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Briefings in Functional Genomics and Proteomics Advance Access published online on July 4, 2007
Briefings in Functional Genomics and Proteomics, doi:10.1093/bfgp/elm011
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© Oxford University Press, 2007, All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

The flow of cytometry into systems biology

John P. Nolan and Loretta Yang

Corresponding author. John P. Nolan, La Jolla Bioengineering Institute, 505 Coast Boulevard South, La Jolla, CA 92037, USA. Tel: 858-456-7500; Fax: 858-456-7540; E-mail: jnolan{at}ljbi.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 FLOW CYTOMETRY: KEY ANALYTICAL...
 CELL SYSTEMS ANALYSIS
 CELL-BASED MOLECULAR ENGINEERING
 SPANNING SIZE SCALES: WHOLE...
 MICROARRAYS IN SUSPENSION
 CONCLUSIONS AND PROSPECTS
 Acknowledgements
 References
 
Biomedical research is evolving to address biological systems as molecular pathways integrated into complex networks. Tools for molecular and cell analysis are also evolving to address the new challenges and opportunities of this approach. Flow cytometry is a versatile analytical platform, capable of high speed quantitative measurements of cells and other particles. These capabilities are being exploited and extended in a range of new applications stemming from opportunities presented by the advances of genomics, proteomics and systems biology, which are in turn impacting clinical diagnosis, vaccine development and drug discovery. In this review, we highlight some of these advances and consider the future evolution of flow cytometry technology.

Keywords: fluorescence, screening, high throughput, cytomics, nanotechnology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
FLOW CYTOMETRY: KEY ANALYTICAL...
 CELL SYSTEMS ANALYSIS
 CELL-BASED MOLECULAR ENGINEERING
 SPANNING SIZE SCALES: WHOLE...
 MICROARRAYS IN SUSPENSION
 CONCLUSIONS AND PROSPECTS
 Acknowledgements
 References
 
A key goal of biomedical research is to develop a predictive understanding of biological systems that can be used to develop new preventative, diagnostic and treatment approaches. The large scale molecular analysis of biological systems, embodied in the notions of genomics, proteomics and systems biology represents the latest evolution of this endeavour. Developments in analytical technologies are critical to this effort, and a number of tools, both novel and familiar, are playing central roles in the study of biological systems. Among these, flow cytometry, and the principles underlying it, has emerged as one of most versatile, owing to its ability to rapidly make quantitative molecular measurements of individual objects on size scales from nanometers to hundreds of micrometers, and time scales down to the sub-millisecond. In this article, we review some of the recent developments in flow cytometry technologies and new applications that are addressing the challenges of large scale analysis in systems biology.


   FLOW CYTOMETRY: KEY ANALYTICAL FEATURES
TOP
 ABSTRACT
 INTRODUCTION
 FLOW CYTOMETRY: KEY ANALYTICAL...
CELL SYSTEMS ANALYSIS
 CELL-BASED MOLECULAR ENGINEERING
 SPANNING SIZE SCALES: WHOLE...
 MICROARRAYS IN SUSPENSION
 CONCLUSIONS AND PROSPECTS
 Acknowledgements
 References
 
Flow cytometry rapidly measures the fluorescence and other optical properties of individual particles. Most often those particles are cells, but as described further may range from whole organisms to single molecules. In a typical flow cytometer (Figure 1), sample is carried in a sheath stream through a laser beam where fluorescent dyes are excited. The emitted fluorescence is collected, spectrally filtered and detected using photomultiplier tubes. The basic principles of flow cytometry, as well as the numerous variations, have been well described in the text by Shapiro [1]. In this review, we will highlight the features that make it uniquely powerful for the large scale applications characteristic of genomics, proteomics and systems biology.


Figure 1
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  		Figure 1: Schematic diagram of a typical flow cytometer showing arrangement of the light source, flow cell, collection optics, filters and detectors, as well as a close up diagram of the point of measurement or probe volume, where the illuminating light, sample stream and collection optics focus intersect.

 
High speed single particle analysis and selection
In flow cytometry, samples are hydrodynamically focused to a very thin sample stream, typically on the order of 10 µm in diameter. This focused sample stream is passed through a focused laser beam on the order of 10 µm in height. The intersection of the sample stream and laser beam (Figure 1, inset), often called the probe volume, has dimensions of ~10 µm3, or about 1 pl. Under these conditions, in a typical mammalian cell (diameter ~10 µm) suspension, cells will be lined up single file and will pass one at a time through the probe volume, where fluorescence and light scatter signals are collected. Typical transit times through the probe volume are 10 µs or less for many commercial flow cytometers, enabling sample analysis rates of thousands of cells per second. High speed cell sorters are capable of analysing tens of thousands of cells per second [2], and sorting selected sub-sets of cells into tubes or microwell plates.

Sensitive and quantitative detection
Because the measurement probe volume is small, background signal, which often limits sensitivity, is low, making flow cytometry an especially sensitive fluorescence detection platform. While custom instruments have reported single molecule sensitivity [3, 4], most commercial cytometers have detection limits of a few hundred molecules of a small organic fluorophore such as fluorescein. In practice, cellular autofluorescence will often limit measurement sensitivity. Importantly, intensity standards and calibration protocols have been developed that allow fluorescence measurements to be expressed in absolute units of molecules per cell [4–8]. These approaches consider instrument response, the properties of reagents used (the fluorophore to protein ratio of an antibody, for example), and spectral matching between calibrators and unknowns. Such absolute quantification facilitates assay development and mechanistic studies, and is critical for certain clinical applications.

Multiparameter measurements
Flow cytometry can make high speed, quantitative optical measurements of multiple fluorophores simultaneously. The simplest bench top instruments typically measure three or four colours of fluorescence excited by a single laser. Additional lasers and detectors enable the detection of additional fluorophores, and the past decade has seen a steady increase in the number of parameters measured [9, 10], such that three laser eight colour experiments are not uncommon, and 19 parameter (fluorescence plus light scatter) measurements have been reported [11]. The high information content provided by multiparameter measurements not only allows for more efficient analysis of samples, it is required to identify key sub-populations present in a complex mixture of cells.

Homogeneous assays
Because the probe volume in the flow cytometry measurement is small, signal from free fluorophore is often negligible [12], allowing samples to be measured without a wash step. In addition, homogeneous assays enable continuous kinetic resolution, allowing flow cytometry to be exploited for real-time mechanistic studies of biochemical processes. Such wash-less assays enable streamlined sample processing and are especially amenable to automated analysis.

High speed, high throughput sample handling
The analytical advantages discussed earlier are enhanced when coupled to sample handling hardware to provide speed, automation or both. Rapid mixing devices [13–15] have been developed to enable sub-second kinetic resolution of biochemical processes occurring on cells [16] or microspheres [17]. Automated samplers are now available for many commercial instruments, providing throughput on the scale of two to four samples per minute. High throughput approaches have been developed that improve on this by more than an order of magnitude [18, 19], and these too are making their way to the market, making flow cytometry a viable compound screening platform for drug discovery [18, 20].

The capabilities of flow cytometry are co-evolving with the emerging challenges and opportunities presented by modern biomedical science. In the following sections, we highlight some aspects of this co-evolution, with an emphasis on those areas that are contributing to our understanding of complex biological systems.


   CELL SYSTEMS ANALYSIS
TOP
 ABSTRACT
 INTRODUCTION
 FLOW CYTOMETRY: KEY ANALYTICAL...
 CELL SYSTEMS ANALYSIS
CELL-BASED MOLECULAR ENGINEERING
 SPANNING SIZE SCALES: WHOLE...
 MICROARRAYS IN SUSPENSION
 CONCLUSIONS AND PROSPECTS
 Acknowledgements
 References
 
Flow cytometry was developed for the measurement of cells, and over the past 30 years has established itself as the method of choice for the quantitative fluorescence analysis of single cells. As systems-level cellular analyses take form from various genomics and proteomics efforts, flow cytometry has emerged as a key tool for understanding the regulation and interactions of cell systems. The measurement of cell surface markers, intracellular epitopes and fluorescence reporters of a range of biochemical activities are well established, and the integration of these measurements on an increasingly greater scale is revolutionizing our understanding of the functions of cell systems.

The best examples to date come from the analysis of the mammalian immune system, where multi-parameter analysis of cell surface markers to identify immune cell sub-sets is being combined with functional measurements of antigen recognition [21, 22] and cytokine production. These approaches can reveal the fine details of individual responses to infection and vaccination that enable the improved assessment of candidate vaccines [23] and will lead to a better mechanistic understanding of the immune response and increase the efficiency of vaccine development.

Another example is the analysis of signalling pathways in immune cells, where multiparameter surface markers are combined with analysis of the phosphorylation states of intracellular signalling molecules [24, 25]. Analysis of the basal states of signalling molecules in specific cells from healthy individuals and cancer patients can reveal the presence of constitutively active signalling pathways, while measurement of the perturbation of these pathways by various activators and inhibitors reveals connections among pathways not observable in the analysis of the basal signalling state [26, 27]. These single cell proteomics approaches to understanding the networks of signalling pathways inside the individual cells provide a new window on the function of the immune system that has exciting implications for disease diagnosis and treatment [28, 29].

A final example is in the diagnosis and treatment of cancer, especially leukaemia and lymphoma, where multiparameter immunophenotyping plays a major role in disease detection [30] as well as treatment monitoring [31]. Coupled with genetic and chemical analysis, single cell cytometry provides a rich set of data with which to make clinical decisions for individual patients, if the data could be integrated and understood. This integration of diverse information-rich data sets to make predictive assessments of cell systems to aid in the diagnosis and treatment of disease has been described by the term ‘cytomics’ in recognition of the expanded scope of these efforts relative to traditional cytometry [32–34]. These goals will be tightly coupled to the development of new computational methods, and the practice of cytometry is evolving to reflect this.


   CELL-BASED MOLECULAR ENGINEERING
TOP
 ABSTRACT
 INTRODUCTION
 FLOW CYTOMETRY: KEY ANALYTICAL...
 CELL SYSTEMS ANALYSIS
 CELL-BASED MOLECULAR ENGINEERING
SPANNING SIZE SCALES: WHOLE...
 MICROARRAYS IN SUSPENSION
 CONCLUSIONS AND PROSPECTS
 Acknowledgements
 References
 
The high speed cell sorting capabilities of flow cytometry are being used to exploit the inherent diversity present in immune system genes and molecular evolution to develop proteins and other molecules with new and improved properties. Libraries of single chain variable fragments (scFv) of immunoglobulin molecules have been displayed on the surface of either bacteria [35, 36] or yeast [37, 38], and cells that bind a fluorescence-labelled target are physically sorted to select for clones with the desired binding properties. This is essentially analogous to phage display, where selections are carried out by panning against an immobilized target. Flow cytometric selections offer more control over affinity and specificity than is achievable through simple panning methods.

Cell-surface display is not limited scFvs, but has been extended to larger antibody Fab fragments, and even whole immunoglobulins [39], as well as cell surface receptors [40–43], their ligands [44], protease enzymes [45, 46], protease substrates [47] and other protein scaffolds [48]. The binding properties of selected molecules can be further optimized through molecular evolution techniques such as error prone PCR [49] and chain shuffling [50] to generate higher affinity binding. The selectivity of binding proteins for different targets can be tuned by employing multiple differently-labelled binding targets during the selection [51]. Such high throughput multiparameter selections are a unique capability of flow cytometry.

While cell-surface display is the most widely practiced mode of cell-based molecular engineering, any molecular feature that results in a fluorescent readout can serve as the basis of a flow cytometric selection. A good example is the development of a new family of fluorescent proteins based on the Discosoma red fluorescent protein [52]. Somatic hypermutation [53] was used to drive molecular evolution in conjunction with flow cytometry-based selection to produce new fluorescent proteins with a range of emission spectra. In another example, fluorescent proteins were evolved to optimize fluorescence resonance energy transfer (FRET) [54]. Selections based on reporter gene synthesis [55] and FRET [56] in two-hybrid based screens are additional examples of cell-based fluorescence screens.


   SPANNING SIZE SCALES: WHOLE ORGANISMS TO SINGLE MOLECULES
TOP
 ABSTRACT
 INTRODUCTION
 FLOW CYTOMETRY: KEY ANALYTICAL...
 CELL SYSTEMS ANALYSIS
 CELL-BASED MOLECULAR ENGINEERING
 SPANNING SIZE SCALES: WHOLE...
MICROARRAYS IN SUSPENSION
 CONCLUSIONS AND PROSPECTS
 Acknowledgements
 References
 
Flow cytometry, as conventionally performed, is well-suited to the study of single mammalian cells in suspension. However, the principles of flow cytometry are equally adaptable to both larger and smaller particles. On the upper end of the size spectrum, instruments employing wide bore flow cells have enabled the analysis and sorting of particles over 100 µm in diameter, including multicellular spheroids [57], pancreatic islets [58] and whole nematodes [59–61] and fly larvae [62, 63]. Smaller cells, in the range of a micron or so in diameter, are also commonly analysed by flow cytometry. These include platelets [64, 65] and bacteria [66, 67] that, with care, can be analysed in a manner comparable to larger cells, although fluorescence and light scatter intensities tend to be closer to the optical and electronic noise floor of conventional commercial flow cytometers.

Below the level of single cells, there are many biologically important particles, both intracellular and extracellular, for which our understanding would benefit from a single particle analysis approach. A good case in point is the so-called ‘microparticles’ that are released from platelets and many other cell types upon activation [68–71]. Typically 100–200 nm in diameter [72–77], as indicated by electron microscopy, these cell-derived membrane vesicles have been postulated to play roles in intercellular communication and are potential biomarkers for a host of diseases. Flow cytometry has been used frequently to study these particles [78, 79], but a critical review of the primary literature indicates that the vast majority of these studies report the analysis of coincident occurrence of multiple vesicles, electronic noise or both. Specially configured high sensitivity instruments have been used to measure single lipid vesicles in this size range [80, 81], but these were custom-built one or two colour instruments and were not suitable for the routine analysis of complex samples. A robust multiparameter instrument for the analysis of individual nanoparticles remains an unmet need.

Flow cytometry, in custom high sensitivity configurations, has been used to measure single fluorescent molecules. The best developed biologically-relevant application is the fluorescence-based sizing of single DNA fragments [4, 82–84], but the complexity of most biological samples and challenges of labelling with specific reagents have prevented the general application of flow cytometry to the study of individual soluble molecules. However, the challenge of large scale analysis of soluble molecules is being addressed using optically-encoded microparticles and conventional flow cytometry [85, 86].


   MICROARRAYS IN SUSPENSION
TOP
 ABSTRACT
 INTRODUCTION
 FLOW CYTOMETRY: KEY ANALYTICAL...
 CELL SYSTEMS ANALYSIS
 CELL-BASED MOLECULAR ENGINEERING
 SPANNING SIZE SCALES: WHOLE...
 MICROARRAYS IN SUSPENSION
CONCLUSIONS AND PROSPECTS
 Acknowledgements
 References
 
The use of microspheres as solid supports for the analysis of soluble analysis dates back decades in the flow cytometry literature [87–90]. The increased demands for multiplexed molecular analysis spurred the resurgence of this approach and the development of fluorescence-encoded microsphere reagents to support simultaneous analysis of dozens of analytes. Popularized by the Luminex brand microspheres and instruments [91, 92], optically encoded microspheres and assay kits are now available from numerous commercial sources and are compatible with most commercial flow cytometers. The combination of a microarray-type multiplexed analysis and the high speed particle analysis of flow cytometry is revolutionizing existing assays and inspiring new ones.

The prototypical multiplexed bead-based analysis is the antibody sandwich assay. Essentially, an ELISA performed on a microparticle instead of a microwell bottom, an immobilized antibody captures an analyte from a complex sample, and a labelled reporter antibody completes the sandwich allowing the analyte to be quantified via the fluorescence intensity of the microsphere. The principles and considerations for developing such multiplexed assays have been described in detail [93–95], but the numbers of commercial assay reagents is increasing steadily and include kits to simultaneously measure a dozen or more soluble signalling molecules such as cytokines and chemokines. In general, the bead-based assays offer sensitivity comparable to the standard colorimetric ELISA, with the advantages of smaller sample size, fewer processing steps, which combined with the efficiency of multiplexing constitute an extremely powerful approach to the detection of soluble proteins. However, it should be noted that the multiplexing levels typically employed in antibody sandwich-type assays do not test the limits of optical encoded beads. Rather, multiplexing is limited by the availability of antibodies with suitable specificity and non-cross-reactivity.

Genetic and genomic analysis is another area where microparticle arrays have been implemented for large scale analysis. Robust chemistries for analysing single nucleotide polymorphisms (SNPs), including primer extension [96] and ligation [97], have been adapted to the microsphere platform and used for large scale genotyping studies [98]. More recently, microsphere arrays have been used to profile miRNAs in cancer cells, providing a new tool for the molecular classification of human cancer [99]. With a multiplexing capacity near 100, microsphere arrays do not challenge the multiplexing capabilities of the more conventional flat surface microarrays, which can analyse tens of thousands of targets simultaneously. However, microsphere arrays use less sample, are less expensive and more flexible than planar arrays, making them better suited for large scale analyses involving numerous samples. Reagents and software for molecular genetic analysis are becoming commercially available.

The notion of performing multiplexed solid phase assays on microspheres is not limited to ELISA and hybridization-based assays. Molecular assemblies ranging from enzymes and substrates [100–103] to receptors and G-proteins [104–108] have been analysed via microsphere-based flow cytometry. Purified antibody fragments [109], and even whole bacteriophage [110], selected from phage display libraries can be screened in multiplex. Finally, as combinatorial chemical libraries are often synthesized on polymer microparticle supports, flow cytometric sorting is being exploited for on-bead screening of chemical libraries [111–116].


   CONCLUSIONS AND PROSPECTS
TOP
 ABSTRACT
 INTRODUCTION
 FLOW CYTOMETRY: KEY ANALYTICAL...
 CELL SYSTEMS ANALYSIS
 CELL-BASED MOLECULAR ENGINEERING
 SPANNING SIZE SCALES: WHOLE...
 MICROARRAYS IN SUSPENSION
 CONCLUSIONS AND PROSPECTS
Acknowledgements
 References
 
In the 40 years since its introduction, flow cytometry technology has evolved in response to the biological problems of the day. Driven by early studies on the cell cycle and the immune system and the challenges of diagnosis and treatment of diseases such as leukaemia and HIV/AIDS, flow cytometry matured from a field characterized by home-built instruments and home-brewed reagents in a few labs to one supported by an array of commercial instruments, reagent kits and assay protocols, in centralized core facilities. The large scale analyses inspired by the notion of systems biology described previously are driving a new round of technology development in flow cytometry. In conclusion, we describe a few of developments that are characterizing the next stage of evolution of flow cytometry.

Multiparameter measurements
There is a continuing push to expand the number of unique labels that can be used as reporters for antibodies or other tags. The fundamental limitation to increasing the number of labels is the broad excitation and emission spectra of the fluorophores conventionally used in flow cytometry. Fluorescent semi-conductor nanoparticles (quantum dots) offer somewhat narrower emission bands but, while these have demonstrated some utility for flow cytometric analysis [117], they do not offer the opportunity for a large increase in the number of reporter labels. Spectral analysis-based approaches to single particle analysis [118, 119], coupled with new types of nanoparticle labels [120–122], offer the potential for significant increase in the levels of multiparameter analysis.

Flow cytometry automation
Sample loading from microwell plates is becoming more common in flow cytometry, with several hardware options from commercial cytometer manufacturers and third-party providers. Software has lagged behind the hardware for automated analysis, due in large part to a lack of bioinformatics standards to support large-scale applications. Efforts are underway [123, 124] to standardize the descriptions of experimental design, measurement and data analysis, and provide a standard for capturing and sharing this information. Procedures for instrument and measurement calibration and standardization are also being developed [7, 125–127] to help ensure proper instrument set up and to allow researchers to more readily take advantages of the quantitative capabilities of flow cytometry.

Convergence with image cytometry
Historically, flow cytometry was employed when quantitative fluorescence measurements were needed from large numbers of cells for statistical analysis, and microscopic imaging was used when qualitative 2D or 3D localization information was needed. To fit this paradigm, adherent cells were often dispersed for flow cytometry by physical scraping or enzymatic digestion, and suspension cells were ‘stuck’ onto coated cover slips. Today, increases in the speed of imaging platforms and the introduction of flow cytometers with imaging capabilities are driving a revision of this paradigm. High speed automated microscopy and image analysis of adherent cells and tissues allows researchers to perform statistically robust image analysis [128–130], and laser scanning cytometry [131, 132] can provide quantitative analysis formerly provided only by flow cytometry. Conversely, commercially available flow imaging capabilities (for example, the Amnis ImageStream) are enabling a new generation of image-based analysis to be applied to suspension cells [133–136]. In the future, the decision about whether to use flow- or image-based cytometry will be based primarily on the cell type: adherent or suspension.


Key Points

  • Flow cytometry is unique in its ability to rapidly make sensitive and quantitative multiparameter measurements of individual cells.
  • Flow cytometry is having a major impact on the study of cellular systems, especially the immune system, as well as in the screening of biological and chemical libraries for drug discovery.
  • The use of optically encoded microparticles as solid supports for molecular assembly greatly expands the use of flow cytometry for genomics and proteomics beyond cell-based applications.
  • Flow and image cytometry are complementary, with the speed and optical resolution of each converging to provide powerful fluorescence analysis capabilities for both suspension and adherent cells.

 


   Acknowledgements
TOP
 ABSTRACT
 INTRODUCTION
 FLOW CYTOMETRY: KEY ANALYTICAL...
 CELL SYSTEMS ANALYSIS
 CELL-BASED MOLECULAR ENGINEERING
 SPANNING SIZE SCALES: WHOLE...
 MICROARRAYS IN SUSPENSION
 CONCLUSIONS AND PROSPECTS
 Acknowledgements
References
 
Work in the authors’ lab is supported by the National Institute of Bioimaging and Bioengineering of the National Institutes of Health (EB003824).


   FOOTNOTES
 
John Nolan is a Professor at the La Jolla Bioengineering Institute. His research is focused on quantitative molecular analysis of cells and cell function, and includes both instrument and assay development.

Loretta Yang is a Research Scientist at the La Jolla Bioengineering Institute. Her research interests are in the area of protein engineering using phage and yeast display.


   References
TOP
 ABSTRACT
 INTRODUCTION
 FLOW CYTOMETRY: KEY ANALYTICAL...
 CELL SYSTEMS ANALYSIS
 CELL-BASED MOLECULAR ENGINEERING
 SPANNING SIZE SCALES: WHOLE...
 MICROARRAYS IN SUSPENSION
 CONCLUSIONS AND PROSPECTS
 Acknowledgements
 References
 

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