Basics of Fluorescence Tom Brumett Turner Designs June 20th 2017 - - PowerPoint PPT Presentation

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Basics of Fluorescence Tom Brumett Turner Designs June 20th 2017 - - PowerPoint PPT Presentation

Jun 12, 2023 371 likes •479 views

Basics of Fluorescence Tom Brumett Turner Designs June 20th 2017 Fluorescence The molecular absorption of light energy at one wavelength and its nearly instantaneous re-emission at another wavelength. Emitted light is always a longer

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Basics of Fluorescence

Tom Brumett Turner Designs June 20th 2017

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Fluorescence

  • The molecular absorption of light energy at one wavelength and its nearly

instantaneous re-emission at another wavelength.

  • Emitted light is always a longer wavelength than the absorbed light due to

energy loss by the molecule prior to emission.

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Excitation and Emission

  • Fluorescent compounds have two characteristic spectra
  • Excitation spectrum (the wavelength and amount of light absorbed)
  • Emission spectrum (the wavelength and amount of light emitted).
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Light source Many wavelengths of light Excitation Filter Specific wavelengths of light Cuvette or Sample Cell Emission Filter Light Detector Digital Readout Wavelengths specific to compound Wavelengths created by compound, plus stray light

Filter Fluorometer

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Light Source

  • Provides the energy that excites the compound of interest by

emitting light of the desired wavelength

  • Most common are Light Emitting Diodes (LED)
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Optical Filters

  • Colored glass filters have high energy transmittance which enhances

sensitivity of fluorometers. However, they are broad bandwidth, typically allowing bandwidths of more than 100 nm to pass and provide marginal wavelength discrimination.

  • Interference filters can provide narrow bandwidths, typically 5 to 25 nm wide,

which makes them superior for wavelength selection in a fluorometer.

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Light Detectors

  • Photomultipliers or photodiodes
  • Emitted light intensity produces a proportional electrical current in the
  • detector. This current is converted to a voltage providing a digital readout.
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Some Fluorescence Applications

  • Environmental monitoring
  • Assess health of the ocean
  • Identify harmful algal blooms (HABs)
  • Oil spill tracking
  • Aquaculture
  • Monitor fish food
  • Determine fish freshness
  • Drinking water monitoring
  • Assess health of the intake water
  • Wastewater monitoring
  • Identify disposal sites
  • Dye tracing
  • Track water flow
  • DNA detection
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Some Fluorescence Wavelengths

Excitation Emission

  • Chlorophyll

465nm 700nm

  • Phycocyanin

609nm 643nm

  • Crude Oil

365nm 550nm

  • Refined Fuels

285nm 340nm

  • Optical Brighteners

365nm 445nm

  • DOM

365nm 500nm

  • Rhodamine Dye

530nm 610nm

  • Fluorescein Dye

442nm 530nm

  • Hoechst Dye

365nm 510nm