Beyond conventional cell analysis: the latest science and technology in flow cytometry

The field of flow cytometry has enabled a greater understanding of cell biology and immune function over the past 50 years. The power of the technology is used in basic cell biology research, pharmaceutical discovery, and routine clinical diagnosis, as well as in agricultural and environmental applications. This progress has been made possible by a higher number of analytical parameters to measure cells in suspension. The first cytometers were systems capable of merely three or four parameters, using a single laser and four detectors, and were the size of a small car. Today, however, flow cytometers (including cell sorters) can analyze more than 30 parameters, and new technology in benchtop analyzers can deliver exponentially better performance in a footprint less than 45 cm².

Shifting paradigms

This paradigm shift, of higher performance in a small, less expensive instrument, is driven by investigators who want to capture the power of flow cytometric analysis, but don’t want to devote a career to learning the instrumentation. The democratization of flow cytometry is enabled by key advances in technology. Prominent concepts in other scientific fields such as the telecommunications industry are being leveraged to allow the subsystems to be miniaturized while at the same time providing even better performance. These compact high-performance systems not only deliver better performance than historically expensive systems, but they are also easy to set up, operate, and maintain, enabling a greater number of laboratory scientists to leverage the power of flow cytometry.

Seeing the light

Performance of flow cytometers is typically measured in their capacity to resolve and sensitivity to detect dim and/or rare populations. In this regard, efficient light management for optimal excitation and emission of fluorochrome-tagged cells is critical to performance.

With conventional analyzers, laser excitation sources are optimized by shaping and focusing light through a series of lenses and filters onto a flow cell where cells are hydrodynamically focused. However, newer systems use unique laser designs that are focused onto a flow cell with integrated optics. These systems can ensure both maximum excitation of the dyes not only on (and within) cells, but also maximum collection of the emitted light for integration and measurement. When designing a compact flow cytometer, the use of fiber optics to carry light is an efficient way of transmission, providing flexibility in laying out system components. These cables capture emitted light to deliver it onto a unique detector array, reducing cross talk between channels, which improves performance.

Another recent development is a key concept borrowed from the telecommunications industry, the wavelength division multiplexor (WDM), which is used for light detection and measurement. The WDM is the method used to deconstruct and measure multiple wavelengths of light as signals that relate to analytical parameters. These detectors are highly sensitive semiconductor devices used to measure each parameter. By contrast, conventional cytometers to date have (and continue to use), photomultiplier tubes (PMTs). The major advantages of the use of alpha photodiodes (APDs) over PMTs include but are not limited to 1) perfect linearity; 2) 4-5x the quantum efficiency; 3) higher dynamic range, 10⁶ vs 10³; and 4) significantly smaller size and about one-tenth the cost.

The WDM’s innovative and simple design uses a single bandpass filter to select the various colors of light. This contrasts with traditional systems, which use a series of dichroic steering- and bandpass-filters that bounce the light along an array leading to successively less available light, resulting in diminishing light collection efficiency and ultimately compromising fluorescence sensitivity and resolution.

Simplifying the complex

Leveraging the linearity of detection systems that use APDs in the operation of the cytometer can be dramatically simplified due to the predictability of the signals. The linear gain and the normalization performed during the daily QC routine takes care of the relative variations during instrument setup typically seen in instruments. Further, setting up a multicolor assay is simplified by using a software gain-only adjustment. The linearity of gain adjustment also simplifies the typically arduous task of spectral compensation which has been the barrier for many to push to higher number of colors/parameters. Leveraging the APD linearity, new software algorithms have been developed to facilitate setup and analysis of multi-color experiments by simplifying compensation.

It is now possible to create a compensation library which stores the APD gain settings and spectral spillover coefficients for every parameter and multicolor combination. This allows users to make a virtual spectral compensation matrix selecting various single colors from the library. In addition, the library can intelligently adjust the compensation values when gains are adjusted due to the predictive responses of linear APDs. The result is a dramatically simplified and intuitive method of setting up multicolor applications.

The size factor

Along with multicolor performance, flow cytometry has proven to be a valuable tool in small particle research, which is growing exponentially along with the medical research field’s understanding of microvesicle and exosome native biology and their potential applications in diagnostics and therapeutics. These extracellular nanoparticles are heterogeneous cell-derived particle populations in a size range between 50 nm and 500 nm. Flow cytometry is unique in that it is not only able to measure size but can deliver much more information about the characteristics and function of these nanoparticles using multiple fluorescent markers.

For most cytometers, however, measuring less than 300 nm is difficult if not impossible because of the fact that they deliver relative sizing information using forward scattered light off of the 488 nm Blue laser. For these systems, particles of less than 1 mm (1,000 nm) usually fall below the noise threshold of the laser and detector sub-systems. In contrast, newer systems use principles of Mie scattering which predicts that with lower wavelengths of excitation there will be an increased amount of scattered light and improved resolution. Therefore, measuring scattered light from a shorter-wavelength 405 nm Violet laser versus a longer-wavelength 488 nm Blue laser will allow the system to resolve smaller particles. The use of the Violet Side Scatter parameter enables systems to detect particles of less than 0.2 mm (200 nm) in size, enabling excellent resolution of microparticles.

In summary

Combining powerful performance and innovative design and technology, it is possible to deliver a compact, easy-to-use flow cytometry system. Pushing the ‘norms’ of conventional flow cytometry, today’s—and tomorrow’s—systems enable complex research into high-content applications in cell biology, as well as a deeper understanding of the advantages gained from the emerging nanoparticle frontier. Flow cytometry is a powerful tool for interrogating complex biological questions at the forefront of biomedical and life science research and increasingly for clinical laboratory applications. Today’s investigators want to harness that power and are demanding smaller and more powerful instruments that are more affordable and easier to use. Using innovation, engineers are able to deliver solutions to meet the challenge.