Cytometer (Cyto:cells; meter: measuring device) is a device used to count the different types of cells. The hemocytometer is the most basic device used to count the different types of cells, preferably blood cells. Hemocytometers, though still in use, are time consuming and cannot be fully-automated.
Then in 1950s, discovery of flow cytometry, changed the way the cells were analysed. It is a much rapid process, that can be fully automated.
Flow cytometer can be used to measure and analyze various characteristics of particles, like cells, as they flow through a beam of laser. The cells or particles scatter the incident laser beam. This scattered light is detected and determined using optical-to-electronic coupling system.
The flow cytometers on an average can be used to analyze particles at a rate of up to 20,000 cells per second (some devices are even faster). The different characteristics that the flow cytometer can analyze are the size, granularity and fluorescence intensity of the particles. It can also sort the particles based on these characteristics.
– Design:
A flow cytometer is made up of three main systems (fig 3):
1. The fluidics system
2. Lasers
3. Detectors
Let’s understand each component of the design.
1. The fluidics system:
The fluidics system is used to align the sample particles in a single line. It consists of a central core, through which the sample fluid is injected, enclosed by a tube, through which the outer sheath fluid is passed (Fig 4). The narrow inner sample tube ends after a short distance and allows sheath fluid to interact with the sample.
The sheath fluid flows under high pressure in the outer tube. At optimum pressure, the outer fluid pulls the inner sample fluid along, without mixing, in a way that sample is narrowed down to create a thin stream, allowing single line of particles. This phenomenon is called hydrodynamic focusing.
(Just for info: Read about the hydrodynamic focusing (the physics aspects) more in detail.)
Few examples of the sheath fluids used in the flow cytometer are phosphate buffered saline, Hepes-buffered saline and water with 2-phenoxyethanol (0.1%).
2. Optics:
The optics involves the incident light and scattering. The most preferred light source for the flow cytometer is the laser.
Lasers:
Lasers are the most commonly used light sources in modern flow cytometers. They emit light which is coherent (synchronized) and monochromatic (single wavelength). Flow cytometers may have one or more lasers, with a range of options of wavelength, from ultraviolet to far red, to be chosen.
The laser beam interacts with the particles in the sample and causes scattering of light. It may cause fluorescence emission if the sample has been labeled with a fluorophore. The pattern of light scattering or fluorescence by the particles provide information about the particles (fig 5).
• Scattering of laser beam:
There are two types of scattering occurring when the laser beam interacts with it.
1. Forward scatter (FSC)
2. Side scatter (SSC)
Let’s read the two in more detail:
1. Forward scatter (FSC)
This is the light scattered in the forward direction when the particle pass through the beam of laser light (see fig 6). The forward light is collected by a photodetector known as forward scatter channel (20 ͦ angle). The forward scatter gives information about the particle’s size. The larger cells refract more light than smaller ones.
2. Side scatter (SSC):
The side scatter is the amount of light scattered at around right angle (see fig 6). It is measured at an approximately 90° angle to the laser beam. The side scatter gives information about the granularity and internal structures.
Each particle in the heterogenous mixture will have a unique combination of FSC and SSC. This unique combination can be used to differentiate between different types of particles in a heterogenous sample, eg. blood.
The FSC and SSC are effected by various factors like the sample type and sample preparation procedure. Therefore, these factors can effect the results considerably and fluorescent labeling is preferred.
• Fluorescence emission:
As mentioned the particles can be labelled with fluorophores. Fluorophores are conjugated to antibodies against cell surface receptors or intracellular molecules such as DNA and cytokines and hence can be used to differentiate between different cell types.
The fluorophore accepts light energy at a particular wavelength and re-emit it at a longer wavelength. Several fluorophores can be excited by a single laser, but each may emit light of different wavelength (or colour). Hence by using combination of such fluorochromes, several parameters of the sample can be simultaneously determined. Hence allowing multicolor fluorescence studies.
Hence, the fluorescence measurements can provide the quantitative and qualitative data about labeled cells or particles.
3. Detection system:
The detection system consists of the optical filters and the detectors. Proper optical filters can segregate the fluorescent light from different fluorophores. These fluorescent emissions are then deflected to and detected by the detectors (see fig 3). The number of lasers and detectors vary according to the instrument and its manufacturer. Let’s read more on them.
• Optical Filters:
Optical filters play a very important role in the detection of the fluorescent light emitted by fluorescent labels. The
optical filters block certain wavelengths while transmitting others. Hence particular optical filters only allow a subset of wavelength(s) to pass through them.
Depending on the wavelengths they allow to pass or block, there are three major types of filter:
Long pass filters: allow light above a cut-off wavelength
Short pass filters: allow light below a cut-off wavelength
Band pass filters: allow light within a specific range of wavelengths or light with specific band width.
Dichroic filter/mirror is a filter placed at an angle to the incoming light. These filters allow specific wavelengths in the forward direction and deflect blocked light at around 90° angle.
To detect multiple fluorescent signals simultaneously, the choice and order of optical filters is very critical decision (see their placement in fig 3).
• Detectors:
The detectors used in flow cytometer are either silicon photodiodes or photomultiplier tubes (PMTs).
Silicon photodiodes were used earlier for the measurement of forward scatter. However, now-a-days PMTs are used for FSC channel alongwith SSC.
PMTs are more sensitive detectors and are ideal for scatter and fluorescenc readings.
As a particle passes through the laser beam, it will result in light scattering and fluorescence signals (if labelled). The photons of light hits the photocathode.
The single electron pulse from photocathode is amplified by passing the signal through different dynodes, which eventually hits the anode.
The electrons when reach anode, and current is created (fig 11).
(Read about PMT in our previous post on Scintillation Counter.)
The magnitude of the current is proportional to the number of photons hitting the photocathode. Hence the scatters are measured.
The values FSC and SSC are plotted on the X and Y axes of a graph, respectively. Each cell population will form different regions on the graph.
For eg: given below is the representative scatter plot of lysed normal whole blood. FSC is plotted on X-axis (provides information on the relative size of the analyzed events), while SSC is plotted on Y-axis ( provides estimates of the granularity).
Granulocytes, monocytes, and lymphocytes can be referred to as FSC-high/SSC-high, FSC-high/SSC-med, and FSC-low/SSC-low, respectively (Ossowski et al. 2015).
~ Cell Sorting:
Many flow cytometer have cell sorters, which helps in separating the cells of different types. When the desired particle is detected, droplets are formed using high-frequency vibration of the nozzle over a period of time. The particle of interest is then captured in a drop.
Later, the droplet is charged electrically. Unwanted cells or empty droplets will not be charged. The droplet then passes through an electrical field, and is deflected into a collection tube or plate. Uncharged droplets pass into the waste (see fig 13). Some of the new sorters can sort 6 populations.
Applications:
The flow cytometer has found a number of applications in different fields. Few of them are listed below:
– Identification and quantification of different types of cells in a heterogenous population. This also includes identification of microbes, abnormal cells, etc in the sample.
– The cells can be analysed based on their sizes, granularity, cell surface receptors or even intracellular components.
– The different types of cells can be sorted and isolated.
– Immunophenotyping, protein expression study in cells.
(Just for info: Here’s a paper on immunophenotyping of peripheral blood and bone marrow cells by flow cytometry.)
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References:
Rowley (2012). Flow Cytometry – A Survey and the Basics. Materials and Methods. 2. 10.13070/mm.en.2.125.
Ossowski et al. (2015) Differentiation of morphotic elements in human blood using optical coherence tomography and a microfluidic setup. Optics Express 23(21):27724-27738.
Wei et al. (2015). In Vivo Flow Cytometry Combined with Confocal Microscopy to Study Cancer Metastasis. 10.1007/978-94-007-6174-2_17-2.
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