9/25/2023 0 Comments Define cytometry![]() However, most absorption occurs with very narrow light wavelengths represented by the peak spectra. Soon after excitation, the electrons revert to their ground states, emitting fluorescent light with a wavelength that is longer than that of laser light.Įach fluorochrome can absorb a specific range of light, called the excitation spectra. In addition to light scattering, the laser beam can excite the electrons of a fluorochrome, which is used to stain or interact with the sample cells. FSC is generally used to determine the surface size or area of the cells while SSC typically reflects the particle’s internal complexity. When the laser light illuminates the sample cells, it ricochets in different directions depending on their physical properties.įorward scatter lights (FSC) refers to the diffracted light, while the refracted light is called side scatter lights (SSC). Light scatteringįigure 2: Light scattering when the laser beam illuminates a cell in a flow cytometer Hydrodynamic focusing occurs at the nozzle tip where the sample stream is released in the air so the laser beam can excite it. In stream-in-air models, sheath and sample fluids are pressurized downward into a small narrow aperture. At the optically clear region or cuvette, the cells are illuminated by the laser beam, sending off fluorescence and light scattering. In benchtop cytometers, the sample fluids are injected upward into the flow chamber, called the flow cell. The sheath stream accelerates the sample fluid and its suspending cells, causing the cells to become focused on the sample core where they can be struck individually by the laser beam. Instead, it flows along the same axis as the sheath fluid but with different pressure and flow rate. Subsequently, hydrodynamic focusing is created by interjecting the sample fluid into the steady stream of the sheath fluid. The sheath fluid is typically an isotonic buffer and is first injected into the fluidics system, creating a laminar flow of a smooth fluid stream with steady pressure. The three systems in flow cytometers achieve their tasks based on the following principles: Laminar flow and hydrodynamic focusing The electronics system collects light signals from the optics system and scattered light resulting from cells. The optics system directs fluorescent light to the detectors, including scattered light caused by cells. Sample cells are transported by sheath fluid into the flow chamber via the fluidics system. Some flow cytometers also possess a sorting function where the illuminated cells are captured and collected based the on user’s definition.įigure 1:How a flow cytometer works. These numbers are collected and stored in a computer before they are displayed as histograms and graphs such as dot displays. The electronics systemĬonverts the light signals from detectors into electrical signals, which are subsequently converted into numbers. The lenses are beam splitters and filters that collect and direct the fluorescence and scattered lights onto detectors like photodiodes and photomultiplier tubes (PMTs). The system also contains optical lenses and detectors. Houses the light source for the laser beam, which is used to measure the suspending cells. Transports a sheath fluid and the sample fluid containing the target cells, collectively called the sample core, into the flow chamber and collects the waste fluids after the sample has been illuminated by the laser beam. The instrument can be benchtop or stream-in-air devices, but both consist of these systems: The fluidics system What is Flow Cytometry?įlow cytometry is a high-throughput instrument that measures the physical properties of cells suspended in fluid based on their abilities to absorb and scatter light. Thus, flow cytometry is useful for analyzing biological samples such as blood, serum, dividing, and cancerous cells in research and clinical diagnosis. The technique provides the physical and chemical characteristics of every individual cell excited by the laser beam, providing information on the properties of the mixed cell population in minutes. ![]() These lights are split and filtered onto detectors and converted into electrical signals. The illumination causes fluorescence and scattered lights, which are emitted and reflected from the examining cell. It uses a built-in laser beam to illuminate individual cells as the fluid passes through. The Definitive Guide to Flow Cytometry for Scientistsįlow cytometry is an analytical technique that examines cells suspended in fluids. To reference this article: The Definitive Guide to Flow Cytometry, ConductScience. Balances, Scales and Weighing Equipment.
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