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Biochemical instrumental analysis-3. Dr. Maha Al- Sedik. Emission spectroscopy. LUMINESCENCE. Florescence : The light emission of a given wavelength by a substance that had absorbed light of a different wavelength. OR

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slide4

Florescence :

  • The light emission of a given wavelength by a substance that had absorbed light of a different wavelength.
  • OR
  • Absorption of radiant energy and re-emission of some of this energy in the form of light.
  • The light emitted is of longer wave length than the one absorbed ( Stokes law ).
slide5

Fluorescence occurs when an orbital electron of a molecule, atom or nanostructure relaxes to its ground state by emitting a photon of light after being excited to a higher quantum state by some type of energy.

slide8

The most striking examples of fluorescence occur when the absorbed radiation is in the ultraviolet region of the spectrum, and thus invisible to the human eye, and the emitted light is in the visible region..

slide10

Fluorometer

  • Principle:
  • The appropriate wave length is selected from the light source and then falls onto the sample.
  • The substance absorbs part from this light.
  • Measurement of fluorescence (light emitted from substance after being stimulated by light energy).
  • The amount of emitted light is directly proportional to the concentration of substance.
slide12

COMPONENTS:

  • 1- light source.
  • 2- monochromator.
  • 3-cuvet.
  • 4- monochromator.
  • 5-detector.
slide14

COMPONENTS:

  • The light source is high intensity lamp as xenon-arc lamp.
  • Monochromator.
  • Cuvet: all faces must be optically clear.
  • Monochromator.
  • Detector: fluorescence is measured at right angle to the existing source.
slide16

Applications of fluorimetry:

  • 1-measurement of naturally fluorescent compounds:
  • such as tyrosine, tryptophan, bilirubin and vitamins.
  • 2-measurment of chemically induced fluorescence:
  • Tagging the compounds with fluorescence label.

Labeling of antigen with fluorescence label:

Antibody to these antigen are labeled with fluorescence so allowing its binding to the Ag.

slide17

When a fluorescent dye is conjugated to a monoclonal antibody, it can be used to identify a particular cell type based on the individual antigenic surface markers of the cell.

  • In a mixed population of cells, different fluorochromes can be used to distinguish separate subpopulations.
slide21

what happens to the laser light as it strikes the single-file particles.

Light scattering

slide23

Light scattering:

  • It occurs when a particle deflects incident laser light.
  • Factors that affect light scattering are the cell's membrane, nucleus, and any granular material inside the cell. Cell shape and surface topography also contribute to the total light scatter.
  • Types of light scattering:
  • Forward-scattered light (FSC).
  • Side-scattered light (SSC).
slide25

Forward-scattered light (FSC):

  • FSC is proportional to cell surface area or size.
  • FSC is a measurement of mostly diffracted light.
  • FSC is detected just off the axis of the incident laser beam.
  • FSC provides a suitable method of detecting particles greater than a given size independent of their fluorescence.
  • FSC is often used in immunophenotyping .
slide28

Side scattered light (SSC):

  • SSC is proportional to cell granularity or internal complexity.
  • SSC is a measurement of mostly refracted and reflected light that occurs at any interface within the cell.
  • SSC is collected at approximately 90 degrees to the laser beam by a collection lens.