FLOW CYTOMETRY

FLOW CYTOMETRYMOHAMED NIJAS VSEMESTER 7I.S.P.

INTRODUCTIONA flow cytometer is an optical diagnostic device which is used in research and clinical laboratories for disease profiling by measuring the physical and/or chemical characteristics of cells. also suitable for rapid and sensitive screening of potential sources of deliberate contamination, an increasing source of concern of bioterrorism. also emerges as a powerful technique for agriculture research and livestock development.

WHAT IS CYTOMETRY???The term cytometry refers to the measurement of physical and/or chemical characteristics of cells or, in general, of any biological assemblies. In flow cytometry, such measurements are made while the cells, the biological assemblies, or microbeads (as calibration standards) flow in suspension, preferably in a single file, one by one, past a sensing point. The sensing is conducted by using an optical technique where the beam from a light source interacting with each individual cell, a bio assembly, or a microbead produces scattering or fluorescence.The optical response is used to determine cellular features and organelles, providing counts and ability to distinguish different types of cells in a heterogeneous population

A LITTLE MOREAnother name used for ow cytometry and thus applied interchangeably is uorescence-activated cell sorting (FACS), which emphasizes the utilization of uorescence detection and the ability of the instrument to sort cells that meet specic measured criteria. In a regular microscope with point detection (such as in a confocal microscope),the light (laser) beam moves (scanned) to detect and image cells, but the cells are moving (owing in a single le) in a ow cytometer.

THE COMPONENTS OF A FLOW CYTOMETER Light Source. A ow cytometer may use a single excitation wavelength or a number of excitation wavelengths from different laser sources.Flow Cell. The ow cell is designed to hydrodynamically focus the sampleIllumination Optics. Optical elements between the laser and the sample are referred to as illumination optics and are used to shape and focus the laser beam. Collection Optics. The collection optics consists of a train of optical ports to separate and collect various optical responses produced by illumination of each owing cell. Detection and Electronics. In most ow cytometers, the photodetectors used for ourescence detection are photomultiplier tubes. Cell Sorter. The two types of cell sorting devices used in ow cytometry are (a) electrostatic sorting, (b) mechanical sorting

LIGHT SOURCEA ow cytometer may use a single excitation wavelength or a number of excitation wavelengths from different laser sources. The laser beams can be coaxial or separated so that one or more interrogation point occurs. The critical requirements for a laser in ow cytometry are power stability, high-quality beam characteristics, and low-level high-frequency noise.Currently used ones are compact solid-state lasers. Another new prospect is the use of near IR lasers (700800nm) to excite IR dyes, whereby the problem due to autouorescence interference is considerably reduced.

FLOW CELLThe ow cell is designed to hydrodynamically focus the sample stream.A cell suspension is introduced through a core inlet which has an inner diameter of 20mm and is surrounded by a larger (~200mm) stream of owing saline (sheath liquid). Therefore,in this arrangement,a core stream of the cell suspension is injected into the center of the sheath stream.During the ow,the sheath uid produces hydrodynamic focusing of coaxial ow of the core uid, whereby the two streams maintain their relative positions and do not mix signicantly and move at the velocity of the sheath uid.

The hydrodynamic focusing produces the ow of cells in a single le (one cell at a time). The core and sheath streams are driven by syringe pumps or by sources of pressure that deliver a known volume of sample per unit time with minimum pulsation. From the sample ow rate, one can easily derive the cell count per unit volume.

ILLUMINATION OPTICSOptical elements between the laser and the sample are referred to as illumination optics and are used to shape and focus the laser beam.The beam shaping optics most frequently used in current ow cytometers utilize a pair of anomorphic prisms or two crossed cylindrical lenses that provide an elliptical spot of 10 20mm in dimension parallel to the direction of cell ow and 60mm in dimension perpendicular to the ow dimensions. .The elliptical beam provides a wider illumination eld across the width of the ow At the same time, the smaller (20mm) dimension of the elliptical beam parallel to the ow direction allows cells to pass in and out of the light illumination quickly and avoids simultaneous

COLLECTION OPTICSThe collection optics consists of a train of optical ports to separate and collect various optical responses produced by illumination of each owing cell.These optical responses are (i) forward scattering count (FSC), (ii) side scattering count (SSC),and (iii) various uorescence signals at different wavelengths collected at 90 to the laser beam.The number of optical responses detected (hence the number of photodetectors used) dene what is known as the number of measured parameters in a ow cytometer.

DETECTION AND ELECTRONICS.In most ow cytometers, the photodetectors used for ourescence detection are photomultiplier tubes.An electrical preamplier following the photodetector is required because a dc offset voltage that establishes zero baseline is used to account for steady-state stream uorescence. This stream uorescence is the result of uorochromes remaining in solution. Subsequent ampliers can be either logarithmic or linear. The logarithmic amplier allows one to process signals over a wide range of intensities, while a linear amplier restricts sensitive measurements to signals in a small linear range.The logarithmic ampliers also have an offsetcontrol to select an intensity range to be analyzed without changing the amplication.

CELL SORTERMost commercial ow cytometers are not equipped with a cell sorter capability.

In the electrostatic sorting device, the cells of a specic type, after passing the interrogation point, are charged and electrostatically deected to a collection point. The electrostatic sorting method can be operated at rates up to 50,000 cells per second. The necessary time delay to trigger the vibrating nozzle and the charging collar can be determined from the ow rate of the cells.

The second method utilizes a mechanical gate that swings back and forth to direct a particular type of cell into a desired pathway. While this method is considered to be more gentle on cells ,it has only a maximum rate of 300 sorted cells per second.

BASICS OF FLOW CYTOMETRY

APPLICATIONSHIV monitoring Leukaemia or lymphoma immunophenotyping Organ transplant monitoringDNA analysis for tumour ploidy and SPFPrimary and secondary immunodeciencyHematopoietic reconstitution Paroxysmal nocturnal haemoglobinuriaMultiplexing immunoassays Multiparameter immunophenotyping Measurement of intracellular cytokines Signal transduction pathways Cell cycle analysis Measuring cellular functionMultiplexing oligonucleotide assays Measuring gene expression In situ hybridization Drug discoveryClinical Applications Research Applications Molecular Flow Cytometry

IMMUNOPHENOTYPINGIdentication of cells using uorochrome conjugated antibodies as probes for proteins (antigens) expressed by cells. The reasons for using the term immunophenotyping are twofold (I) it relates to the activities of immunological species, namely, antibodies (Ii) it is primarily used to identify lymphoid and hematopoietic cells, which are constituents of immune systems.

So it deals with classication of normal or abnormal white blood cells according to their multiparameter surface antigen characteristics, which can then be used as a prole for a specic disease or malignancy. Hiv immunophenotyping is another common clinical application of ow cytometry.

FUTURE DIRECTIONSFlow cytometry is a rapidly expanding eld worldwide where an enormous increase in its capability can be expected over the coming years. There has been renewed interest in ow cytometry from the point of view of research where a major impetus is derived from its applications to genomics and proteomics. Recent advances in solid-state lasers, microuidics, microarray technology, micro-optics, and miniaturized detectors provide challenging technological opportunities for developing small and compact ow cytometers with enhanced capabilities to simultaneously monitor many more parameters than currently possible. It is refuelling the expectation that perhaps a ow cytometer-on-a-chip is not such a distant dream.

REFERENCESIntroduction To Biophotonics : P.N. Prasad

Castro, A., Fairdield, F. R., and Shera, E. B., Fluorescence Detection and Size Measurement of Single DNA-Molecules, Anal. Chem. 65, 849852 (1993).Givan,A. L.,Flow Cytometry:First Principles,2nd edition,Wiley-Liss,New York,2001.

Thank You

Thank You(Basic)

To reproduce the video effects on this slide, do the following:

On the Home tab, in the Slides group, click Layout, and then click Blank.On the Insert tab, in the Media group, click Video, and then click Video from File. In the left pane of the Insert Video dialog box, click the drive or library that contains the video. In the right pane of the dialog box, click the video that you want and then click Insert.Under Video Tools, on the Format tab, in the Sizing group, click the arrow to the right of Size launching the Format Video dialog box, select Size from the left pane and under Size in the right pane do the following:Click the Lock Aspect Ratio box.In the Height box, enter 6.03.In the Width box enter 8.03. Also in the Format Video dialog box, click Border Color in the left pane, under Border Color in the right pane select Solid Line, and then click the arrow to the right of Color, and under Theme colors select Black, Text 1, Lighter 25% (fourth row, second option from left).Also in the Format Video dialog box, select Border Style in the left pane, under Border Style in the right pane set the Width to 15 pt.Also in the Format Video dialog box, select Shadow in the left pane, under Shadow in the Right pane, click the arrow to the right of Colors and under Theme Colors, select Black, Text 1 (first row, second option from left), and then do the following:In the Transparency box, enter 60%.In the Size box, enter 100%.In the Blur box, enter 21 pt.In the Angle box, enter 40 degrees.In the Distance box, enter 19 pt.Also in the Format Video dialog box, select 3-D Format in the left pane, under Bevel in the right pane click the arrow to the right of Top and under Bevel, select Relaxed Inset (first row, second option from left), and then do the following:To the right of Top, in the Width box, enter 6 pt.To the right of Top, in the Height box, enter 16.5 pt.On the Home tab, in the Drawing group, click Arrange, point to Align, and then do the following: Click Align Center.Click Align Middle. Under Video Tools, on the Playback tab, in the Video Options group, select Loop until Stopped.On the Animations tab, in the Animation group, select Play.On the Animations tab in the Timing group, click the arrow to the right of Start and select With Previous.

To reproduce the text effects on this slide, do the following:

On the Insert tab, in the Text group, click Text Box, and then on the slide drag to draw a text box.Type text in the text box (Thank You or whatever text suits your message).Select the text, on the Home tab, in the Font group, select Garamond from the Font list, select 88 pt from the Font Size list, and then click on the Bold icon.Also in the Home tab, in the Font group, select the arrow to the right of the Font Color Icon, and then under Theme Colors, select White, Background 1 (first row, first option from left).With the text box selected, under Drawing Tools, on the Format tab, click the arrow in the bottom right corner of the WordArt Styles group, click the arrow opening the Format Text Effects dialog box. In the Format Text Effects dialog box, click 3-D Format on the left pane, under Bevel on the right pane, click the arrow next to Top and under Bevel select Relaxed Inset (first row, second option from left). Set the Width to 5 pt and the Height to 3 pt.Also in the 3-D Format right pane, under Surface, click the arrow next to Material and under Special Effect select Dark Edge (first row, first option from left).Also in the 3-D Format right pane, under Surface, click the arrow next to Lighting and under Neutral select Soft (first row, third option from left).Also in the 3-D Format right pane, under Surface, set the Angle to 290 Degrees.Close the Format Text Effects dialog box.

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On the Design tab, in the bottom right corner of the Background group, click the arrow at the bottom right corner launching the Format Background dialog box.In the Format Background dialog box, select Fill in the left pane, and under Fill in the right pane select Solid fill, then click the arrow to the right of Color and under Theme Colors select White, Background 1, Darker 50% (sixth row, first option from left).Close the Format Background dialog.

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