Dr Gihan Gawish Hydrodynamic focusing is a technique used to provide more accurate results from flow cytometers or Coulter counters for determining the size of bacteria or cells.bacteriacells

Dr Gihan Gawish Measuring particles Counting cells happens by forcing them to pass through a small tunnel, causing disruptions in a laser light beam or electricity flow. laser These disruptions are being analyzed by the instruments. It is hard to create these small tunnels for these cells using ordinary manufacturing processes, as: 1.the diameter should be in the magnitude of micrometers 2.the length of the tunnel should exceed several millimeters.

Dr Gihan Gawish Focusing with a fluid Hydrodynamic focusing solves this by building up the walls of the tunnel from fluid, using the effects of fluid dynamics. There is a wide (hundreds of micrometers in diameter) tube created of glass or plastic, and a "wall" fluid called the sheath fluid is being pumped through.

Dr Gihan Gawish The sample is injected into the middle of the sheath flow. If the two fluids differ enough in their velocity or density, they do not mix: they form a two-layer stable flow, with the sheath enveloping the sample in a linear, steady flow.steady flow

Dr Gihan Gawish Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus. Hydrodynamic & Spectroscopy

Dr Gihan Gawish

Principles of Flow cytometry A beam of light (usually laser light) of a single wavelength is directed onto a hydro- dynamically focused stream of fluid.hydro- dynamically focused A number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors).

Dr Gihan Gawish Principles of Flow cytometry Each suspended particle from 0.2 to 150 micrometers passing through the beam scatters the light in some way. Fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a higher wavelength than the light source.

Dr Gihan Gawish Principles of Flow cytometry 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)fluorescent emission peak Then, 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 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) apoptosis

Dr Gihan Gawish Flow cytometers Modern flow cytometers are able to analyze several thousand particles every second, in "real time” It can actively separate and isolate particles having specified properties. To analyze solid tissues single-cell suspension must first be prepared.tissues

Dr Gihan Gawish A flow cytometer has 4 main components : 1.Fluidic system: The fluidic system is the heart of a flow cytometer and is responsible for transporting cells or particles from a prepared sample through the instrument for data acquision

Dr Gihan Gawish The flow cell is the functional core of the fluidic system because it presents cells in a single file for interrogation by the cytometer illumination system. A typical flow cell consists of a converging nozzle in which sample is introduced at low flow rates into a larger laminar flow of isotonic saline or sheath fluid. The cells in the sample follow the converging streamlines and are hydrodynamically focused into alignment.

Dr Gihan Gawish A flow cytometer has 4 main components : 2.Illumination system: Flow cytometers use laser beams that intersect a cell or particle that has been hydro dynamically focused by the fluidic system. Light from the illumination source passes through a focusing apparatus before it intersects the sample stream. This apparatus is a lens assembly that focuses the laser emission into a beam with an elliptical cross- section that ensures a constant amount of particle illumination despite any minor positional variations of particles within the sample stream

Dr Gihan Gawish 2.Illumination system:

Dr Gihan Gawish A flow cytometer has 4 main components : 3.Optical and electronics system: The excitation optics consists of the light source and the optical components that serve to interrogate or excite the hydrodynamically focused sample stream in the flow cell. Optical components are used to expand, shape and focus the light which then interacts with the sample in the flow cell.

Dr Gihan Gawish

A flow cytometer has 4 main components 4- Data storage and computer control system: After light scattering and fluorescence is converted to electrical signals by the optical and electronics system, the information is converted into digital data that the computer can interpret. The signals generated from cells or particles are referred to as events and are stored by the computer

Dr Gihan Gawish Early flow cytometers were generally experimental devices, but recent technological advances have created a considerable market for the instrumentation, as well as the reagents used in analysis, such as fluorescently-labeled antibodies and analysis software.fluorescently-labeled Modern instruments usually have multiple lasers and fluorescence detectors (the current record for a commercial instrument is 4 lasers and 18 fluorescence detectors).

Dr Gihan Gawish Becton, Dickinson and Company (BD) (NYSE: BDX), is a medical technology company that manufactures and sells medical devices, instrument systems and reagents. Founded in 1897NYSEBDX