How fluorescence works Adele Marston. Topics covered X The nature of light and colour X Colour...
-
Upload
edwin-powell -
Category
Documents
-
view
220 -
download
4
Transcript of How fluorescence works Adele Marston. Topics covered X The nature of light and colour X Colour...
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
Colour detection in the human eye - 2
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
Colour detection in the human eye - 3
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
Colour detection in the human eye - 4
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
The physical basis of fluorescence - 2
Filter set
emission
excitationdichromatic mirror
FITC filter set (Chroma)
Light in
To detector (eyepiece/camera)
to objective
Emission intensity depends on the excitation wavelength
The physical basis of fluorescence - 3
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)
Fluorescent probes and dyes - 2
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
Fluorescent probes and dyes - 3
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
Photobleaching and Quenching - 2
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
Photobleaching and Quenching - 3
FRAP (Fluoresence recovery after photobleaching)
Photobleaching and Quenching - 4
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
Photobleaching and Quenching - 5
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)
Photobleaching and Quenching - 6
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