Principle of fluorescence

Outline• Luminescence : fluorescence or phosphorescence?• Jablonski diagram• Characteristics of fluorescence emission• Fluorescence lifetime and quantum yield• Quantum mechanic behind• Quenching• Beer-Lambert law• Biochemical fluorescence

phosphorescence• Phosphorescence – electron go back to

ground state from triplet excited state (which is forbidden). Thus, it has lower rate about 103~100 s-1 (life time≈ms~s)

Pic. from : http://www.glassner.com/andrew/cg/research/fluphos/fluphos.htm

Fluorescence

• Fluorescence – electron go back to ground state from singlet excited state. Fluorescence has emission rate about 108 s-1 (lifetime≈ns).

Pic. From : http://en.wikipedia.org/wiki/Image:Fluorescent_minerals_hg.jpg

Jablonski diagram

• Jablonski diagram can schematically tell us the fluorescence activity. It is proposed by Professor Alexander Jablonski in 1935 to describe absorption and emission of light.

Time scale (s)

Absorption 10-15

IC 10-12

ISC 10-8

F 10-8

P 10-3-100

π-bond

Mirror image

Exception of mirror image

• Relaxation time is much smaller than emission, ΔE is much bigger than emission.

• Excimer – excited state dimer.• Influence of solvent – pH, O2…et

fluorescein

Emission characteristic – Stoke’s shift

• Obviouly, form the Jablonski diagram of previous page, we know energy of emission light is less than energy of absorption light.

• This energy shift is called “Stoke’s shift”, usually shown in diagram by wavelength or wavenumber difference.

• Q1 and Q0 are energies of vibration taken by surround molecules.Q1≈Q0

hvF hvA (Q1 Q0) /NA

Stoke’s shift

Fluorescence lifetime

• First order rate equation!

• Unfortunately, there’s also contained nonradiative decay in nature.

• ko-1 is called natural lifetime, (ko+knr)-1 is real

lifetime.

d[ f ]

dt k0[ f ] [ f ] [ f ]0

k0te

d[ f ]

dt (k0 knr)[ f ] [ f ] [ f ]0

(k0 knr )t

e

Quantum yield

• Definition is is the ration of the number of photons emitted to the number of photons absorbed. That is emission efficiency.

• Quantum yield can be calculated from standard quantum yield.

Qphotons emitted

photons absorbed

k0

k0 knr

QQRI

IR

ODROD

n2

nR2

Quantum yield of some fluorephores

Quantum Yield [Q.Y.] Standards

Q.Y.[%] Conditions for Q.Y. Measurement

Excitation [nm]

Cy3 4 PBS 540

Cy5 27 PBS 620

Cresyl Violet 53 Methanol 580

Fluorescein 95 0.1 M NaOH, 22°C 496

POPOP 97 Cyclohexane 300

Quinine Sulfate 58 0.1 M H2SO4, 22°C 350

Rhodamine 101 100 Ethanol,25°C 450

Rhodamine 6G 94 Ethanol 488

Rhodamine B 31 Water 514

Tryptophan 13 Water, 20°C 280

L-Tyrosine 14 Water 275

Quenching

• Enery of excited state could be taken by other substance, this process is called fluorescence quenching.

• Collisional (dynamics) quenching and static (complex- forming) quenching are most often process in quenching.

Collisional quenching

• Oxygen, halogen, amines, and electron-deficient molecule often act as quenchers.

• In simplest quenching, stern-volmer equation holds

KD is stern-volmer quenching constant, kq is bimolecular constant, τ0 is unquenched lifetime.

F0

F1KD[Q] 1 kq 0[Q]

Static quenching

• Energy is taken by forming complex.

• Combine with collisional quenching

Ks [FQ]

[F][Q]

[F]0 [F]

[F][Q]

F0

F1Ks[Q]

F0

F(1KD[Q])(1Ks[Q])

Modify Stern-Volmer plots

• Some of fluorphores are accessibile and some aren’t for quenchers.

F0 Fa Fb F Fa

1Ka[Q] Fb

F0

F F0

1

faKa[Q]

1

fa

, where fa FaF0

F0 F FaKa[Q]

1Ka[Q]

Time scale of molecular processes in solution

• Is quenching rapidly happened? • Ex. quenching by O2, which has diffusion

coefficient 2.5 x 10-5 cm2/s. The average distance of an O2 can diffuse in 10ns is given by Eistein equation

• About 7 nm. Concentration of quenching would process is

• In 25 oC water, oxygen dissolve is about 10-4 M

x 2 2D 5*10 13cm

C Liter

(7nm)3 3.3*1021 0.5 *10 2M

Optical density

• In optics, density is the transmittance of an optical element for a given length and a given wavelength.

• In fluorescence, opticaldensity indicates us the absorption of fluorescentsolution.

Optical density log10

I0I

d

Beer-Lambert law

• Absorption of light go through a substance is proportional to the effective cross section(σ), concentration of molecules(n) and intensity(I).

• Rewrite the Beer-Lambert law

where c is concentration (M) and ε is the extinction coefficient (M-1cm-1)

dI

dx In ln

I0I

nd

lnI0I

nd log10

I0I

cd optical density

Extinction coefficient

• Extinction coefficient is calibrated by a fluorescent solution with width 1 cm and concentration 1 mole per liter.

fluorphores Extinction coefficient (M-1cm-

1)

Rhodamine 6G 105,000

Rhodamine B 123,000

Cell tracker (BLue) 16,000

SYTOX 38,000

IF Iincantilog( cd) * (Quantum Yield)

Inner filter effect (IFE)

• Solution with optical density absorbs not only excitation light but also emission light.

• Excitation IEF• Emission IEF – absorbs by solute or fluorphores• Correction of IFE could be wrote down in the

following formula

• Usually, solution with OD<0.05 avoids IFE.

IF Iabsorbanti logODex ODem

2

Biochemical fluorophores

• Intrinsic fluorphores• Extrinsic fluorphores• DNA probes• Chemical sensing probes• Fluorscent protein

Intrinsic fluorphores

Intrinsic fluorphores

• Vitamine A – Retinol, in liver stellate cell and retina.

Retinol

Extrinsic fluorphores

• Eg. FITC, rhodamine – conjugate with protein, dextran, antibody…etc. for labeling specific target.

wavelength

fluor

esce

nce

Extrinsic fluorphores

• Different Stoke’s shift of rhodamine derivatives.

wavelength1.Fluorescein 2.Rhodamine 6G

3.Tetramethylrhodamine 4.Lissamine rhodamine B 5.Texas Red

DNA probes

• Hoechst33342 (binding to minor groove of DNA)

Red: rhodamine dextran blue: hoechst33342

Fluorescent protein

• GFP – Green fluorescence protein• Extracted from jellyfishAequorea victoria.• Vector contained DNA of GFP is used in celltransfection.

GFP

As a reporter

• GFP vector• Put in liposome• Place into cells by injection or fusion

• Use as NFkB reporter

EGFP vector