Fluorescence%20101

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Fluorescence 101 Steve Lee MiraiBio Inc. STR
2003
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Outline

• Introduction to Fluorescence
• Principles and Definitions
• Stokes shifts, Jablonski diagrams, excitation
  and emission, extinction coefficient, quantum
  efficiency
• Excitation and Emission Spectra
• Choosing Exicitation Wavelengths III, III plus
• Choosing Emission Filters
• Chemistry The Dyes
• Structure- Big Greasy Blobs
• Effects of structure on fluorescence
• Other factors
• Effects of rigidity, pH and temperature
• Effects of Fluorophores on Oligos and visa versa

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Why Fluorescence?
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Advantages of Fluorescence

• Easy, Fast (eg. vs silver staining)
• Visualize tagged primer strand
• Multiplexing
• High Sensitivity
• Dynamic Range

Detection of 25 pg of dsDNA with PicoGreen Reagent
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Principles and DefinitionsWhat is Fluorescence?
Fluorescence is a molecular phenomenon in which a
substance absorbs light of some color
(excitation) and almost instantaneously radiates
light of another color, one of lower energy and
thus longer wavelength (emission). Primary
fluorescence- intrinsic property of a
substance Secondary or indirect fluorescence uses
dyes Fluorochromes dyes Fluorescent probes or
fluorophores are dyes conjugated to substances
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How does it work?
laser beam
1. laser strikes fluorophore
2. fluorophore absorbs laser energy
3. fluorophore emits light at a Longer wavelength
Light is collected? CCDs or PMTs
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Three-Stage Process of Fluorescence
- Jablonski
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The 3 stage Fluorescence Process- Jablonski
diagram

• 1- Excitation Photon of energy (hvEX) strikes a
  fluorophore ? excited state
• 2- Excited State Lifetime Energy dissapated by
• a. Relaxed state ? emission
• b. Quenching, energy transfer
• Quantum yield
• fluor photons emitted
• photons absorbed
• Most efficient are 0.3 values reduced by
  quenching- eg photobleaching
• 3- Fluorescence Emission Photon of energy (hvEM
  ) is emitted
• Due to energy dissapation in 2, emitted photon
  is of lower energy and longer wavelength- Stokes
  Shift

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Excitation and Emission Spectra
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Choosing Excitation Wavelengths
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Effect of Excitation Wavelength on Fluorescence
Emission
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Excitation Wavelength Choice

• Fluorescence intensity is directly affected
• Emission wavelength is not directly affected
• Excitation can occur over a distribution of
  wavelengths, not just at one wavelength
• Selecting dyes with larger Stokes shifts allows
  for excitation closer to the absorbance maximum
• Choice exists with the III and III plus (no
  choice for ABI, II or II e)

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Spectral Match of Fluorophore Labels with the
FMBIO (coherent) II and II e - 532nm YAG lasers
http//www.cohr.com/Products/- note the second
line at 532/2262
II
II
Fluorophores in Powerplex 16 Bio
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Spectral Match of Fluorophore Labels with the
ABI and the FMBIO III and III plus
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Emission Wavelength Choice

• The percentage of the signal that is captured
  depends in great part on emission filter
  wavelength choice.
• Emission filters are selected to
• maximize fluorescent signal emission
• attenuate (block) the excitation light- laser
  light

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• Factors in emission filter selection
• Spectral performance of Optical filters
• Laser excitation wavelength (need to block it)
• Dye emission spectra (need to collect it)
• Fluorescence emission occurs over a
  distribution of wavelengths (blocking)
• Spectral bandwidth of dyes (need to isolate
  them)
• Spectral overlap when multiplexing

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Spectral Performance of Optical Filters

• Band Pass
• Center wavelength- CWL- mean of wavelength at 50
  peak transmission
• Band width- FWHM is the bandwidth at 50 peak
  transmission
• Longpass and short pass cut-on or cut-off
  filters (LP, SP)
• Denoted by their cut-on or cut-off wavelengths
• Attenuation (blocking) level and range

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Spectral Performance of Optical Filters in the
FMBIO II, II e and III Traditionally for II and
II e (532 nm laser only), the band pass worked
by reflection for attenuation. Enhanced optics
in the FMBIO III- 3 lasers, new PMT, etc.
required filter design optimization
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Considerations when multiplexing fluorophores -
Discriminating Multiple Signals

• Spectral bandwidth
• Spectral overlap with other dye emissions
• Blocking capability of filters
• Usefulness of large Stokes shifts

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Comparison of Emission Bandwidths
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Spectral overlap -Multiplexing
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Discriminating Multiple Fluorophores
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Effects of Fluorophore Labels on Oligonucleotides

• Solubility
• Electrophoretic mobility distortion

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Comparison of Sequencing Using JOE or BODIPY
523/547 Primers
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Structures of the BODIPY Dyes Used in DNA
Sequencing
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DNA Sequence Obtained Using Four BODIPY Dye
Labeled Primers Without Mobility Correction
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Effects of Oligonucleotides on Fluorophores

• Most dyes are quenched upon conjugation.
• The extent of the quenching varies from dye to
  dye.
• The extent of quenching can vary from sequence to
  sequence
• Observation of difference in spectral properties
  of one green locus in Profiler plus- D8S1179
  appears to have more spectral overlap into blue
  than other green loci)

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Other Effects on Fluorescence Emission

• Structural rigidity and quantum yield
• Thermostability
• Photostability
• pH sensitivity

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Fluorophore Structural Rigidity
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Temperature Dependence of Fluorescence
Some RFI ToC --- Some RFI 1/ToC In particular
Tamra is very ToC sensitive
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Photostability Comparison of two dyes
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pH Sensitivity of Oregon Green 488, FAM and
Rhodamine Green
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Summary

• Introduction to Fluorescence
• Principles and Definitions
• Stokes shifts, Jablonski diagrams, excitation
  and emission, extinction coefficient, quantum
  efficiency
• Excitation and Emission Spectra
• Choosing Exicitation Wavelengths III, III plus
• Choosing Emission Filters
• Chemistry The Dyes
• Structure- Big Greasy Blobs
• Effects of structure on fluorescence
• Other factors
• Effects of rigidity, pH and temperature
• Effects of Fluorophores on Oligos and visa versa

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Resources and Acknowledgements Molecular Probes-
Vicki Singer www.probes.com Excellent resource
for fluorescent dye information- see Intro to
Fluorescence- http//www.probes.com/servlets/publi
cations?id144 or http//www.probes.com/handbook/
sections/0001.html Chroma- Jay Reichman
www.chroma.com FMBIO filter supplier
Handbook http//www.chroma.com/handbook.html
Coherent- www.coherent.com- FMBIO laser
provider Hammamatsu- http//usa.hamamatsu.com/cmp
-detectors/pmts/Default.htm PMT provider Univ.
of Maryland Medicine- Center for Fluorescence
Spectroscopy http//cfs.umbi.umd.edu/ Peer
reviewed literature, publications, courses on
fluorescence Fluorescence microsphere resource
center U Washington http//fmrc.pulmcc.washin
gton.edu/fmrc.shtml Excellent references on
standards, controls, instrumentation,
etc. Fluorescence spectrum viewer
http//www.bdbiosciences.com/spectra/ View up to
3 dyes simultaneously Salk flow cytometry table
of fluorochromes http//pingu.salk.edu/flow/fluo.
html Lists dyes with excitation and emission max