In terms of construction, flow cytometers (Figure 1) comprise three main components:

 • Fluidics system – this is critical to draw particles, such as blood cells, into the machine and channel them into single file

• Optical system – this system consists of lasers and the lenses that are used to focus the laser beam

• Signal detection and processing system – this converts the light signals to voltages that are then recorded.

 Basic knowledge of how these components function aids the user to ensure not only optimal, but appropriate, set-up of the flow cytometer for different applications.

Fig1. The photo shows a typical flow cytometry instrumentation with the cytometer itself and attached computer used for data acquisition. Underneath the flow cytometer are the sheath fluid tank and the waste tank.

Fluidics System

 Perhaps the key benefit of flow cytometry over many other widely used techniques is the capability to simultaneously assess multiple characteristics of individual cells or particles. Initially, when particles enter the flow cytometer, they pass through the sample injection port (SIP; Figure 2) and subsequently become distributed randomly in the space provided by the sample line, since the diameter of the latter is larger than that of most particles that are being analysed. In order to characterise each particle individually, the particles are fi rstorganised into a single stream that is then allowed to pass through the ‘interrogation point’ where it intersects the focussed light from the laser beams.

Fig2. Sample injection port (SIP). This is the port through which the cells are brought into the flow cytometer and subsequently channelled into a single stream of cells.

The fluidics system has two components: a central core, through which the sample fluid is injected, and an outer sheath fluid. These two components are pushed through the system at slightly different pressures. Of note, the sheath fluid is pushed through at higher pressure than the sample under laminar flow. A drag effect is created, causing the central core of sample to become narrower, aligning the particles in single fi le and allowing for uniform illumination. This is termed hydrodynamic focussing (Figure 3).

Fig3. Hydrodynamic focussing to generate a stream of single particles. Following entry into the flow cytometer via the SIP, cells are focussed into a single stream.

It is possible to manipulate the flow rate of cells passing the laser beam, depending on the purpose for which the flow cytometer is being used. For example, high flow rates are appropriate for immunophenotyping cells; in contrast, if the DNA content of the cell is assessed then a slower injection rate may be required to ensure sufficiently high resolution. A slow flow rate decreases the diameter of the sample stream and increases the uniformity and accuracy of illumination. It is important to note that proper functioning of the fluidics system is critical to allow for appropriate interception between the particle and the laser beam. Importantly, the user needs to ensure that the fluidics system is free of air bubbles and debris, and is properly pressurised at all times.

Optical System

Figure 8.3 Hydrodynamic focussing to generate a stream of single particles. Following entry into the flow cytometer via the SIP, cells are focussed into a single stream. Following alignment of the particles into a single fi le they pass through the interrogation point where they are intercepted by a laser beam, providing the incident light. Together with the lenses and collection optics, these components form the optical system of the flow cytometer.

Lenses are used to focus the laser light that hits the particle resulting in light scat tering around the edges of the particle ( Figure 4 ). Two types of scattered light are analysed:

• Forward scatter (FSC) is the light collected along the same direction as the incident laser beam (and up to about 20° offset from that direction). The intensity of forward-scattered light is proportional to the cell surface area and thus provides an estimate of cell size. Larger cells tend to diffract and refract more light than smaller cells. Diffracted light arises due to bending of the incident light when it hits the edge of the cell. In contrast, refracted light occurs due to bending when it enters the trans lucent cell and then enters the air again. For either phenomenon, a larger cell results in more scattered photons.

• Side scatter (SSC) is a measure of reflected light (i.e. light bouncing off the sur face) that is collected at a direction perpendicular to the incident laser beam. SSC is related to the cell granularity or internal complexity (more granules result in more pronounced reflection of light). For example, eosinophils that are highly granular will have a higher SSC than lymphocytes.

Fig4. Light scattering by a cell flowing past the interrogation point. As the cell passes through the laser beam, scattered light is acquired along the same direction as the laser beam (forward scatter, FSC; information about the cell size) and at a perpendicular direction (side scatter, SSC; information about granularity or internal complexity).

FSC and SSC properties can be used to differentiate specific cell types within a heterogeneous cell population such as the blood.

If fluorescent dyes or fluorescently conjugated antibodies are used to detect specifi c attributes of the particle, then, depending on the dyes employed, different wavelengths of incident light and laser types (gas lasers, diode lasers) can be used to excite the fluorescent moiety by either red, yellow-green, blue, violet or ultra violet light. Collection of the emitted light from the fluorophore uses a system of optical filters and mirrors that separate and point light of defined wavelengths towards the appropriate detectors (Figure5). Depending on the required band width and the fluorescence signal to be detected, different types of filters are used (Figure6 ):

 • Band pass – these allow a narrow range of wavelengths, close to the emission peak of the fluorescent dye to reach detector

 • Short pass – transmits wavelengths of light equal to or less than a specific wavelength

• Long pass – filters transmit light equal to or longer than the specified wavelength.

Fig5. Typical flow cytometer set-up for signal detection. The light scattered by particles passing the interrogation point is reoriented and filtered to be ultimately detected by photodiodes or photomultiplier tubes (PMT).

Fig6. Different types of optical filter are employed, depending on the fluorescence signal to be detected.

Signal Detection and Processing

The light emitted and refracted/reflected from the single stream of particles is sensed by photodetectors. These photodetectors convert the light signal into a stream of electrons (current). Two different types of detectors are commonly used in flow cytometers: silicon photodiodes (PDs) and photomultiplier tubes (PMTs). PDs convert light into photoelectrons with greater efficiency than PMTs, but PMTs have a greater sensitivity. Historically, the stronger FSC light signals have been detected by PDs, while the weaker SSC and fluorescence signals have been detected by PMTs. In contemporary instruments, PMTs are frequently used, even for the FSC channel.

In response to incoming photons, the photodetector will generate a current, the extent of which is proportional to the number of photons detected, and thus the scatter or fluorescence signal emitted by the particle. When the particle enters the cross-section of the interrogation point, the current produced by the detector will start to increase. Ultimately, when the cell is at the centre of the incident laser beam, the current will reach maximum. As the particle leaves the cross-section of the interrogation point, the electrical current/signal will return to baseline levels. Such a pulse of signal is termed an event (Figure 7).

Fig7. Parameters used to quantify a signal pulse (event) as a cell moves through the interrogation point.

The initial signal measured is an analogue signal, but this is converted to a digital signal by an analogue-to-digital converter (ADC) to allow for subsequent computer analysis. Additionally, prior to conversion, the electrical current can be amplified by a linear or logarithmic amplifier. The choice of linear or logarithmic amplification is dependent on the application for which the flow cytometer is being used and the dynamic range required. For example, log amplification is typically used for fluorescence studies of immune cell populations as it expands weak signals and reduces strong signals allowing them to be easily displayed on a histogram. In contrast, something like DNA analysis typically employs linear scaling as here it is important to be able to detect very small differences.

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مواضيع ذات صلة

Fluorescence Labels

Fluorescence-Activated Cell Sorting (FACS)

Antibody Preparation

Harnessing the Immune System for Antibody Production

Electrophoretic Techniques : Support Media and Buffers

Principles of Liquid Chromatography

Preliminary Purification Steps

Cell Disruption and Production of Initial Crude Extract

Determination of Protein Concentrations

Molecular Biotechnology and Applications

The Biology of Restriction Enzymes

The Isolation of DNA