FlowCytometry

Flow Cytometery as the name suggests is a technique for cell counting, inter-complexity, shape and measurement of different properties of the cell. In this technique there is a column in which cells are placed can be more granulated or agranulated when these cells are passed through the column the beam is passed through the cell which is detected by the detector. 

Adding saline solution to the column gives the directional focusing. The cells which have no granules, passing through the column shows forward scattering. While the cell with granules show side scattering.These detectors are connected by the computer. 

A flow cytometer is made up of three main systems:

·        FLUIDICS - the fluidics system transports particles  in a stream to the laser beam for interrogation

·        OPTICS-The optics system consists of lasers to illuminate the particles in the sample stream and optical filters to direct the resulting light signals to the appropriate detectors.

·        ELECTRONICS-The electronics system converts the detected light signal into electronic signals that can be processed by the computer. This is system is also capable of initiating sorting decisions to charge and deflect particles.

A fluorescent compound absorbs light energy over a range of wavelengths that is characteristic for that compound. This absorption of light causes an electron in the fluorescent compound to be raised to a higher energy level. The excited electron quickly decays to its ground state, emitting the excess energy as a photon of light. This transition of energy is called fluorescence.

The argon ion laser is commonly used in flow cytometry because the 488-nm light that it emits excites more than one fluoro-chrome. Light signals are generated as particles pass through the laser beam in a fluid stream. These light signals are converted to electronic signals (voltages) by photo detectors and then assigned a channel number on a data plot. 

There are two types of photodetectors in BD flow cytometers:

 Photodiodes and Photomultiplier tubes (PMTs). 

The photodiode is less sensitive to light signals than the PMTs and thus is used to detect the stronger FSC signal. PMTs are used to detect the weaker signals generated by SSC and fluorescence. A voltage pulse is created when a particle enters the laser beam and starts to scatter light or fluoresce. Once the light signals, or photons, strike one side of the PMT or the photodiode, they are converted into a proportional number of electrons that are multiplied, creating a greater electrical current. The electrical current travels to the amplifier and is converted to a voltage pulse. The highest point of the pulse occurs when the particle is in the center of the beam and the maximum amount of scatter or fluorescence is achieved. As the particle leaves the beam, the pulse comes back down to the baseline

Data Collection and Display

Once light signals have been converted to electronic pulses and then converted to channel numbers by the ADC, the data must be stored by the computer system. Flow cytometric data is stored according to a standard format, the flow cytometry standard (FCS) format, developed by the Society for Analytical Cytology. According to the FCS standard, a data storage file includes a description of the sample acquired, the instrument on which the data was collected, the data set, and the results of data analysis.

 A single cell analyzed for four parameters (FSC, SSC, FITC, and PE fluorescence) generates 8 bytes of data. When multiplied by the approximately 10,000 events collected for a single sample, an FCS data file typically contains 80 kB of data. Once a data file has been saved, cell populations can be displayed in several different formats. A single parameter such as FSC or FITC (FL1) can be displayed as a single parameter histogram, 

where the horizontal axis represents the parameter’s signal value in channel numbers and the vertical axis represents the number of events per channel number. Each event is placed in the channel that corresponds to its signal value. Signals with identical intensities accumulate in the same channel. Brighter signals are displayed in channels to the right of the dimmer signals. A subset of data can be defined through a gate. A gate is a numerical or graphical boundary that can be used to define the characteristics of particles to include for further analysis this is known as Gating.