Flow Cytometer

Muhammad MusaddiqPresentation on FLOW CYTOMETRY : 

Muhammad MusaddiqPresentation on FLOW CYTOMETRY

What is Flow Cytometry? : 

What is Flow Cytometry? Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. The properties measured include a particle’s relative size, relative granularity or internal complexity, and relative fluorescence intensity.These characteristics are determined using an optical-to-electronic coupling system that records how the cell or particle scatters incident laser light and emits fluorescence.

Instrument Overview : 

Instrument Overview This view shows the primary systems of the flow cytometer schematically. These are: the fluidic system, which presents samples to the interrogation point and takes away the waste; the lasers, which are the light source for scatter and fluorescence; the optics, which gather and direct the light; the detectors, which receive the light; and, the electronics and the peripheral computer system, which convert the signals from the detectors into digital data and perform the necessary analyses. LASER SOURCE Fluidic system Detector Optical filters Computer

Interrogation Point : 

Interrogation Point The interrogation point is the heart of the system. This is where the laser and the sample intersect and the optics collect the resulting scatter and fluorescence. First, let’s talk about how the sample is delivered to the laser. Interrogation Point

Fluidics System : 

Fluidics System One of the fundamentals of flow cytometry is the ability to measure the properties of individual particles. When a sample in solution is injected into a flow cytometer the particles are randomly distributed in three-dimensional space. The sample must therefore be ordered into a stream of single particles. That can be interrogated by the machine’s detection system. This process is managed by the fluidics system.

Hydrodynamic Focusing : 

Hydrodynamic Focusing Here we see how the sample is transported through the interrogation point. For accurate data collection, it is important that particles or cells are passed through the laser beam one at a time. Most flow cytometers accomplish this by injecting the sample stream containing the cells into a flowing stream of sheath fluid or saline solution. As you can see, the sample stream becomes compressed to roughly one cell in diameter. This is called hydrodynamic focusing. Sheath fluid Central Core Single flow

Optics and Detection : 

Optics and Detection After hydrodynamic focusing, each particle passes through beams of light. Light scattering or fluorescence emission (if the particle is labeled with a fluorochrome) provides information about the particle’s properties. The laser are the most commonly used light sources in modern flow cytometry. It give information about the particle’s by forward scattering Channel and Side scattering channel The detectors Used in flow cytometry is Photomultiplier Tube(PMTs).

Forward Scatter : 

Forward Scatter As a cell passes through the laser, it will refract or scatter light at all angles. Forward scatter, or low-angle light scatter, is the amount of light that’s scattered in the forward direction as laser light strikes the cell. The magnitude of forward scatter is roughly proportional to the size of the cell, and this data can be used to quantify that parameter.

Forward Scatter Detector : 

Forward Scatter Detector But how can we record this scattered light? Light is quantified by a detector that converts intensity into voltage. In most cytometers, a blocking bar (called an obscuration bar) is placed in front of the forward scatter detector. The obscuration bar prevents any of the intense laser light from reaching the detector. As a cell crosses the laser, light is scattered around the obscuration bar and is collected by the detector.

Forward Scatter Detector(cont) : 

Forward Scatter Detector(cont) Small cells produce a small amount of forward scatter and large cells produce a large amount of forward scatter, the magnitude of the voltage pulse recorded for each cell is proportional to the cell size.

Side Scatter : 

Side Scatter The side scatter channel (SSC) provides information about the granular content within a particle A cell traveling through the laser beam will scatter light at all angles. Light scattering at larger angles, for example to the side, is caused by granularity and structural complexity inside the cell. This side-scattered light is focused through a lens system and is collected by a separate detector, usually located 90 degrees from the laser’s path.

Side Scatter (cont) : 

Side Scatter (cont) This side-scattered light is focused through a lens system and is collected by a separate detector, usually located 90 degrees from the laser’s path. The signals collected by the side-scatter detector

Fluorescence Detection : 

Fluorescence Detection The fluorescent light coming from labeled cells as they pass through the laser travels along the same path as the side scatter signal. As the light travels along this path, it is directed through a series of filters and mirrors, so that particular wavelength ranges are delivered to the appropriate detectors.

Optical Filters : 

Optical Filters The specificity of detection is controlled by optical filters, which block certain wavelengths while transmitting (passing) others. There are three major filter types. ‘Long pass’ filters allow through light above a cut-off wavelength, ‘short pass’ permit light below a cut-off wavelength and ‘band pass’ transmit light within a specified narrow range of wavelengths (termed a band width). All these filters block light by absorption

Signal Processing : 

Signal Processing When light hits a photodetector a small current (a few microamperes) is generated. Its associated voltage has an amplitude proportional to the total number of light photons received by the detector. This voltage is then amplified by a series of linear or logarithmic amplifiers, and by analog to digital convertors (ADCs), into electrical signals large enough (5–10 volts) tobe plotted graphically.

Computer System : 

Computer System which convert the signals from the detectors into digital data and perform the necessary analyses by software and get final results.

Thanks : 

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