Basics of fluorescence

Microscopy Course IST Austria, 24.09.- 1.10. 2019Anna Hapek-Sikora

Emission of light- LUMINESCENCE

Luminescence is the emission of light from any substance not resulting from heat and occurs from electronically excited states.

According to excitation mechanisms:

- chemiluminescence

- thermoluminescence

- electroluminescence

and... PHOTOLUMINESCENCE

PHOTOLUMINESCENCE

fluorescence phosphorescence

occures from excited singlet states

emission rates very fast 109 s-1

lifetimes 10 ns

occures from excited triplet states

emission rates slow 103 – 100 s-1

lifetimes miliseconds to seconds

Common fluorophores

Common fluorophores

• organic molecules with aromatic ring structures

• delocalized electrones in binding π-orbitals

• π-electrons interact with the environment

What we see, what we cannot see

E = hν = hc/λ

Few people you should know

Max PlanckNobel prize in 1918

Sir John Herschel

Sir George Stokes

Aleksander Jabłoński

But we can try

Good enough ;)

Jabłoński Energy Diagram

Stokes Shift

Different deactivation pathways, through which an excited molecule can return to its ground-state.

Characteristic of fluorescence emission

● the Stokes' shift

● emission spectra are independent of excitation λ

● fluorescence quantum yield

● fluorescence lifetime

● fluorescence quenching

Stokes' shift

Absorption and emission spectra of Rhodamine with ~25 nm Stokes shift

Emission spectra are independent of excitation λ

The emission spectrum is independent of the excitation energy as a consequence of rapid internal conversion from higher initial excited statesto the lowest vibrational energy level of the S(1) excited state.

Quantum yield

Substances with the largest quantum yield display the brightest emissions.

hϑA hϑA

Relaxation (10 -12s)

S0

S1

S1

ᴦ knr

• ᴦ - emissive rate• knr - non radiative decay rate to S0

Q= ΓΓ +k nr

Ratio of the number of emitted to absorbed photons

• Q is close to unity when knr ˂ ᴦ• substances with largest quantum yields (rhodamins) display brightest emissions• Q is highly dependent on the solvent of the fluorophore

Quantum yield of different fluorophores

Fluorescence lifetime (τ) - τ is the average value of the time spent in the excited state

- formally, the fluorescence lifetime is defined as the time in which the initial fluorescence intensity of a fluorophore decays to 1/e (approximately 37 percent) of the initial intensity

- determines time avaiable for molecular interactions, diffusion into environment and hence the info avaiable from its emission

τ= 1

ᴦ + knr

• ᴦ - emissive rate• knr - non radiative decay rate to S0

τ

F(t) = F0 • exp(-t/τ)

Fluorescence quenchingRefers to any process which decreases the fluorescence intensity of a given substance.

Different mechanisms:

Collisional quenching (dynamic)- excited state fluorophore is deactivated by other molecules (quenchers)- O2; I-, Cl-(halogens); amines, e- deficient molecules- mechanisms: e- transfer ( Indole → Acrylamide)- intersystem crossing to triplet state (halogens, heavy atoms)

Static quenching- fluorophore forms nonfluorescent complexes with other molecules- occures in the ground state independent of diffusion or moleculecollision (dye aggregation )

Nonmolecular mechanisms- attenuation of the incident light

Fluorescence Anisotropy

Photoselectedfluorophores

Rotationaldiffusion

Randomizedfluorophores

Effects of polarized excitation and rotational diffusion on the polarization oranisotropy of the emission.

polarizedexcitation(laser light)

polarized emission

unpolarized emission

III

I

Resonance energy transfer

- occurs in excited state

- whenever emission spectrum of fluorophore called DONOR overlaps with absorption spectrum of another molecule called ACCEPTOR

Förster resonance energy transferFRET

FRET occurs when DONOR and ACCEPTOR are within the Förster distance (0.5-10 nm)

Donor-Acceptor spectral overlap

FRET efficiency

R0 50% of E

EFRET=1

1+(r /R0)6

EFRET – efficiencyr – distance donor-acceptorR0 - the distance at which the energy transfer efficiency is 50%

The FRET efficiency (E) is the quantum yield of the energy transfer transition, i.e. the fraction of energy transfer event occurring per donor excitation event.

FRET efficiency

The efficiency of the energy transfer decreases to the 6th power of the distance between donor and acceptor.

FluorescentProteinPair

DonorExcitationMaximum(nm)

AcceptorEmissionMaximum(nm)

DonorQuantumYield

AcceptorExtinctionCoefficient

FörsterDistance(nm)

EBFP2-mEGFP 383 507 0.56 57,500 4.8

ECFP-EYFP 440 527 0.40 83,400 4.9

Cerulean-Venus 440 528 0.62 92,200 5.4

MiCy-mKO 472 559 0.90 51,600 5.3

TFP1-mVenus 492 528 0.85 92,200 5.1

CyPet-YPet 477 530 0.51 104,000 5.1

EGFP-mCherry 507 510 0.60 72,000 5.1

Venus-mCherry 528 610 0.57 72,000 5.7

Venus-tdTomato 528 581 0.57 138,000 5.9

Venus-mPlum 528 649 0.57 41,000 5.2

FRET applications● can be used to measure distances between domains in a single protein

and therefore to provide information about protein conformation

● detect interaction between proteins

● applied in vivo, FRET has been used to detect the location and interactions of genes and cellular structures including intergrins and membrane proteins

● is also used to study lipid rafts in cell membranes

SummaryMolecular information from fluorescence

Emission spectra and Stoke’s shift

- location of the fluorophore

- binding to macromolecules (DNA, membrane)

- chemical sensing dyes

Quenching of fluorescence

- accessibility of fluorophores to quenchers (location on macromolecule, porosity of proteins)

FRET

- distance measurment between sites on macromolecules. Protein-protein interaction.

Fluorescence polarization and anisotropy

- used to measure molecular weight of proteins or protein binding

10 minutes break!

Fluorophores

Intrinsic

natural

aromatic aaNADHflavinschlorophyll

Extrinsic

added to the sample

fluoresceinrhodamine,DAPI

Cells are autofluorescent!

Important characteristics of fluorophores

● Absorbtion and emission maxima (nm)● Fluorescensce intensity of the emitted light● Molar Extinction Coefficient ● Quantum Yield (0-1)● Solubility in water or other substances● Lifetime of the excited state● Photostability

Absorbtion and emission maxima

Molar extinction coefficientsM-1 cm-1

The extinction coefficient is determined by measuring the absorbance at a reference wavelength

(characteristic of the absorbing molecule) for a one molar (M) concentration (one mole per

liter) of the target chemical in a cuvette having a one-centimeter path length.

Extinction coefficients are a direct measure of the ability of a fluorophore to absorb light, and

those chromophores having a high extinction coefficient also have a high probability of

fluorescence emission.

Molecules exhibiting a high extinction coefficient have an excited state

with a short intrinsic lifetime.

Photostability and photobleaching

Fluorophores permanently loses ability to fluorescence. Why?

The average number of excitation and emission cycles that occur for a particular fluorophore before photobleaching is dependent upon the molecular structure and the local environment.

- due to photon induced chemical damage- covalent modifications- transition from singlet to triplet state (longer time in excited state to interact with others molecules)

Dye + dye

Dye+light+oxygenphotobleaching

How to control photobleaching

• reduction of light intensity and time span of light exposure

• increase of fluorophore concentration

• remove oxygen

• stop (compete with) photooxidation

use antioxidants „antifade reagents“ (e.g. DABCO)

• 2-photon excitation

• employment of more robust fluorophores (Alexa Fluor dyes)

• employment of dyes with longer excitation wavelength

FRAP – application of photobleaching

Used to investigate the diffusion and motion of biological macromolecules.

Phototoxicity

Cell damage as a result of exciting exogenous or endogenous fluorophores with lasers or high-intensity arc-discharge lamps

How to fight with it?

Reasons:

• In their excited state, fluorescent molecules tend to react with molecular oxygen to produce free radicals that can damage subcellular components

• particular constituents of standard culture media, including the vitamin riboflavin and the amino acid tryptophan, may also contribute to adverse light-induced effects on cultured cells

• interaction between light and DNA intercalating dyes causes denaturation of the polymer, Bernas T. et al. 2005

Fighting with photoxicity

• reduction of light intensity and time span of lightexposure

• addition of scavengers into the medium (ascorbic acid)

• fluorescent proteins are generally not phototoxic

• fluorophores that exhibit the longest excitation wavelengths possible should be chosen to minimize damage to cells by short wavelength illumination

• many synthetic fluorophores, such as the MitoTracker and nucleic acid stains (Hoechst, SYTO cyanine dyes, and DRAQ5) are highly toxic to cells.

Fighting with photoxicity

Good to read

And many more, just google it

Antibody labeling

Covalent binding:• free NH2-groups• free SH-Groups• carbohydrate side chains

Noncovalent binding:• Fc-portion of antibodies( Zenon® labeling system)

Antibody labeling

Protein labeling reagents

FITCRhodamine

480/510600/615

Why this two?

- high quantum yield (0.90, 0.95)- display longer excitation and emission λ than aa- high molar extinction coefficient (80 000 M-1 cm-1)- lifetime ~ 4ns- good for antibody labeling

Stokes shift in protein labeling

FITC 494/519

Small stoke shift leads to self-quenching events

FRET can occur between two molecules attached to the same protein if they are within Förster distance

More fluoresceine molecules attached to protein ≠ brighter signal

DNA probes

Ethidium bromide, PI, 7-AAD

TOTO familly

DAPI, Hoechst

Very weak intrinsic emission is observed from unlabeled DNA

Number of probes bind spontaneously to DNA and display enhanced emission up to 1000 fold upon binding

DNA staining

Applications: counterstains for nucleic acids and chromosomes,cell cycle analysis, dead cell staining.

DAPI stainedNIH 3T3 mouse fibroblast

PI stainedHEK 293

Membrane probes

Mouse fibroblastsMembranes-Texas red DHPEActin-FluorescinDNA-DAPI

B. subtilis PY79 Stained with TMA-DPH

Texas Red DHPE

Other cell organella stainings

Bovine pulmonary artery endothelial cell labeled with probes to visualize mitochondria, peroxisomes, and the nucleus.

Immunocytochemistry/Immunofluorescence:ABCA7 Antibody.Staining of human cell line A-431 shows positivity in plasma membrane and golgi apparatus

Chemical sensing probes

Example: Ca+2 indicators

chemical indicators

small molecules able to chelate ions

genetically encoded indicator

fluorescent proteins derived from GFP or its variants + calmodulin

genes encoding these proteines transfected to cell lines

Cameleons

BAPTA basedfuncional carboxylix acid gr.

fura-2, indo-1, fluo-3, fluo-4, Calcium Green-1

Wavelength-ratiomeric dyes which allow the analysis of kationconcentrations like Ca2+, H+, Na2+, K+, Mg2+ by determining the ratioof intensities at different excitation or emission wavelengthsDyes are trapped in cells by lysis of cell permeable esters or bymicroinjection.

GFP- Green Fluorescent Protein238 aa protein isolated from Aequorea victoriabarrel of β-sheets

WT GFP ex.max. near to UV→Mutation S65T ex.max. shifted to 488 nm (Tsien)→extraordinary photostability and high quantum yieldNobel prize in chemistry in 2008 for Martin Chalfie, Osamu Shimomura, Roger Y. Tsien

Pallette of fluorescent proteins

there is a wide variety of fluorescating proteins availabe (Emmax 448 – 600 nm) derived from modified GFP (YFP, CFP) or corals RFPs.

Proteins can be fused to any protein of interest to study protein geography,movement, lineage and biochemistry in living cells

Roger Y. Tsien Nobel Lecture 2008

www.fpbase.org

Phycobiliproteins

• intensely fluorescent proteins from cyanobacteria and algae

• chromophores (bilins) are covalently bound to the protein subunit

• very stable (10x more than FITC), water soluble, display high quantum yields

• do not form autocatalytically, use them as a chemical tags (bind with antibodies)

• conjugatable to other proteins

PhycoerythrobilinRed algae - Rhodophyta HeLa cells stained with PElabeled antibody

Quantum dots - nanocrystals made of semiconductor - small enough to exhibit quantum mechanical properties- coated with a hydrophilic polymer- can be conjugated to antibodies, peptides, carbohydrates - energy of emitted photon depends on the particel size

Significant benefits:

long term photostability, high fluorescense intensity levels, multiple

colors with single wavelength excitation for all emission profiles, narrow

emission profile.

Optical Highlighters fluorescent proteins which undergo a variety of

light-induced switching characteristics

photoactivatable

photoconvertable

photoswitchable

Photoactivatable

An optical highlighter derived from aceGFP, isolated from Aequorea coerulescens, has been reported to transition from cyan (468 nanometers) to green fluorescence (511 nanometers) upon illumination at 405 nanometers.Used for superresolution techniques like PALM

Photoconvertable

One of the most useful classes of optical highlighters encompasses the growing number of fluorescent proteins reported to undergo photoconversion from one emission wavelength to another.

First isolated from the stony Open Brain Coral, Trachyphylla

geofrey.

Kaede photoconverts from green to red emission by illumination

with ultraviolet light.

Photoconversion of Kaede is stable and irreversible.

Mechanism is the light catalyzed cleavage of the the His 62-Tyr63-

Gly64 backbone

Photoswitchable

the ability to switch between fluorescent and dark states

- Dronpa is most prominent, the monomeric variant derived from a stony

coral tetramer

- illumination at 488 nanometers drives Dronpa to the dark species,

which can be switched back on by brief illumination at 405 nanometers

- this cycle can be repeated several hundred times without significant photobleaching

- used for superresolution techniques like RESOLFT

Take care: excitation efficiency

FITC (Emax 480) excitation with 488 nm laser (87%), 477 nm laser (56%), 514 laser (28%)Alexa Fluor 546 excitation with 568 laser (84%), 543 laser ( 43%), 594 (4%)Take care when double labeling with FITC and Alexa Fluor 546, 7% of Alexa Fluor 456are excited with 488 nm laser.

Take care: bleedthrough artifacts

- are due to very broad bandwidths and asymetrical spectral profiles exhibited by many common fluorophores which lead to spectral overlap

- fundamental problem of both widefield and laser scanning microscopy- manifested by the emission of one fluorophore being detected in the PMT or filtercombination reserved for another fluorophore

- artifacts can complicate the interpretation of the results especially wheninvestigating the colocalization of fluorophores, quantitative measurements

Simultaneously

Sequentialy

Scanning of a thin sectionof mouse intestine Labeledwith Alexa fluor 488and Cy3 with 488 nm argonlaser and 543 nm helium-neolaser

Spectra analyzer

Remember!

Choose:

● optical filters which can match absorption and emission maxima of your fluorophore optimally!

● fluorophores which have absorption max. which closely match your laser lines

● dyes with high quantum yield for target which you have less

Always know properties of your fluorophore.

It's good to spend some time to design your experiment properly.

...and good luck!