dr gihan gawish hydrodynamic focusing is a technique used to provide more accurate results from flow...

Dr Gihan GawishDr 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.


Measuring particles• Counting cells happens by forcing them to pass

through a small tunnel, causing disruptions in a laser light beam or electricity flow.

• 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.


• 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.


• 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.


• 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


Principles of Flow cytometryPrinciples of Flow cytometry

• A beam of light (usually laser light) of a single wavelength is directed onto a hydro-dynamically focused stream of fluid.

• 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).



• 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.



• 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)

• 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


Flow cytometersFlow 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.


A flow cytometer has 4 main A flow cytometer has 4 main componentscomponents: :

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


• 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.


A flow cytometer has 4 main A flow cytometer has 4 main componentscomponents::

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


22..Illumination systemIllumination system::


A flow cytometer has 4 main A flow cytometer has 4 main componentscomponents::

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.


A flow cytometer has 4 main A flow cytometer has 4 main componentscomponents

• 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


• 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.

• Modern instruments usually have multiple lasers and fluorescence detectors (the current record for

a commercial instrument is 4 lasers and 18 fluorescence detectors).


• Becton, Dickinson and Company (BD) (NYSE: BDX), is a

medical technology company that manufactures and sells medical devices,

instrument systems and reagents. Founded in 1897

dr gihan gawish hydrodynamic focusing is a technique used to provide more accurate results from flow...

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