MiraiBio Inc.
A Hitachi Software Company
Fluorescence 101
Steve Lee
MiraiBio Inc.
STR 2003
© MiraiBio Inc., 2003
Outline
MiraiBio Inc.
• Introduction to Fluorescence
• Principles and Definitions
• Stoke’s 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
© MiraiBio Inc., 2003
A Hitachi Software Company
MiraiBio Inc.
A Hitachi Software Company
Why Fluorescence?
© MiraiBio Inc., 2003
Advantages of Fluorescence
MiraiBio Inc.
A Hitachi Software Company
•
•
•
•
•
Easy, Fast (eg. vs silver staining)
Visualize tagged primer strand
Multiplexing
Detection of 25 pg of dsDNA
with PicoGreen Reagent
High Sensitivity
Dynamic Range
© MiraiBio Inc., 2003
Principles and Definitions
What is Fluorescence?
MiraiBio Inc.
A Hitachi Software Company
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
© MiraiBio Inc., 2003
MiraiBio Inc.
A Hitachi Software Company
How does it work?
1. laser strikes fluorophore
2. fluorophore absorbs laser energy
3. fluorophore emits light at a
Longer wavelength
Light is collected CCDs or PMTs
© MiraiBio Inc., 2003
MiraiBio Inc.
A Hitachi Software Company
Three-Stage Process
of Fluorescence
S0
2
1
Photon Absorption
Energy
S1’
Relaxed
Excited
State
3
S1
Photon Emission
Excited State
of Fluorophore
Ground State
of Fluorophore
- Jablonski
© MiraiBio Inc., 2003
MiraiBio Inc.
The 3 stage Fluorescence Process- Jablonski diagram
A Hitachi Software Company
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- Stoke’s Shift
© MiraiBio Inc., 2003
MiraiBio Inc.
A Hitachi Software Company
Excitation and Emission Spectra
© MiraiBio Inc., 2003
MiraiBio Inc.
A Hitachi Software Company
Choosing Excitation Wavelengths
© MiraiBio Inc., 2003
MiraiBio Inc.
A Hitachi Software Company
Effect of Excitation Wavelength on
Fluorescence Emission
© MiraiBio Inc., 2003
Excitation Wavelength Choice
MiraiBio Inc.
A Hitachi Software Company
• 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)
© MiraiBio Inc., 2003
Spectral Match of Fluorophore Labels with the
MiraiBio Inc.
FMBIO (coherent) II and II e - 532nm YAG lasers
http://www.cohr.com/Products/- note the second line at 532/2=262
A Hitachi Software Company
II
II
Fluorescein
JOE
TAMRA
BODIPY R6G
BODIPY 564/570
BODIPY 581/591
ROX
Rhodamine Red
Texas Red
200
Fluorophores in Powerplex 16 Bio
© MiraiBio Inc., 2003
300
400
500
600
700
Spectral Match of Fluorophore Labels
with the ABI and the FMBIO III and III plus
MiraiBio Inc.
A Hitachi Software Company
Fluorescein
JOE
TAMRA
BODIPY R6G
BODIPY 564/570
BODIPY 581/591
ROX
Rhodamine Red
Texas Red
200
© MiraiBio Inc., 2003
300
400
500
600
700
MiraiBio Inc.
A Hitachi Software Company
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
© MiraiBio Inc., 2003
Factors in emission filter selection:
MiraiBio Inc.
A Hitachi Software Company
• 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
© MiraiBio Inc., 2003
Spectral Performance of Optical Filters
MiraiBio Inc.
A Hitachi Software Company
• 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
© MiraiBio Inc., 2003
MiraiBio Inc.
A Hitachi Software Company
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
© MiraiBio Inc., 2003
MiraiBio Inc.
A Hitachi Software Company
Considerations when multiplexing fluorophores Discriminating Multiple Signals
•
•
•
•
Spectral bandwidth
Spectral overlap with other dye emissions
Blocking capability of filters
Usefulness of large Stokes shifts
© MiraiBio Inc., 2003
MiraiBio Inc.
A Hitachi Software Company
Comparison of Emission Bandwidths
© MiraiBio Inc., 2003
MiraiBio Inc.
Spectral overlap -Multiplexing
400
© MiraiBio Inc., 2003
450
500
550
A Hitachi Software Company
600
650
700
MiraiBio Inc.
Discriminating Multiple Fluorophores
© MiraiBio Inc., 2003
A Hitachi Software Company
MiraiBio Inc.
A Hitachi Software Company
Effects of Fluorophore Labels on Oligonucleotides
• Solubility
• Electrophoretic mobility distortion
© MiraiBio Inc., 2003
MiraiBio Inc.
A Hitachi Software Company
Comparison of Sequencing Using JOE or
BODIPY 523/547 Primers
© MiraiBio Inc., 2003
MiraiBio Inc.
A Hitachi Software Company
Structures of the BODIPY Dyes Used in
DNA Sequencing
© MiraiBio Inc., 2003
MiraiBio Inc.
DNA Sequence Obtained Using Four
BODIPY Dye Labeled Primers Without
Mobility Correction
A Hitachi Software Company
© MiraiBio Inc., 2003
MiraiBio Inc.
A Hitachi Software Company
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)
© MiraiBio Inc., 2003
MiraiBio Inc.
A Hitachi Software Company
Other Effects on Fluorescence Emission
•
•
•
•
Structural rigidity and quantum yield
Thermostability
Photostability
pH sensitivity
© MiraiBio Inc., 2003
Fluorophore Structural Rigidity
© MiraiBio Inc., 2003
MiraiBio Inc.
A Hitachi Software Company
MiraiBio Inc.
A Hitachi Software Company
Temperature Dependence of Fluorescence
Some RFI ~ ToC --- Some RFI ~ 1/ToC
In particular Tamra is very ToC sensitive
Relative Fluorescence
Intensity
120
100
80
60
FAM
40
JOE
20
TAMRA
ROX
0
10
20
30
40
50
60
Temperature
© MiraiBio Inc., 2003
70
80
90
MiraiBio Inc.
A Hitachi Software Company
Photostability Comparison of two dyes
© MiraiBio Inc., 2003
MiraiBio Inc.
A Hitachi Software Company
pH Sensitivity of Oregon Green 488, FAM
and Rhodamine Green
© MiraiBio Inc., 2003
Summary
• Introduction to Fluorescence
MiraiBio Inc.
A Hitachi Software Company
• Principles and Definitions
• Stoke’s 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
© MiraiBio Inc., 2003
Resources and Acknowledgements
MiraiBio Inc.
A Hitachi Software Company
Molecular Probes- Vicki Singer: www.probes.com
Excellent resource for fluorescent dye information- see:
* Intro to Fluorescence- http://www.probes.com/servlets/publications?id=144 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.washington.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
© MiraiBio Inc., 2003