Mirai Bio Inc A Hitachi Software Company Fluorescence

Mirai. Bio Inc. A Hitachi Software Company Fluorescence 101 Steve Lee Mirai. Bio Inc. STR 2003 © Mirai. Bio Inc. , 2003

Outline Mirai. Bio Inc. A Hitachi Software Company • 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, p. H and temperature • Effects of Fluorophores on Oligos and visa versa © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. A Hitachi Software Company Why Fluorescence? © Mirai. Bio Inc. , 2003

Advantages of Fluorescence • • • Easy, Fast (eg. vs silver staining) Visualize tagged primer strand Multiplexing Detection of 25 pg of ds. DNA with Pico. Green Reagent High Sensitivity Dynamic Range © Mirai. Bio Inc. , 2003 Mirai. Bio Inc. A Hitachi Software Company

Principles and Definitions What is Fluorescence? Mirai. Bio 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 © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. A Hitachi Software Company 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 © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. A Hitachi Software Company Three-Stage Process of Fluorescence S 0 2 1 Photon Absorption Energy S 1’ Relaxed Excited State 3 S 1 Photon Emission Excited State of Fluorophore Ground State of Fluorophore - Jablonski © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. The 3 stage Fluorescence Process- Jablonski diagram A Hitachi Software Company 1 - Excitation: Photon of energy (hv. EX) 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 (hv. EM ) is emitted Due to energy dissapation in 2, emitted photon is of lower energy and longer wavelength- Stoke’s Shift © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. A Hitachi Software Company Excitation and Emission Spectra © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. A Hitachi Software Company Choosing Excitation Wavelengths © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. Effect of Excitation Wavelength on Fluorescence Emission © Mirai. Bio Inc. , 2003 A Hitachi Software Company

Excitation Wavelength Choice • • • Mirai. Bio 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) © Mirai. Bio Inc. , 2003

Spectral Match of Fluorophore Labels with the Mirai. Bio Inc. FMBIO (coherent) II and II e - 532 nm YAG lasers http: //www. cohr. com/Products/- note the second line at 532/2=262 A Hitachi Software Company II Fluorophores in Powerplex 16 Bio © Mirai. Bio Inc. , 2003 II

Spectral Match of Fluorophore Labels with the ABI and the FMBIO III and III plus © Mirai. Bio Inc. , 2003 Mirai. Bio Inc. A Hitachi Software Company

Mirai. Bio 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 © Mirai. Bio Inc. , 2003

Factors in emission filter selection: Mirai. Bio 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 © Mirai. Bio Inc. , 2003

Spectral Performance of Optical Filters Mirai. Bio 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 © Mirai. Bio Inc. , 2003

Mirai. Bio 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 © Mirai. Bio Inc. , 2003

Mirai. Bio 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 © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. A Hitachi Software Company Comparison of Emission Bandwidths © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. Spectral overlap -Multiplexing 400 © Mirai. Bio Inc. , 2003 450 500 550 A Hitachi Software Company 600 650 700

Mirai. Bio Inc. Discriminating Multiple Fluorophores © Mirai. Bio Inc. , 2003 A Hitachi Software Company

Mirai. Bio Inc. A Hitachi Software Company Effects of Fluorophore Labels on Oligonucleotides • • Solubility Electrophoretic mobility distortion © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. A Hitachi Software Company Comparison of Sequencing Using JOE or BODIPY 523/547 Primers © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. A Hitachi Software Company Structures of the BODIPY Dyes Used in DNA Sequencing © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. DNA Sequence Obtained Using Four BODIPY Dye Labeled Primers Without Mobility Correction A Hitachi Software Company © Mirai. Bio Inc. , 2003

Mirai. Bio 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- D 8 S 1179 appears to have more spectral overlap into blue than other green loci) © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. A Hitachi Software Company Other Effects on Fluorescence Emission • • Structural rigidity and quantum yield Thermostability Photostability p. H sensitivity © Mirai. Bio Inc. , 2003

Fluorophore Structural Rigidity © Mirai. Bio Inc. , 2003 Mirai. Bio Inc. A Hitachi Software Company

Mirai. Bio Inc. Temperature Dependence of Fluorescence A Hitachi Software Company Some RFI ~ To. C --- Some RFI ~ 1/To. C In particular Tamra is very To. C sensitive © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. A Hitachi Software Company Photostability Comparison of two dyes © Mirai. Bio Inc. , 2003

Mirai. Bio Inc. A Hitachi Software Company p. H Sensitivity of Oregon Green 488, FAM and Rhodamine Green © Mirai. Bio Inc. , 2003

Summary • Introduction to Fluorescence Mirai. Bio 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, p. H and temperature • Effects of Fluorophores on Oligos and visa versa © Mirai. Bio Inc. , 2003

Resources and Acknowledgements Mirai. Bio 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 © Mirai. Bio Inc. , 2003