1. Fluorescence 101
Steve Lee
MiraiBio Inc.
STR 2003

2. Outline Introduction to Fluorescence Chemistry: The Dyes
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

3. Why Fluorescence?

4. 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

5. Principles and Definitions 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

6. 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

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

8. 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

9. Excitation and Emission Spectra

10. Choosing Excitation Wavelengths

11. Effect of Excitation Wavelength on Fluorescence Emission

12. 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)

13. Spectral Match of Fluorophore Labels with the FMBIO (coherent) II and II e - 532nm YAG lasers note the second line at 532/2=262 II II
Fluorophores in Powerplex 16 Bio

14. Spectral Match of Fluorophore Labels with the ABI and the FMBIO III and III plus

15. 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

16. 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

17. 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

18. 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

19. Considerations when multiplexing fluorophores - Discriminating Multiple Signals
Spectral bandwidth
Spectral overlap with other dye emissions
Blocking capability of filters
Usefulness of large Stokes shifts

20. Comparison of Emission Bandwidths

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

22. Discriminating Multiple Fluorophores

23. Effects of Fluorophore Labels on Oligonucleotides
Solubility
Electrophoretic mobility distortion

24. Comparison of Sequencing Using JOE or BODIPY 523/547 Primers

25. Structures of the BODIPY Dyes Used in DNA Sequencing

26. DNA Sequence Obtained Using Four BODIPY Dye Labeled Primers Without Mobility Correction

27. 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)

28. Other Effects on Fluorescence Emission
Structural rigidity and quantum yield
Thermostability
Photostability
pH sensitivity

29. Fluorophore Structural Rigidity

30. Temperature Dependence of Fluorescence
Some RFI ~ ToC --- Some RFI ~ 1/ToC
In particular Tamra is very ToC sensitive

31. Photostability Comparison of two dyes

32. pH Sensitivity of Oregon Green 488, FAM and Rhodamine Green

33. Summary Introduction to Fluorescence Chemistry: The Dyes
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

34. Resources and Acknowledgements
Molecular Probes- Vicki Singer:
Excellent resource for fluorescent dye information- see:
* Intro to Fluorescence- or
Chroma- Jay Reichman: FMBIO filter supplier
* Handbook:
Coherent- FMBIO laser provider
Hammamatsu- PMT provider
Univ. of Maryland Medicine- Center for Fluorescence Spectroscopy:
Peer reviewed literature, publications, courses on fluorescence
Fluorescence microsphere resource center – U Washington:
Excellent references on standards, controls, instrumentation, etc.
Fluorescence spectrum viewer:
View up to 3 dyes simultaneously
Salk flow cytometry table of fluorochromes:
Lists dyes with excitation and emission max