flow cytometry and facs

FLOW CYTOMETRY : 

FLOW CYTOMETRY M-3908

INTRODUCTION : 

INTRODUCTION Flow – act of moving Cytometry – measurement of physical and chemical characteristics of cells Flow cytometry –measurement of single cells as they pass in a fluid stream through a measuring point surrounded by an array of detectors.

History : 

History Flow cytometry developed from microscopy. Thus Leeuwenhoek is often cited in any discussion regarding it’s history. Moldaven (1934) - attempted photoelectric counting of cells flowing through a capillary tube. L. Kamentsky and M. Fulwyler (1965) - experimented with fluidic switching and electrostatic cell sorters respectively. Both described cell sorters. M. Fulwyler utilized Pulse Height Analyzers to accumulate distributions from a Coulter counter. This feature allowed him to apply statistical analysis to samples analyzed by flow.

History : 

History Van Dilla, Fulwyler and others (1969) - developed the first fluorescence detection cytometer that used the principle of hydrodynamic focusing, 90oC optical configuration and argon ion laser excitation source. In 1972 L. Herzenberg (Stanford Univ.), developed a cell sorter that separated cells stained with fluorescent antibodies.The Herzenberg group coined the term Fluorescence Activated Cell Sorter (FACS). (KYOTO prize in 2006) Kohler and Milstein (1975) –introduced monoclonal antibody technique, provide basis for specific immunological reagents for use in cell studies.

Principle : 

Principle FC is a laser based technique in that light is scattered and the fluorescence is emitted as light from the excitation source strikes the moving particles. Light scattering and fluorescence is measured for individual particle that passes an excitation source. Scattering and emission data can be used to examine characteristics of particles.

Slide 6: 

Optics FL2 PE SSC FSC Fluidics Electronics Computer FL3 PerCP, Cy5 FL1 FITC Light detectors Components of FC Laser

Working : 

Working Prepare the sample Suspend in medium Label with fluorochrome and introduce in chamber Single Cell pass in a flow – hydrodynamic focusing Each cell is then intersected by a laser beam Cell produce a short flash of fluorescence in all directions

Working : 

Working Different fluorochrome produce fluorescence of different wavelength Forward flashes and flashes in 90o direction to the cell surface are then collected by series of optics Flashes are then focused on sensitive photodetectors (PMT) PMT transforms the flashes into electric pulses which are recorded by electronic converters These are then transferred to computer for assimilation and interpretation.

Working : 

Working It is possible to label the cell with more than one label at a time Simultaneous measurement of two cellular constituents can be integrated as ++, +-, -+, -- FC can measure- 1)Cell size 2)Cell density This can be used in hematology

Sample preparation : 

Sample preparation Density of suspension must be 105-107 cells/ml in PBS buffer Whole blood lyse RBC lymphocyte count Solid tissue disaggregate cells mechanically or enzymatically To study intracellular components, make the cell membrane permeable for dyes or EDTA or antibodies by using low concentration of non-ionic detergents

Fluorochromes and light : 

Fluorochromes and light Fluorochromes are dyes which accept light at given wavelength and re-emit at higher lambda Light used is laser light from the source such as argon and krypton-argon lasers

Probes for Proteins : 

Probes for Proteins FITC 488 525 PE 488 575 APC 630 650 PerCP™ 488 680 Probe Excitation Emission Peridinin-chlorophyll-protein complex

Specific Organelle Probes : 

Specific Organelle Probes BODIPY Golgi 505 511 NBD Golgi 488 525 DPH Lipid 350 420 TMA-DPH Lipid 350 420 Rhodamine 123 Mitochondria 488 525 DiO Lipid 488 500 diI-Cn-(5) Lipid 550 565 diO-Cn-(3) Lipid 488 500 Probe Site Excitation Emission BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH – diphenylhexatriene TMA - trimethylammonium

Probes for Nucleic Acids : 

Hoechst 33342 (AT rich) (uv) 346 460 DAPI (uv) 359 461 POPO-1 434 456 YOYO-1 491 509 Acridine Orange (RNA) 460 650 Acridine Orange (DNA) 502 536 Thiazole Orange (vis) 509 525 TOTO-1 514 533 Ethidium Bromide 526 604 PI (uv/vis) 536 620 7-Aminoactinomycin D (7AAD) 555 655 Probes for Nucleic Acids

Hydrodynamic focusing : 

Hydrodynamic focusing

Optical Filters : 

Optical Filters Specificity of detection is controlled by optical filters These block the certain wavelength by absorption and transmit others There are three types

Types of Optical Filters : 

Types of Optical Filters

Photodetectors : 

Photodetectors These are the fluorescence channel to detect light emitted Silicon photodiodes, PMT When light hit, small current is generated, amplified by amplifier into electrical signals(5-10 volts)

Slide 19: 

Creation of a Voltage Pulse 1. Cells enter the laser beam, the FS detector picks up the scattered light and the pulse begins. When a minimum threshold (blue line) is exceeded, the timing window opens. 2. The cell is fully in the laser beam. The pulse reaches the highest level. A bigger particle gives a higher voltage. 3. As the cell exits the laser beam the voltage drops. As it falls below the threshold, the timing window closes. Picks highest value and digitizes. This is the intensity of the signal

Electronics : 

Electronics Purpose is to monitor and control the operation of FC The amplified signals from PMT are presented to the processor Threshold is set Signals are then sent to computer where it undergo digital conversion through analogue-to digital (ACD) converter which is then plotted as histogram or dot plot This data is then stored in a FC standards (FCS)

Data analysis : 

Data analysis Histogram

Data analysis : 

Data analysis Dot plot

Types of flow cytometers : 

Types of flow cytometers Coulter epics elite Partec PAS III Microcyte FACS

Slide 24: 

FACS

Application of FC : 

Application of FC Molecular and cell biology Clinical immunology Plant biology Marine biology Cell cycle kinetics Genetics Parasitology Bioterrorism Chemotherapy Microbiology

Advantages : 

Advantages 1. Distinct cell populations are defined by their size (forward light scatter) and granularity (side light scatter). 2. Dead cells may be gated out of the analysis. 3. Weakly expressed surface antigens may be detected. 4. Multicolor (2-, 3-, 4-) analysis may be performed, allowing for an accurate definition of the surface antigen profile of specific cells. 5. Two simultaneous hematologic malignancies may be detected within the same tissue site. 6. Tissue biopsy may be obviated by the relatively non-invasive diagnostic evaluation of body fluids.

Disadvantages : 

Disadvantages High speed FC- expensive Difficult to keep sorted cell gnotobiotic (can be done but increases complexity of operation) Sclerotic tissue may be difficult to suspend for individual cellular analysis There is loss of architectural relationships

REFERENCE : 

REFERENCE Thrope R. and Thrope S.Immunochemical techniques,in : principles and techniques of biochemistry Molecular biology by Wilson and Walker(2005) 6th edition,cambridge university press,pg no: 159,345-346,737. Henry john B.(1996),Flow cytometry ; in: clinical diagnosis and management by laboratary method. 19th edition, A prism edition,pg no: 62-63. Harrigan wilkie F. (1995) in: laboratary methods in food microbiology,3rd edition,academic press london,pg 74. I. M. Roitt, (2005)Immunological methods and application in: essential immunology,11th edition,academic press london, pg :123-125. Ascacher ronald and et. al. in: Wedmens clinical interpretation of laboratarhy test 11th edition pg: 352-55.

Reference : 

Reference H:\ppt fc\Fluorochrome Table.htm H:\ppt fc\ad and disad.htm http://www3.interscience.wiley.com/journal/48689/abstract?CRETRY=1&SRETRY=0 http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/ http://en.wikipedia.org/wiki/Flow_cytometry http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html http://www.google.co.in/imgres?imgurl=http://ase.tufts.edu/biomedical/research/georgakoudi/images/flowCytometry http://www.abdserotec.com/uploads/Flow-Cytometry.pdf