How fluorescence works Adele Marston. Topics covered X The nature of light and colour X Colour detection in the human eye X The physical basis of fluorescence

How fluorescence works

Adele Marston

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

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

The Electromagnetic Spectrum

Wavelengths 400nm-750nm are visible to the human eye

The nature of light and colour - 2

The Human Eye

SensitivityPeak sensitivity is at 555nm (yellow-green)In bright light, 3 orders of magnitudeAfter time to accommodate, 10 orders of magnitude!

Resolution~0.1mm for an object 25mm from the eye

Composed of Rod and Cone cells

Can detect differences in light intensity and wavelength (colour)

Colour detection in the human eye - 1

Rod cell photoreceptors

comprise 95% of photoreceptors in the retina active in dim light but provide no colour sense peak sensitivity at 510nm (blue-green) contain Rhodopsin

Bright light temporarily bleaches Rhodopsin(20-30 min recovery time)

Best high visual sensitivity in a darkened room

Retinal


Cone cell photoreceptors

comprise only ~5% of photoreceptors in the retina contained nearly exclusively in fovea (0.5mm spot) 3 types: red, green and blue Action spectra differ for the different cone cells


Positive and negative colours

Positive colours are generated by combining different colour wavelengths

--> Yellow perceived by stimulating red and green cones individually with 2 different wavelengths

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


Fluorescence Occurs following excitation of a fluorescent molecule upon absorption of a photon Energy is released as light as the molecule decays to its ground state

The physical basis of fluorescence - 1

absorptionEmission

Typical fluorochrome:100,000 cycles per second for 0.1-1 seconds

excitation

energy loss (rapid 10-9-10-12s) excited states

ground state

emitted light (longer wavelength)

Jablonski diagram

Fluorochrome “a molecule that is capable of fluorescing”

Excitation and Emission Spectra

Stoke’s shift

For FITC (fluorescein-5-isothiocyanate) coupled to IgG

wavelength


Filter set

emission

excitationdichromatic mirror

FITC filter set (Chroma)

Light in

To detector (eyepiece/camera)

to objective

Emission intensity depends on the excitation wavelength


Properties of fluorophores

Stokes shift - difference between excitation and emission maxima (large advantageous) Molar extinction coefficient - potential of a fluorophore to absorb photons Quantum efficiency (QE) of fluorescence emission -fraction of absorbed photons that are re-emitted Quantum yield - how many photons emitted by a fluorophore before it is irreversibly damaged

Quenching - quantum yield (but not emission spectrum) altered by interactions with other molecules Photobleaching - permanent loss of fluorescence by photon-induced chemical damage

Fluorescent probes and dyes - 1

Choice of Fluorophore will depend on the application

Protein localization (Immunofluorescence microscopy or GFP-tagging). organelle marking (e.g. DAPI to label nucleus) protein dynamics (FRAP ) protein interactions (FRET) ion concentration (using ratiometric dyes) enzyme reactions (“caged” fluorescent compounds) cell viability (viability-dependent permeabilization)


Some applications of fluorescence microscopy

Fluorochromes in microscopy

Biologically active fluorescent compounds - bind directly to cellular structures

Chemical dyes - most need to be coupled to a macromolecule to be useful in microscopy

Fluorescent proteins - can be fused genetically to a protein of interest


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

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

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

Epitope tags in Fluorescence microscopy

Common epitopes = Myc, HA

Gene X 6xHA

Fuse protein of interest to an epitope “tag”

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 functionalProblems of specificity of antibodies to tag

Chemical Dyes - 2

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

Improved dyes

CyDyes (Cyanine dye-based) Amersham-Pharmacia Inc

Alexafluor (molecular probes/invitrogen)

(brighter, more stable)

Chemical Dyes -4

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

Multicolour labeling

can simultaneously image multiple fluorophores e.g to localize multiple proteins in the same cell

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 channelsuitable not suitable

Chemical Dyes -6

Fluorescent proteinsGreen Fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria

My protein

GFP

Short flexible linker

Fusion protein

Advantages: can use in live cellsfixing artefacts avoideddynamics

Disadvantages: photobleachingfolding environment

dependentfunctionality of fusion

protein

Fluorescent proteins -1Mutagenisation of GFP --> more stable

--> spectrally shifted variants

Other fluorescent proteins from other organismse.g. DsRed from Discosoma (26% homology with GFP)

GFP variants

Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods. 2(12):905-9.

GFP (wt) 395/475 509Green Fluorescent Proteins

EGFP 484 507

AcGFP 480 505

TurboGFP 482 502

Emerald 487 509

Azami Green 492 505

ZsGreen 493 505Blue Fluorescent Proteins

EBFP 383 445

Sapphire 399 511

T-Sapphire 399 511Cyan Fluorescent Proteins

ECFP 439 476

mCFP 433 475

Cerulean 433 475

CyPet 435 477

AmCyan1 458 489

Midori-Ishi Cyan 472

mTFP1 (Teal) 462 492

Orange and Red Fluorescent Proteins

Kusabira Orange 548

mOrange 548 562

dTomato 554 581

dTomato-Tandem 554

DsRed 558 583

DsRed2 563 582

DsRed-Express (T1) 555

DsRed-Monomer 556

mTangerine 568 585

mStrawberry 574 596

AsRed2 576 592

mRFP1 584 607

JRed 584 610

mCherry 587 610

HcRed1 588 618

mRaspberry 598 625

HcRed-Tandem 590

mPlum 590 649

Yellow Fluorescent Proteins

EYFP 514 527

Topaz 514 527

Venus 515 528

mCitrine 516 529

YPet 517 530

PhiYFP 525 537

ZsYellow1 529 539

mBanana 540 553

Fluorescent proteins -2

PhotobleachingPhotobleaching: 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)

Internalconversion(heat)

Phosphorescence(102 - 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

How to reduce photobleaching

chemical reactivity of the fluorophore intensity and wavelength of the excitation light intracellular chemical environment

Photobleaching influenced by:

Reduce photobleaching by:

choice of fluorophore limit exposure time (but will reduce emission) use of antifade reagents


Antifade Reagents

Act by scavenging reaction oxygen species

Common Antifade Reagents

DIY (buy from Sigma)p-phenylenediaminen-propyl gallateDABCO

ProprietySlowFade Molecular Probes (Invitrogen)ProLong Antifade kit Molecular Probes (Invitrogen)Vectashield Vector laboratories


FRAP (Fluoresence recovery after photobleaching)


phenomenon of photobleaching is exploited in FRAP FRAP- learn how dynamic a protein is by monitoring recovery of fluoresence after photobleaching

bleachTime taken to recover

Quenching


Quenching - reduced fluoresence intensity as a result of the presence of oxidizing agents or the presence of salts of heavy metals or halogen compounds

Quenching reduces emission

Quenching sometimes results from the transfer of energy to other “acceptor molecules” close to the excited fluorophore = Resonance energy transfer

Resonance energy transfer has been exploited to measure the proximity of two molecules in a technique called FRET (Fluoresence energy transfer)

FRET (Fluoresence resonance energy transfer)


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 Donor and acceptor molecules must be in close proximity (10-100Å) Fluoresence at emission wavelength of acceptor indicates that FRET has occurred (donor and acceptor are close)

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.ukClontech (GFP vectors)www.clontech.comVector laboratories (antifade)www.vectorlabs.comOlympus (excellent general info and tutorials)www.olympusmicro.comChroma (filter sets)www.chroma.comMolecular Expressions (general info)www.microscopy.fsu.edu/Nikon (general info - good for GFP)http://www.microscopyu.com

BookFundamentals of light microscope and electronic imagingDouglas B. Murphy. Wiley-Liss 2001 ISBN 0-471-25391-X

Documents

How fluorescence works Adele Marston. Topics covered X The nature of light and colour X Colour detection in the human eye X The physical basis of fluorescence