Flow Cytometry: The Next Revolution

Abstract

Unmasking the subtleties of the immune system requires both a comprehensive knowledge base and the ability to interrogate that system with intimate sensitivity. That task, to a considerable extent, has been handled by an iterative expansion in flow cytometry methods, both in technological capability and also in accompanying advances in informatics. As the field of fluorescence-based cytomics matured, it reached a technological barrier at around 30 parameter analyses, which stalled the field until spectral flow cytometry created a fundamental transformation that will likely lead to the potential of 100 simultaneous parameter analyses within a few years. The simultaneous advance in informatics has now become a watershed moment for the field as it competes with mature systematic approaches such as genomics and proteomics, allowing cytomics to take a seat at the multi-omics table. In addition, recent technological advances try to combine the speed of flow systems with other detection methods, in addition to fluorescence alone, which will make flow-based instruments even more indispensable in any biological laboratory. This paper outlines current approaches in cell analysis and detection methods, discusses traditional and microfluidic sorting approaches as well as next-generation instruments, and provides an early look at future opportunities that are likely to arise.

Keywords: fluorescence-based cytomics; high-throughput analysis; microfluidics; multiomics; single-cell analysis; spectral flow cytometry.

Figure 1

A general outline of a flow cytometer showing a sorting instrument capable of isolating individual cells. On the right side of the figure are examples of possible analyses.

Figure 2

A comparison between polychromatic cytometry (A) and spectral cytometry (B). In (A), the fluorescence from each dye is collected by a single detector, while in (B), many detectors are used to collect the entire spectrum of all dyes, enabling a process called spectral unmixing that can identify each dye. There are many advantages to the spectral concept.

Figure 3

Flow cytometry applications include cellular DNA/RNA analysis, phenotyping, ion flux, microbial analysis, cytokines, plant DNA, and a variety of additional assays.

Figure 4

Overview of directed evolution approaches for protein engineering. (A) Thousands to millions of individual protein variants can be produced. (B) Types of cell-based assays for protein evolution compatible with flow cytometry.

Figure 5

Single bacterial cell sorting using the Bigfoot cell sorter on two selective media, XLT4 and Salmonella Shigella (S.S) agar. (A) Screenshot of the 96-well plate map from the Bigfoot cell sorter software used as a reference to deposit single bacterial colonies at desired spots on the agar plate. (B) Colonies grown on XLT4 and S.S agar after 24 h of incubation.