2002). The History and Future of the Fluorescence Activated Cell Sorter and Flow Cytometry: A View from Stanford. Clinical Chemistry, 48(10), 1819–1827.

[2] Adams, D. (n.d.). LESSON 1: FLOW CYTOMETRY SIGNALS (SCATTER AND FLUORESCENT). Retrieved from https://brcf.medicine.umich.edu/cores/flow-cytometry/training-education/lessons/lesson-one-flow-cytometry-signals-scatter-and-fluorescent/

[3] Bakke, A. C. (2001). The Principles of Flow Cytometry. Laboratory Medicine, 32(4), 207­–211.

[4] McKinnon K. M. (2018). Flow Cytometry: An Overview. Current protocols in immunology, 120, 5.1.1–5.1.11. doi:10.1002/cpim.40