Fluorescence 101 Steve Lee MiraiBio Inc. STR 2003

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

Why Fluorescence?

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

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

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

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

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

Excitation and Emission Spectra

Choosing Excitation Wavelengths

Effect of Excitation Wavelength on Fluorescence Emission

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)

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 532/2=262 II II Fluorophores in Powerplex 16 Bio

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

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

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

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

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

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

Comparison of Emission Bandwidths

Spectral overlap -Multiplexing 700 400 500 600 550 650 450

Discriminating Multiple Fluorophores

Effects of Fluorophore Labels on Oligonucleotides Solubility Electrophoretic mobility distortion

Comparison of Sequencing Using JOE or BODIPY 523/547 Primers

Structures of the BODIPY Dyes Used in DNA Sequencing

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

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)

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

Fluorophore Structural Rigidity

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

Photostability Comparison of two dyes

pH Sensitivity of Oregon Green 488, FAM and Rhodamine Green

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

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/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