FLUORESCENCE ACTIVATED CELL SORTER

PRESENTED BY

Sudhanshu S Aayush Jain Ankit Mishra Pramendra K

: Introduction : Principle : Components & Data : Applications

 Discovery: The Fluorescence Activated Cell Sorter (FACS) was invented in the late 1960s by Bonner, Sweet, Hulett, Herzenberg for cell sorting of viable cells at University of Stanford, USA. The term FACS was coined by Len Herzenberg who won the Kyoto Prize in 2006 for his work in flow cytometry. It is based on the Coulter principle which can be applied to count and size the various cells that make up whole blood.

What is fluorescence???
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. A wide range of fluorophores can be used as labels in flow cytometry. These each have a characteristic peak excitation and emission wavelength. Also, the emission spectra of the labels often overlap. Consequently, the combination of labels which can be used depends on the wavelength of the lamp(s) or laser(s) used to excite the fluorochromes and on the detectors available.

Introduction: Fluorescence-activated cell sorting is a specialized type of flow cytometry in which microscopic particles such as cells and chromosomes has been counted and examined by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. It is a powerful tool which enumerated lymphocytes by detecting and counting individual cells passing in a stream through a laser beam.

 Fluorescence-activated cell sorter provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument, as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.

Multiparametric analysis Total DNA content (cell cycle analysis, cell kinetics, proliferation etc. Total RNA content DNA copy number variation (by Flow-FISH) Protein expression and localization Protein modifications, phospho-proteins Cell surface antigens (CD markers) Intracellular antigens (cytokines, secondary mediators) Nuclear antigens Apoptosis (measurement of DNA degradation) Cell viability Membrane fluidity Protein expression, localization and Protein modifications Characterizing multidrug resistance (MDR) in cancer cells

 Principle: A beam of light, usually laser light, of a single wavelength is directed onto a hydrodynamically focused stream of fluid. The purpose of fluidics system is to transport particles in a fluid stream for laser beam interrogation, with only one cell or particle moving through the laser beam at any given moment. The flow of sheath fluid accelerates the particles and restricts them to the center of the sample core, in a process of hydrodynamic focusing.

 Two light beams, one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter or SSC) is equipped with and one or more fluorescent detectors. Each suspended particle from 0.2 to 150 m passing through the beam scatters the ray, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a longer wavelength than the light source.

 This combination of scattered and fluorescent light is picked up by the detectors, and, by analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak). Now it is possible to derive various types of information about the physical and chemical structure of each individual particle. FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e., shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness).

COMPONENTS
A flow cell - liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file through the light beam for sensing. An optical system commonly used are measurement of impedance (or conductivity) and optical systems - lamps (mercury, xenon); high-power water-cooled lasers (argon, krypton, dye laser); lowpower air-cooled lasers (argon; 488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV); diode lasers (blue, green, red, violet) resulting in light signals.

 A detector and Analogue-to-Digital Conversion (ADC) system - which generates FSC (Forward Scatter) and SSC (Side Scatter) as well as fluorescence signals from light into electrical signals that can be processed by a computer An amplification system - linear or logarithmic A computer for analysis of the signal

Flow Cell
Injector Tip

Sheath fluid

Fluorescence signals Focused laser beam

Fluorescence Activated Cell Sorting

488 nm laser

FALS Sensor Fluorescence detector

Charged Plates
Single cells sorted into test tubes

Forward Angle Light Scatter

Laser
FALS Sensor

90 Degree Light Scatter

Laser
FALS Sensor

90LS Sensor

DATA ANALYSIS
Gating: The data generated by flow-cytometers can be plotted in a single dimension, to produce a histogram, or in two-dimensional dot plots or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions, termed "gates." Specific gating protocols exist for diagnostic and clinical purposes especially in relation to hematology.

Computational analysis: Recent progress on automated to population gating rare and identification strategies. hidden using computational methods has offered an alternative traditional of Automated populations. include identification systems could potentially help findings Representative Portal (ImmPort). automated methods

FLOCK in Immunology Database and Analysis

APPLICATIONS
It has broad application in medicine (especially in transplantation, hematology, tumor immunology and chemotherapy, genetics and sperm sorting for sex preselection). Sperm sorting is a means of choosing what type of sperm cell is to fertilize the egg cell. It can be used to sort out sperm that are most healthy. The resultant 'sex-sorted' spermatozoa are then able to be used in conjunction artificial insemination or in-vitro fertilization (IVF) to produce offspring of the desired sex.

 In protein engineering: Flow cytometry is used in to identify cell surface-displayed protein variants with desired properties by yeast display. In cell trafficking: Flow cytometry is widely utilized in lymphocyte trafficking by using intravital microscopy (IVM). IVM is a technique by which green fluorescent protein GFP expressed on a CD45 to see all cells with this receptor expressed, and we could compare levels between before and after counting antigen stimulation and experiments, extrapolating. through lymphocytes

 In Blood Cancers and AIDS: Flow cytometry is routinely used in the diagnosis of health disorders, especially blood cancers and AIDS to count the population of lymphocytes. Purification of organelles: Since the organelle is surrounded by each type of organelle-specific proteins, FACS are very useful in purifying organelles and vesicles, particularly those that have a similar size and density.

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