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
Light is a form of electromagnetic radiation
The energy of light is contained in
discrete units or quanta known as photons
Photons have the property of both particles and waves
Light as a wave:
For simplicity, usually only the electrical component is drawn
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
Can detect differences in light
intensity and wavelength (colour)
 Sensitivity
Peak sensitivity is at 555nm (yellow-green)
In bright light, 3 orders of magnitude
After time to accommodate, 10 orders of magnitude!
 Resolution
~0.1mm for an object 25mm from the eye
 Composed of Rod and Cone cells
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
Retinal
 Bright light temporarily bleaches Rhodopsin
(20-30 min recovery time)
 Best high visual sensitivity in a darkened room
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
excited states
excitation
energy loss (rapid 10-9-10-12s)
emitted light (longer wavelength)
Emission
absorption
ground state
Jablonski diagram
Typical fluorochrome:
100,000 cycles per second for 0.1-1 seconds
Fluorochrome “a molecule that is capable of fluorescing”
The physical basis of fluorescence - 1
Excitation and Emission Spectra
Filter set
For FITC (fluorescein-5isothiocyanate) coupled to IgG
To detector (eyepiece/
camera)
Light in
Stoke’s shift
to objective
FITC filter set (Chroma)
excitation
dichromatic
mirror
emission
wavelength
The physical basis of fluorescence - 2
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
Some applications of fluorescence microscopy
 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
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
FM4-64 and DAPI
DAPI
Sporulating Bacillus subtilis
Crystal structure of
DAPI bound to DNA
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
anti-mouse
mouse
Use antibodies raised
against your protein of
interest
OR…
anti-rabbit
rabbit
Chemical Dyes -1
Epitope tags in Fluorescence
microscopy
 Fuse protein of interest to an epitope “tag”
Gene X
6xHA
Common epitopes = Myc, HA
 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
Fluorophores for microscopy
Fluorescein and Rhodamine derivatives
Fluorescein
(IgG-coupled)
(FITC)
Texas Red
(IgG-coupled)
Tetramethylrhodamine
(dextran coupled)
(TRITC)
520nm - green
601 nm - red
573 nm - red
Coupled with Isothiocyanates
- allows attachment via amino groups in proteins
Chemical Dyes -3
Improved dyes (brighter, more stable)
CyDyes
(Cyanine dye-based)
Amersham-Pharmacia Inc
Alexafluor
(molecular probes/
invitrogen)
Chemical Dyes -4
Qdot nanocrystals
Extremely photostable
Small semi-conductors
cadmium/selenium
Zinc sulphide
Different wavelengths
achieved by varying size of
crystal
(molecular probes/ invitrogen)
Chemical Dyes -5
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 channel
suitable
not suitable
Chemical Dyes -6
Fluorescent proteins
Green Fluorescent protein (GFP) isolated from
the jellyfish Aequorea victoria
Short flexible linker
GFP
My protein
Fusion protein
Advantages:
can use in live cells
fixing artefacts avoided
dynamics
Disadvantages: photobleaching
folding environment dependent
functionality of fusion protein
Other fluorescent proteins from other organisms
e.g. DsRed from Discosoma (26% homology with GFP)
Mutagenisation of GFP
--> more stable
--> spectrally shifted variants
Fluorescent proteins -1
GFP variants
GFP (wt)
395/475
509
Green Fluorescent Proteins
Yellow Fluorescent Proteins
EGFP
484
507
EYFP
514
527
Kusabira Orange
548
AcGFP
480
505
Topaz
514
527
mOrange
548
562
TurboGFP
482
502
Venus
515
528
dTomato
554
581
Emerald
487
509
mCitrine
516
529
dTomato-Tandem
554
Azami Green 492
505
YPet
517
530
DsRed
558
583
ZsGreen
493
Blue Fluorescent Proteins
505
PhiYFP
525
537
DsRed2
563
582
ZsYellow1
529
539
DsRed-Express (T1)
555
EBFP
383
445
mBanana
540
553
DsRed-Monomer
556
Sapphire
399
511
mTangerine
568
585
T-Sapphire
399
Cyan Fluorescent Proteins
511
mStrawberry 574
596
ECFP
439
476
AsRed2
576
592
mCFP
433
475
mRFP1
584
607
Cerulean
433
475
JRed
584
610
CyPet
435
477
mCherry
587
610
AmCyan1
458
489
HcRed1
588
618
mRaspberry
598
625
Midori-Ishi Cyan
472
mTFP1 (Teal) 462
492
Shaner NC, Steinbach PA, Tsien RY (2005) A guide
fluorescent proteins. Nat Methods. 2(12):905-9.
Orange and Red Fluorescent Proteins
HcRed-Tandem
590
mPlum
649
590
to choosing
Fluorescent proteins -2
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.
*Triplet state - VERY REACTIVE
may interact with another molecule
to produce irreversible covalent
modifications (photobleaching)
excited state
Fluorescence
(10-9 - 10-12 sec)
(nSec-pSec)
Absorption
(10-15 sec)
ground state
*Triplet state
Internal
conversion
(heat)
Phosphorescence
(102 - 10-2 sec)
(100Sec-0.01Sec)
Photobleaching and Quenching - 1
How to reduce photobleaching
Photobleaching influenced by:
 chemical reactivity of the fluorophore
 intensity and wavelength of the excitation light
 intracellular chemical environment
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-phenylenediamine
n-propyl gallate
DABCO
Propriety
SlowFade
ProLong Antifade kit
Vectashield
Molecular Probes (Invitrogen)
Molecular Probes (Invitrogen)
Vector laboratories
Photobleaching and Quenching - 3
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
bleach
Time taken
to recover
Photobleaching and Quenching - 4
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)
Photobleaching and Quenching - 5
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)
Photobleaching and Quenching - 6
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)
Book
www.chroma.com
Fundamentals of light microscope
Molecular Expressions (general info)
and electronic imaging
www.microscopy.fsu.edu/
Douglas B. Murphy. Wiley-Liss
Nikon (general info - good for GFP)
2001 ISBN 0-471-25391-X
http://www.microscopyu.com