BME 210 Lecture 10 Flow Cytometry

BME 210 Lecture 10 - 10 Flow Cytometry The principle of Flow Cytometry • Flow Cytometry the method allowing to measure multiple optical

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10. Flow Cytometry
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The principle of Flow Cytometry Flow Cytometry - the method allowing to measure multiple optical parameters of cells as they pass through a measuring apparatus in a single file suspended in a fluid stream 2-8 optical parameters are measured simultaneously at rates of thousands of cells per second, which can be used to characterize large cell populations with respect to: cell types and numbers protein content gene expression (RNA content) etc
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Flow Cytometer 1. Laser 2. Fluidics system 3. Optics 4. Detectors 5. Data acquisition system http://www.invitrogen.com/site/us/en/home/support/Tutorials.html
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Fluidics system • Cells pass through a laser beam one a time due to Hydrodynamic Focusing : • 2 coaxial streams: (1) a cell-containing stream is injected into (2) a sheath stream (e.g., saline). The flow is laminar - two streams don’t mix The sheath stream is faster - the cell stream becomes compressed to approximately one cell per stream diameter even before reaching narrow output nozzle (diameter ≈70 µm) • Typically, cells with diameter in the range of 1-15 μm are detected • Flow cytrometry works only on single cell suspensions; hydrodynamic focussing cannot separate cell aggregates Laser beam Hydrodynamic Focusing
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Light scatter When a cell passes through the laser beam, it scatters light (due to reflection and refraction) at many different angles. Measurements are performed at two angles: 2-20 o angle - Forward scatter 90 o angle - Side scatter Side scatter Forward scatter
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Forward scatter A blocking screen is placed in front of a photodetector to block the unscattered light, which is much stronger than the scattered light A single-channel detector (PMT) measures the scattered light and converts it to voltage Voltage signal produced by one cell
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Forward scatter and cell size The larger the cell size, the bigger the voltage pulse Pulse amplitude (or pulse area) of forward scatter is approximately proportional to the cell size Cell size
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This note was uploaded on 04/28/2010 for the course BME eng. biolo taught by Professor Fast during the Spring '10 term at University of Alabama at Birmingham.

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BME 210 Lecture 10 - 10 Flow Cytometry The principle of Flow Cytometry • Flow Cytometry the method allowing to measure multiple optical

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