Dr Gihan Gawish Hydrodynamic focusing is a technique used to provide more accurate results from flow...
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![Page 1: Dr Gihan Gawish Hydrodynamic focusing is a technique used to provide more accurate results from flow cytometers or Coulter counters for determining the.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649eb55503460f94bbec3f/html5/thumbnails/1.jpg)
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.
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Dr Gihan GawishDr 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.
• 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.
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Dr Gihan GawishDr 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.
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Dr Gihan GawishDr 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.
![Page 5: Dr Gihan Gawish Hydrodynamic focusing is a technique used to provide more accurate results from flow cytometers or Coulter counters for determining the.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649eb55503460f94bbec3f/html5/thumbnails/5.jpg)
Dr Gihan GawishDr 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
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Dr Gihan GawishDr Gihan Gawish
![Page 7: Dr Gihan Gawish Hydrodynamic focusing is a technique used to provide more accurate results from flow cytometers or Coulter counters for determining the.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649eb55503460f94bbec3f/html5/thumbnails/7.jpg)
Dr Gihan GawishDr Gihan Gawish
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).
![Page 8: Dr Gihan Gawish Hydrodynamic focusing is a technique used to provide more accurate results from flow cytometers or Coulter counters for determining the.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649eb55503460f94bbec3f/html5/thumbnails/8.jpg)
Dr Gihan GawishDr Gihan Gawish
Principles of Flow cytometryPrinciples 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.
![Page 9: Dr Gihan Gawish Hydrodynamic focusing is a technique used to provide more accurate results from flow cytometers or Coulter counters for determining the.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649eb55503460f94bbec3f/html5/thumbnails/9.jpg)
Dr Gihan GawishDr Gihan Gawish
Principles of Flow cytometryPrinciples 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)
• 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
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Dr Gihan GawishDr Gihan Gawish
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.
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Dr Gihan GawishDr Gihan Gawish
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
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Dr Gihan GawishDr 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.
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Dr Gihan GawishDr Gihan Gawish
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
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Dr Gihan GawishDr Gihan Gawish
22..Illumination systemIllumination system::
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Dr Gihan GawishDr Gihan Gawish
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.
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Dr Gihan GawishDr Gihan Gawish
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Dr Gihan GawishDr Gihan Gawish
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
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Dr Gihan GawishDr 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.
• Modern instruments usually have multiple lasers and fluorescence detectors (the current record for
a commercial instrument is 4 lasers and 18 fluorescence detectors).
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Dr Gihan GawishDr 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 1897