1. How fluorescence works Adele Marston

2. Topics covered X The nature of light and colour X Colour detection in the human eye X The physical basis of fluorescence X Fluorescent probes and dyes X Dyes that bind organelles X Chemical Dyes X Fluorescent proteins X Photobleaching and Quenching

3. The Nature of Light The energy of light is contained in discrete units or quanta known as photons Light is a form of electromagnetic radiation Photons have the property of both particles and waves For simplicity, usually only the electrical component is drawn Light as a wave: The nature of light and colour - 1

4. The Electromagnetic Spectrum Wavelengths 400nm-750nm are visible to the human eye The nature of light and colour - 2

5. The Human Eye X Sensitivity Peak sensitivity is at 555nm (yellow-green) In bright light, 3 orders of magnitude After time to accommodate, 10 orders of magnitude! X Resolution ~0.1mm for an object 25mm from the eye X Composed of Rod and Cone cells Can detect differences in light intensity and wavelength (colour) Colour detection in the human eye - 1

6. Rod cell photoreceptors X comprise 95% of photoreceptors in the retina X active in dim light but provide no colour sense X peak sensitivity at 510nm (blue-green) X contain Rhodopsin X Bright light temporarily bleaches Rhodopsin (20-30 min recovery time) X Best high visual sensitivity in a darkened room Retinal Colour detection in the human eye - 2

7. Cone cell photoreceptors X comprise only ~5% of photoreceptors in the retina X contained nearly exclusively in fovea (0.5mm spot) X 3 types: red, green and blue X Action spectra differ for the different cone cells Colour detection in the human eye - 3

8. Positive and negative colours X Positive colours are generated by combining different colour wavelengths --> Yellow perceived by stimulating red and green cones individually with 2 different wavelengths X Negative colours are generated by the subtraction (absorption) of light of a specific wavelength from light composed of a mixture of wavelengths --> Yellow perceived because a single wavelength stimulates both red and green cones Colour detection in the human eye - 4

9. Fluorescence X Occurs following excitation of a fluorescent molecule upon absorption of a photon X Energy is released as light as the molecule decays to its ground state The physical basis of fluorescence - 1 absorption Emission Typical fluorochrome: 100,000 cycles per second for 0.1-1 seconds excitation energy loss (rapid 10 -9 -10 -12 s) excited states ground state emitted light (longer wavelength) Jablonski diagram Fluorochrome “a molecule that is capable of fluorescing”

10. Excitation and Emission Spectra Stoke’s shift For FITC (fluorescein-5- isothiocyanate) coupled to IgG wavelength The physical basis of fluorescence - 2 Filter set emission excitation dichromatic mirror FITC filter set (Chroma) Light in To detector (eyepiece/ camera) to objective

11. Emission intensity depends on the excitation wavelength The physical basis of fluorescence - 3

12. Properties of fluorophores X Stokes shift - difference between excitation and emission maxima (large advantageous) X Molar extinction coefficient - potential of a fluorophore to absorb photons X Quantum efficiency (QE) of fluorescence emission - fraction of absorbed photons that are re-emitted X Quantum yield - how many photons emitted by a fluorophore before it is irreversibly damaged X Quenching - quantum yield (but not emission spectrum) altered by interactions with other molecules X Photobleaching - permanent loss of fluorescence by photon-induced chemical damage Fluorescent probes and dyes - 1

13. Choice of Fluorophore will depend on the application X Protein localization (Immunofluorescence microscopy or GFP-tagging). X organelle marking (e.g. DAPI to label nucleus) X protein dynamics (FRAP ) X protein interactions (FRET) X ion concentration (using ratiometric dyes) X enzyme reactions (“caged” fluorescent compounds) X cell viability (viability-dependent permeabilization) Fluorescent probes and dyes - 2 Some applications of fluorescence microscopy

14. Fluorochromes in microscopy X Biologically active fluorescent compounds - bind directly to cellular structures X Chemical dyes - most need to be coupled to a macromolecule to be useful in microscopy X Fluorescent proteins - can be fused genetically to a protein of interest Fluorescent probes and dyes - 3

15. Dyes that bind cellular structures or organelles DAPI Crystal structure of DAPI bound to DNA Sporulating Bacillus subtilis FM4-64 and DAPI Dyes that bind organelles - 1

16. Chemical conjugation of fluorescent dyes to chemicals that bind cellular structures Rhodamine-coupled Phalloidin (Phalloidin is a mushroom toxin that binds to F-actin) Dyes that bind organelles -2

17. Immunofluorescence microscopy fluorophore Secondary antibody Primary antibody Use antibodies raised against your protein of interest OR… Chemical Dyes -1 mouse anti-mouse rabbit anti-rabbit

18. Epitope tags in Fluorescence microscopy Common epitopes = Myc, HA Gene X6xHA X Fuse protein of interest to an epitope “tag” X Buy commercially-available antibodies to the epitope and use as primary antibody for IF Advantage: Fast (do not need to raise antibodies) Disadvantages: Protein fusion may not be fully functional Problems of specificity of antibodies to tag Chemical Dyes - 2

19. Fluorophores for microscopy Fluorescein (IgG-coupled) (FITC) 520nm - green Texas Red (IgG-coupled) 601 nm - red Tetramethylrhodamine (dextran coupled) (TRITC) 573 nm - red Fluorescein and Rhodamine derivatives Coupled with Isothiocyanates - allows attachment via amino groups in proteins Chemical Dyes -3

20. Improved dyes CyDyes (Cyanine dye-based) Amersham-Pharmacia Inc Alexafluor (molecular probes/ invitrogen) (brighter, more stable) Chemical Dyes -4

21. Qdot nanocrystals Extremely photostable (molecular probes/ invitrogen) Different wavelengths achieved by varying size of crystal Small semi-conductors Chemical Dyes -5 cadmium/selenium Zinc sulphide

22. Multicolour labeling X can simultaneously image multiple fluorophores e.g to localize multiple proteins in the same cell X need to isolate the signal from each fluorophore individually 1) Choose fluorophores with minimum emission overlap 2) Choose filter sets that minimize “bleed through” into another channel suitablenot suitable Chemical Dyes -6

23. Fluorescent proteins Green Fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria My protein GFP Short flexible linker Fusion protein Advantages:can use in live cells fixing artefacts avoided dynamics Disadvantages: photobleaching folding environment dependent functionality of fusion protein Fluorescent proteins -1 Mutagenisation of GFP--> more stable --> spectrally shifted variants Other fluorescent proteins from other organisms e.g. DsRed from Discosoma (26% homology with GFP)

24. GFP variants Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods. 2(12):905-9. GFP (wt)395/475509 Green Fluorescent Proteins EGFP484507 AcGFP480505 TurboGFP482502 Emerald487509 Azami Green492505 ZsGreen493505 Blue Fluorescent Proteins EBFP383445 Sapphire399511 T-Sapphire399511 Cyan Fluorescent Proteins ECFP439476 mCFP433475 Cerulean433475 CyPet435477 AmCyan1458489 Midori-Ishi Cyan472 mTFP1 (Teal)462492 Orange and Red Fluorescent Proteins Kusabira Orange548 mOrange548562 dTomato554581 dTomato-Tandem554 DsRed558583 DsRed2563582 DsRed-Express (T1)555 DsRed-Monomer556 mTangerine568585 mStrawberry574596 AsRed2576592 mRFP1584607 JRed584610 mCherry587610 HcRed1588618 mRaspberry598625 HcRed-Tandem590 mPlum590649 Yellow Fluorescent Proteins EYFP514527 Topaz514527 Venus515528 mCitrine516529 YPet517530 PhiYFP525537 ZsYellow1529539 mBanana540553 Fluorescent proteins -2

25. Photobleaching Photobleaching: a fluorophore permanently loses the ability to fluoresce due to photon-induced chemical damage and covalent modification. Largely due to the generation of free oxygen radicals that attack and permanently destroy the light-emitting properties of the fluorochrome. Absorption (10 -15 sec) Fluorescence (10 -9 - 10 -12 sec) (nSec-pSec) Internal conversion (heat) Phosphorescence (10 2 - 10 -2 sec) (100Sec-0.01Sec) *Triplet state *Triplet state - VERY REACTIVE may interact with another molecule to produce irreversible covalent modifications (photobleaching) ground state excited state Photobleaching and Quenching - 1

26. How to reduce photobleaching X chemical reactivity of the fluorophore X intensity and wavelength of the excitation light X intracellular chemical environment Photobleaching influenced by: Reduce photobleaching by: X choice of fluorophore X limit exposure time (but will reduce emission) X use of antifade reagents Photobleaching and Quenching - 2

27. Antifade Reagents Act by scavenging reaction oxygen species Common Antifade Reagents DIY (buy from Sigma) p-phenylenediamine n-propyl gallate DABCO Propriety SlowFadeMolecular Probes (Invitrogen) ProLong Antifade kitMolecular Probes (Invitrogen) VectashieldVector laboratories Photobleaching and Quenching - 3

28. FRAP (Fluoresence recovery after photobleaching) Photobleaching and Quenching - 4 X phenomenon of photobleaching is exploited in FRAP X FRAP- learn how dynamic a protein is by monitoring recovery of fluoresence after photobleaching bleach Time taken to recover

29. Quenching Photobleaching and Quenching - 5 X Quenching - reduced fluoresence intensity as a result of the presence of oxidizing agents or the presence of salts of heavy metals or halogen compounds X Quenching reduces emission X Quenching sometimes results from the transfer of energy to other “acceptor molecules” close to the excited fluorophore = Resonance energy transfer X Resonance energy transfer has been exploited to measure the proximity of two molecules in a technique called FRET (Fluoresence energy transfer)

30. FRET (Fluoresence resonance energy transfer) Photobleaching and Quenching - 6 X FRET is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon X Donor and acceptor molecules must be in close proximity (10-100Å) X Fluoresence at emission wavelength of acceptor indicates that FRET has occurred (donor and acceptor are close)

31. Background information and suppliers on the web Molecular probes (invitrogen) (good background and products) probes.invitrogen.com/handbook/ Amersham Biosciences (CyDyes) www.amershambiosciences.com/ Jackson Immunochemicals (secondary antibodies) www.stratech.co.uk Clontech (GFP vectors) www.clontech.com Vector laboratories (antifade) www.vectorlabs.com Olympus (excellent general info and tutorials) www.olympusmicro.com Chroma (filter sets) www.chroma.com Molecular Expressions (general info) www.microscopy.fsu.edu/ Nikon (general info - good for GFP) http://www.microscopyu.com Book Fundamentals of light microscope and electronic imaging Douglas B. Murphy. Wiley-Liss 2001 ISBN 0-471-25391-X