Flow cytometry

Flow cytometry is a method of measuring the cells, or other particles, in a fluid as they pass one at a time through a focused beam of light. Each particle scatters some of the light and may emit fluorescence from dyes or labeled antibodies attached to it, and detectors record both for every particle. An instrument measures thousands of cells a second and stores a set of values for each, so a sample of a million cells becomes a table of a million rows. Some instruments also sort, diverting chosen cells into a tube. The technique is the optical relative of the Coulter counter, which measures each particle as a dip in an electric current, and the first cell sorter was built around a Coulter aperture.

In the instrument, cells suspended in fluid are injected into the center of a faster stream of particle-free sheath fluid in a narrow channel. Sheath fluid squeezes the sample stream to a width of about one cell, so that the cells cross the beam in single file. The method of hydrodynamic focusing was described by Crosland-Taylor in 1953 as a device for counting small particles suspended in a fluid through a tube.^[1]

Light scattered at small angles, forward scatter, rises with the size of the cell. Light scattered to the side reflects its internal structure, such as the granules of a white blood cell. Fluorescence at chosen wavelengths reports on whatever the cell carries: an antibody against a surface protein, a dye that binds DNA, a fluorescent protein the cell makes. Analysis consists of drawing gates around groups of cells on plots of one measurement against another and counting what falls inside. The field's guidelines, in their third edition of 2021, run to 438 pages and cover the phenotypes and functional assays of all the major human and mouse immune cell subsets.^[2]

In 1934 Andrew Moldavan described a photoelectric technique for counting microscopical cells, in which cells pass through a capillary and a photodetector registers each.^[3] In 1947 Gucker and colleagues built a photoelectronic counter for colloidal particles that counted single particles by the light each one scattered.^[4] Between these and the cytometer stands the electrical counter of Wallace H. Coulter, whose patent was granted in 1953 (see Coulter principle).

In 1965 Mack Fulwyler joined the two. His device measured the volume of each cell in a Coulter aperture, then isolated the cell in a droplet of the medium that was charged according to the volume sensed, and an electrostatic field deflected the charged droplets into a collecting vessel. It separated mixtures of mouse and human red blood cells, and cells of an ovary line that went through it survived and grew at their normal rate.^[5] In 1969 a group at Stanford replaced the volume signal with fluorescence. It sorted mouse spleen cells from ovary cells of another species, and highly fluorescent fractions of spleen cells from immunized mice were enriched in antibody-producing cells by factors of 4 to 10.^[6] In the same year Dittrich and Göhde in Germany described impulse fluorometry on single cells in suspension.^[7] The Stanford group's name for sorting by fluorescence, fluorescence-activated cell sorting, was published in 1972.^[8]

Mass cytometry, introduced in 2009, dropped the light. Antibodies carry tags of metal isotopes, and each cell is vaporized and read in an inductively coupled plasma time-of-flight mass spectrometer.^[9]

The two measurements see different things. An impedance counter gives the volume of the cell, because the dip in current is proportional to it, and optical scatter gives a signal that depends on size, shape and structure at once. Hematology analyzers use both (see Hematology analyzer and Complete blood count). The VCS technology of Beckman Coulter combines the volume from impedance with the conductivity of the cell at high frequency and laser light scatter, and has been used for white cell differentials and reticulocyte counts.^[10]

Molecular Reality Corporation belongs to the electrical branch. Its public guide traces the line from the counting of cells to the reading of molecules in one sentence, "Coulter counted cells, the biophysicists threaded DNA, and we are trying to give the whole family mechanical control."^[11] The pore on the company's live molecular stream is 200 µm across, about the size of the apertures that Coulter counters use to count blood cells, and can register cells and particles several micrometers wide (see 200-micron laser-drilled pore). The stream counts and does not sort.

• Resistive pulse sensing
• Complete blood count
• Nanopore