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Cell sorting and flow cytometry

1st April 2013


Flow cytometry was born from printer technology in the 1960s and is used in many and varied applications today. Mark Dessing reports on the unique development of flow cytometry through the years

Since the first half of the past century, scientists have been working on the automation of cell qualification or quantification on microscope systems, usually based on absorption or transmission measurements. During the early 1950s, techniques were developed which accelerated these measurements and which adapted microscopy to automatically analyse cells in flow. These techniques would later come to be known as flow cytometry.

Sorting by flow cytometry

During the mid-1960s and the beginning of the 1970s, the concept of flow cytometry moved on further as scientists at Los Alamos National Laboratory and Stanford University adapted the then-recently developed ink jet technology to be used in an instrument to sort individual cells. Cells were injected into a saline stream, which was usually formed using a 70 or 100-micron orifice. This stream was ultrasonically vibrated causing it to fall apart into equally sized and equally spaced droplets.

The scatter and fluorescence properties of the cells were detected after they emerged from the orifice (but before the stream started forming the droplets) through excitation by one or more focused laser beams and detection by several dedicated photomultiplier tubes. Based on the measured properties of the cell, the droplet containing a specific cell was electrically charged and the droplet pulled out of the stream in a constant electrical field.

By applying differential charge to the droplets it was possible to sort cells into two specific subpopulations, simultaneously.

Flow cytometry grows up

During the mid-1970s, the first commercial sorter became available. At this time cell sorting was still considered to be a stand-alone science and was usually done by dedicated scientists whose research was focused around this technology. Later, many laboratories started to build up core flow cytometry facilities in order to make the, still complex, technology available to other research disciplines.

The possibility to produce monoclonal antibodies in the early 1980s gave a huge boost to flow cytometry and sorting and pushed the technology into the fields of immunology and cell biology. The subsequent efforts to create fluorescent labels of different colours led to the development of multi-colour flow cytometry. AIDS has been a major factor in accelerating these developments.

During the past 30 years cell sorting and flow cytometry have undergone several changes and the trend is clear: the technology is rapidly changing from being a stand-alone science to becoming an enabling scientific tool.

The technology of flow cytometry

Flow cytometry and especially cell sorting technology have always been a complex mixture of electronics, fluidics and optics. Consider the electronics. The first major change was the development of high speed, parallel electronics in the late 1980s, driven by the need to sort individual chromosomes rapidly for the sequencing of the human genome as part of the Human Genome Project. The electronics of the commercial instruments were capable of sorting at rates in the range of a couple of thousand per second.

To get enough chromosomes in a reasonable amount of time, faster electronics were needed. Regarding fluidics, a continuous development from manual to electronic valves and pressure regulators has improved the stability and reproducibility of the droplet break-off, needed for accurate sorting. In optics, improvements in optical components, including optical filters, dichroic mirrors, photo multiplier tubes and lenses, have improved the detection of even weak fluorescent signals.

Recently, more and more development efforts have been invested to create instruments that are more user-friendly. The incorporation of CCD cameras and subsequent image analysis is used to automatically monitor the process of cell sorting, eliminating the need for a full time dedicated operator. The development of small, air-cooled diode lasers, eliminates the need for large, water-cooled lasers. These lasers, combined with modern optical fibre technology, create alignment free instruments, which in turn guarantee reproducible results over extended periods of time.

Flow cytometry today

Now, flow cytometry and cell sorting are used in a large number of scientific areas. The availability of specific antibodies, directly labelled with a variety of fluorochromes has made it one of the most important tools in immunology.

Recently, bead-based assays have been developed as an alternative to ELISA techniques. Soluble molecules are captured onto beads and a detection antibody is used to quantify the amount of secreted protein. The advantage of this flow cytometry technique, combined with automated, high throughput instrumentation, is that by mixing differently labelled beads, one can measure multiple molecules simultaneously (multiplexing).

Naturally occurring fluorochromes have made flow cytometry an interesting tool for marine biology. Fluorescent proteins, isolated from marine organisms, now play an important role as markers for intracellular expression patterns.

Modern instruments are capable of automatically sorting single cells into microtiter plates, onto agar plates or microscope slides, which are used in cloning and in single cell PCR assays.

High speed cell sorting can now be done at over 100 000 cells per second, allowing small populations to be isolated from a large number of cells, making the technique interesting for stem cell sorting and possibly even for therapeutic applications. Flow cytometry has certainly come of age.

Mark Dessing is European Applications Consultant, BD Technology Center, Allschwil, Switzerland. www.bdbiosciences.com





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