Flow Cytometry

Introduction

Flow cytometry is an analytical technique that can rapidly measure the properties of individual cells or particles as they pass through a beam of light, typically a laser. A flow cytometer takes a sample of cells, transitions them into a single stream and uses lasers and/or light sources to excite biomarkers or labels on the cells to count the number of relevant constituents. The properties measured include relative particle size, relative granularity or internal complexity, and relative fluorescence intensity.

The method requires several photonics components, starting with lasers and other light sources with small, tightly focused beams that can illuminate a single size cell or particle (~30 µm diameter). Laser light with specific wavelengths is needed to excite labels or fluorophores on a cell. Dichroic filters with precision coatings are needed for narrow reflection/transmission bands and high optical density for out-of-band rejection. All photonics components must be precisely aligned and controlled with µm spatial resolution.

A flow cytometer (Figure 1) consists of the three major component systems. The first is the fluidics system that transports sample particles to the laser beam in a narrow, single particle wide stream. The second is the optical system composed of lasers or light sources that illuminate the particles in the sample stream as well as optical filters and beamsplitters that direct the post-sample light signals to optical detectors for counting and processing. The third system is the electronics and signal processing equipment that converts the detected optical signals to electronic signals for processing and analysis.

Schematic of a flow cytometry system
Figure 1. Schematic of a flow cytometry system.

Flow Cytometry Systems

Fluidics System

The fluidics system creates a stream of single particles that can be interrogated individually by the instrument's detection system. The sample is focused by the Bernoulli effect (Figure 2), creating a stream of particles in single file using a method called hydrodynamic focusing. Under optimal conditions (laminar flow), there is no mixing of the central fluid stream and the sheath fluid.
Hydrodynamic focusing produces a single stream of particles
Figure 2. Hydrodynamic focusing produces a single stream of particles.

Optics and Detection

Following hydrodynamic focusing, the particle stream passes through one or more focused laser beams and light scattering or fluorescence emission occurs. The optical output is collected as forward or side scattered light (FSC or SSC) by a PMT or photodiode and the optical data is used to characterize the cell properties. FSC data provides an estimate of a particle's size while SSC provides information on the relative internal complexity of a cell. By combining the FSC and SSC information with fluorescence labeling, it is possible to differentiate cell types in a heterogeneous population such as blood.

The detectors in most flow cytometers are usually PMTs. The specificity of detection is controlled by optical filters. There are three major filter types: long pass filters that transmit light above a cutoff wavelength; short pass filters that transmit light below a cutoff wavelength; and band pass filters that transmit light within a narrow band of wavelengths (see Optical Filter Characteristics for details). These are dichroic filters that block light by phased reflection, allowing only certain wavelengths of light to pass through while interfering with other wavelengths (Figure 3).

Dichroic optical filters
Figure 3. Dichroic optical filters.
When placed at an angle to the oncoming light, a dichroic filter acts as a mirror, allowing it to perform two functions: transmitting specific wavelengths in the forward direction and reflecting the remaining light at a 90o angle. This allows the light path to be passed through a series of filters. The precise choice and order of the filters can be arranged so that multiple signals can be detected simultaneously (Figure 4).
Schematic overview of the optical configuration of a typical flow cytometer setup
Figure 4. Schematic overview of the optical configuration of a typical flow cytometer setup.

Signal and Pulse Processing

Every time a particle passes through the interrogation point, a signal pulse is generated in every detector in Figure 4 with the current from each PMT proportional to the intensity of the scatter or fluorescence signal generated by the cell. These pulses can be mapped by plotting signal as a function of time.

Not all signals that are generated correspond to a particle of interest. PMTs are extremely sensitive and detect signals from irrelevant sources such as stray light, dust, very small particles and debris. The number of these irrelevant pulses can be orders-of-magnitude higher than the number of pulses that are generated by particles of interest. It is therefore desirable and necessary to set a threshold below which non-essential data are ignored. This is done by designating a trigger channel, usually a forward scatter detector, and setting a threshold signal intensity in that channel for recording scattering events. Any pulse that fails to exceed the threshold level is ignored in all detectors; any pulse that surpasses the threshold level is fully processed by the electronics.

The electronics process fluorescence signals for display, analysis, and interpretation. The analog current from the PMT is typically digitized and the pulse height, area, and width determined. The height and area, or maximum and integral, respectively, are used to measure signal intensity since their magnitudes are proportional to the number of photons that reach the PMT. The width of the pulse is proportional to the time that the particles spend in the laser and this can be used to distinguish between single particles or closely interacting particles and doublets.

Applications of Flow Cytometry

The ability to simultaneously measure multiple parameters on a cell-by-cell basis is the most powerful attribute of analytical flow cytometry, making it suitable for a wide range of applications. Most commonly, it is used to determine the presence of antigens either on the surface or within cells. In addition, flow cytometry may be used for the analysis of DNA or RNA content, and for functional studies on cells. The following broad range of technological activities employ flow cytometry analytical tools:

  • Medicine
    • Hematology
    • Oncology
    • Immunology
  • Genetic testing
  • Biochemistry and molecular biology, e.g., proteomics, glycomics
  • Marine science
  • Biosynthesis
  • Cell health and biology (including stem cells)
  • Screening
  • Cell cycle analysis
  • Bio Process

Flow Cytometry Products

MKS Instruments provides equipment for flow cytometry applications at the component, sub-assembly and fully integrated systems level. Figure 5 shows a complete flow cytometry system that was designed and manufactured by MKS ISB.
Flow cytometer system
Figure 5. Flow cytometer system.

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