Flow Cytometry M.Pharm (Pharmacology).pptx

Flow cytometry shorts notes.

1. A Presentation on the Topic PRINCIPLES AND APPLICATIONS OF FLOW CYTOMETRY Cellular and Molecular Pharmacology (MPL 104T) Presented by Mohd.Khalid Raza M.Pharm ( 1 st Year) DEPARTMENT OF PHARMACOLOGY

2. Content- Flow Cytometry Principles of Flow Cytometry Application of Flow Cytometry References 2

3. What is flow cytometry Flow cytometry is a laser based technology which is used to identify and analyse independent events, as well as cell counting, the detection of biomarkers and if required cell separation. It allows multiple parameters on each individual cell to be independently analysed in a heterogeneous population in absence of other cells, at a rate of several thousand per second. Cytometers can be used to look at a suspension of particles, for example mammalian cells, organelles such as nuclei, chromosomes or bacteria. 3

4. The three principal components of a flow cytometer are- 1. Fluidics 2. Optics 3. Electronics 4

5. Fluidics System The fluidics system consists of a central channel through which the sample is need closed by an outer sheath that contains faster flowing fluid. As the sheath fluid moves, it creates a massive drag effect on the narrowing central chamber. This alters the velocity of the central fluid whose flow front become parabolic with the greatest velocity at its center and zero velocity at the wall. 5

6. Effect creates a single stream of particles or cell and is called hydrodynamic focusing. 6

7. Optics After hydrodynamic focusing, each cell passes through a beam of light. Light is scattering or fluorescence emission (if the particle is labeled with a fluorochrome) provides information about the cell’s properties. Scattered and emitted light from cells are converted to electrical pulses by optical detectors. Flow cytometers routinely measure light is scattered at two different angles (forward and side) Forward scatter is measured in the same direction as the laser beam is moving. 7

8. Side scatter is measured at the 90 relative to the primary laser beam. 8

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10. Filters and Mirrors Emission optics: A series of filters and mirrors are used to separate and direct the light of different wavelengths to the corresponding detectors. Optical filters are designed such that they absorb or reflect some wavelengths of light, while transmitting other. 3 Types of filters- Long Pass filter Short Pass filter Band Pass filter 10

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12. Electronics When light hits a photo detector 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 analogue to digital converters (ADCs), into electrical signals large enough (5-10 volts) to be plotted graphically. Computer system is used for analysis, presentation and storage of cytometric data. This raw data is computer permits repeated analysis, often with different gating or compensation. 12

13. Application of flow cytometry The most common use of flow cytometry is in immune phenotyping (study of protein present on cell). Cell counting Cell cycle analysis Reticulocyte count Studies on chromosome Analysis of cell death 13

14. References C ytometric evolution of hematopoietic neoplasia Brent L. Wood, Michael J. Borowitz. Henry’s, 22nd edition, Chapter 34. Lovett, E.J., III, Schnitzer, B., Keren, D.F. et al. 1984. Application of flow cytometry to diagnostic pathology.  Laboratory Invest.   50 : 115–140. 14

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