The result is that the reading for any given fluorochrome is actually the sum of that fluorochrome's peak emission intensity, and the intensity of all other fluorochromes' spectra where they overlap with that frequency band. Each particular fluorochrome is typically measured using a bandpass optical filter set to a narrow band at or near the fluorochrome's emission intensity peak. When more than one fluorochrome is used with the same laser, their emission spectra frequently overlap. The rapid increase in the dimensionality of flow cytometry data coupled with the development of high-throughput robotic platforms capable of assaying hundreds to thousands of samples automatically have created a need for improved computational analysis methods. At the same time, microfluidic qPCR methods are providing a flow cytometry–like method of quantifying 48 or more RNA molecules per cell. More recently, the new technology of mass cytometry replaces fluorophores with rare earth elements detected by time of flight mass spectrometry, achieving the ability to measure the expression of 34 or more markers. Through the late 1990s into the mid-2000s, however, rapid development of new fluorophores resulted in modern instruments capable of quantifying up to 18 markers per cell.
Until the early 2000s, flow cytometry could only measure a few fluorescent markers at a time.
The ability to quantify these has led to flow cytometry being used in a wide range of applications, including but not limited to: Schematic diagram of a flow cytometer, showing focusing of the fluid sheath, laser, optics (in simplified form, omitting focusing), photomultiplier tubes (PMTs), analogue-to-digital converter, and analysis workstation.
Open software is most widely available in the form of a suite of Bioconductor packages, but is also available for web execution on the GenePattern platform.įigure 1. Open data is slowly growing with the opening of the CytoBank database in 2010 and FlowRepository in 2012, both of which allow users to freely distribute their data, and the latter of which has been recommended as the preferred repository for MIFlowCyt-compliant data by ISAC. Data standards include the widely adopted Flow Cytometry Standard (FCS) defining how data from cytometers should be stored, but also several new standards under development by the International Society for Advancement of Cytometry (ISAC) to aid in storing more detailed information about experimental design and analytical steps. Open standards, data, and software are also key parts of flow cytometry bioinformatics. Finally, diagnosis using flow cytometry data can be aided by supervised learning techniques, and discovery of new cell types of biological importance by high-throughput statistical methods, as part of pipelines incorporating all of the aforementioned methods. It is also possible to characterize data in more comprehensive ways, such as the density-guided binary space partitioning technique known as probability binning, or by combinatorial gating.
For population identification, tools are available to aid traditional manual identification of populations in two-dimensional scatter plots (gating), to use dimensionality reduction to aid gating, and to find populations automatically in higher dimensional space in a variety of ways. For preprocessing, this includes compensating for spectral overlap, transforming data onto scales conducive to visualization and analysis, assessing data for quality, and normalizing data across samples and experiments. Computational methods exist to assist in the preprocessing of flow cytometry data, identifying cell populations within it, matching those cell populations across samples, and performing diagnosis and discovery using the results of previous steps. The rapid growth in the multidimensionality and throughput of flow cytometry data, particularly in the 2000s, has led to the creation of a variety of computational analysis methods, data standards, and public databases for the sharing of results. Flow cytometry and related methods allow the quantification of multiple independent biomarkers on large numbers of single cells. Flow cytometry bioinformatics requires extensive use of and contributes to the development of techniques from computational statistics and machine learning. Flow cytometry bioinformatics is the application of bioinformatics to flow cytometry data, which involves storing, retrieving, organizing, and analyzing flow cytometry data using extensive computational resources and tools.