Fluorescence 101 - STR 2003mod Handouts

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.
Why Fluorescence?

Advantages of Fluorescence MiraiBio Inc.
• Easy, Fast (eg. vs silver staining)

• Visualize tagged primer strand
• Multiplexing Detection of 25 pg of dsDNA
• High Sensitivity with PicoGreen Reagent
• Dynamic Range

Principles and Definitions MiraiBio Inc.
What 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

MiraiBio Inc.
How does it work?

las
er b
eam
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.
Three-Stage Process
of Fluorescence
Excited State
of Fluorophore Relaxed
Excited
S1’ 2 State
Photon Absorption
S1
Photon Emission
Energy
1 3
S0 Ground State
of Fluorophore
- Jablonski

MiraiBio Inc.
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- Stoke’s Shift
MiraiBio Inc.
Excitation and Emission Spectra

MiraiBio Inc.
Choosing Excitation Wavelengths

MiraiBio Inc.
Effect of Excitation Wavelength on

Fluorescence Emission

MiraiBio Inc.
Excitation Wavelength Choice 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)

Spectral Match of Fluorophore Labels with the MiraiBio Inc.
FMBIO (coherent) II and II e - 532nm YAG lasers A Hitachi Software Company
http://www.cohr.com/Products/- note the second line at 532/2=262

II II
Fluorescein
JOE
TAMRA
BODIPY R6G
BODIPY 564/570
BODIPY 581/591
ROX
Rhodamine Red
Texas Red
200 300 400 500 600 700
Fluorophores in Powerplex 16 Bio

Spectral Match of Fluorophore Labels
with the ABI and the FMBIO III and III plus
MiraiBio Inc.
Fluorescein
JOE
TAMRA
BODIPY R6G
BODIPY 564/570
BODIPY 581/591
ROX
Rhodamine Red
Texas Red
200 300 400 500 600 700

MiraiBio Inc.
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.
Factors in emission filter selection: 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.
Spectral Performance of Optical Filters 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.
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.
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.
Comparison of Emission Bandwidths

Spectral overlap -Multiplexing MiraiBio Inc.
400 450 500 550 600 650 700

MiraiBio Inc.
Discriminating Multiple Fluorophores

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Effects of Fluorophore Labels on Oligonucleotides
• Solubility
• Electrophoretic mobility distortion

MiraiBio Inc.
Comparison of Sequencing Using JOE or

BODIPY 523/547 Primers

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Structures of the BODIPY Dyes Used in

DNA Sequencing

MiraiBio Inc.
DNA Sequence Obtained Using FourA Hitachi Software Company
BODIPY Dye Labeled Primers Without

Mobility Correction

MiraiBio Inc.
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.
Other Effects on Fluorescence Emission
• Structural rigidity and quantum yield

• Thermostability
• Photostability
• pH sensitivity

MiraiBio Inc.
Fluorophore Structural Rigidity A Hitachi Software Company

MiraiBio Inc.
Temperature Dependence of Fluorescence
Some RFI ~ ToC --- Some RFI ~ 1/ToC

In particular Tamra is very ToC sensitive
120
100
Relative Fluorescence
80
Intensity
60
FAM
40
JOE
20 TAMRA
ROX
0
10 20 30 40 50 60 70 80 90
Temperature

MiraiBio Inc.
Photostability Comparison of two dyes

MiraiBio Inc.
pH Sensitivity of Oregon Green 488, FAM

and Rhodamine Green

Summary 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

Resources and Acknowledgements MiraiBio Inc.
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

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MiraiBio Inc.

A Hitachi Software Company

© MiraiBio Inc., 2003

• Choosing Emission Filters

• Chemistry: The Dyes

• Effects of Fluorophores on Oligos and visa versa

© MiraiBio Inc., 2003

© MiraiBio Inc., 2003

• Easy, Fast (eg. vs silver staining)

© MiraiBio Inc., 2003

Primary fluorescence- intrinsic property of a substance

© MiraiBio Inc., 2003

How does it work?

1. laser strikes fluorophore

Light is collected CCDs or PMTs

© MiraiBio Inc., 2003

© MiraiBio Inc., 2003

1- Excitation: Photon of energy (hvEX)

2- Excited State Lifetime: Energy

3- Fluorescence Emission: Photon of

Excitation and Emission Spectra

© MiraiBio Inc., 2003

Choosing Excitation Wavelengths

© MiraiBio Inc., 2003

Effect of Excitation Wavelength on

© MiraiBio Inc., 2003

• Fluorescence intensity is directly affected

© MiraiBio Inc., 2003

http://www.cohr.com/Products/- note the second line at 532/2=262

200 300 400 500 600 700

Fluorophores in Powerplex 16 Bio

© MiraiBio Inc., 2003

200 300 400 500 600 700

Emission Wavelength Choice

• Emission filters are selected to

• attenuate (block) the excitation light- laser light

© MiraiBio Inc., 2003

• Spectral performance of Optical filters

• Spectral bandwidth of dyes (need to isolate them)

• Spectral overlap when multiplexing

© MiraiBio Inc., 2003

• Longpass and short pass cut-on or cut-off filters (LP, SP)

• Attenuation (blocking) – level and range

© MiraiBio Inc., 2003

Spectral Performance of Optical Filters

Traditionally for II and II e (532 nm laser only), the band pass

Enhanced optics in the FMBIO III- 3 lasers, new PMT, etc.

© MiraiBio Inc., 2003

Considerations when multiplexing fluorophores -

© MiraiBio Inc., 2003

Comparison of Emission Bandwidths

© MiraiBio Inc., 2003

400 450 500 550 600 650 700

© MiraiBio Inc., 2003

Discriminating Multiple Fluorophores

© MiraiBio Inc., 2003

Effects of Fluorophore Labels on Oligonucleotides

© MiraiBio Inc., 2003

Comparison of Sequencing Using JOE or

© MiraiBio Inc., 2003

Structures of the BODIPY Dyes Used in

© MiraiBio Inc., 2003

BODIPY Dye Labeled Primers Without