Fluorescence spectroscopy and microscopy for biology and medicine

Martin Hof, Radek Macháň

CZECH TECHNICAL UNIVERSITY IN PRAGUE

FACULTY OF BIOMEDICAL ENGINEERING

Absorption of light and electronic transitionsBasic principles of fluorescence, fluorescence spectraLifetime of fluorescence and its measurementQuenching of fluorescence and its biological applicationsAnisotropy of fluorescence and its biological applicationsInfluence of solvent on fluorescence spectraFoerster resonance energy transfer and excimer fluorescenceFluorescent proteinsFluorescence microscopy, confocal and 2-photon microscopyResolution of fluorescence microscope and its enhancementFluorescence correlation spectroscopyPhotodynamic Therapy

Fluorescence spectroscopy and microscopy for biology and medicine

CZECH TECHNICAL UNIVERSITY IN PRAGUE

FACULTY OF BIOMEDICAL ENGINEERING

Martin Hof, Radek Macháň

Basic literature:

1. Lakowicz J.R.: Principles of Fluorescence Spectroscopy, 3rd edn. Springer 2006 cfs.umbi.umd.edu/

2. Hof M., Hutterer R., Fidler V.: Fluorescence Spectroscopy in Biology. Springer Verlag

3. Gauglitz G., Vo-Dinh T.: Handbook of Spectroscopy. Wiley VCH Verlag, Weinheim 2003

4. Prosser V. a kol.: Experimentální metody biofyziky. Academia,Praha 1989

5. Invitrogen Tutorialswww.invitrogen.com/site/us/en/home/support/Tutorials.html

6. Becker W.: The bh TCSPC Handbook http://www.becker-hickl.com/literature.htm

Why fluorescence?

FluorescentProbe

ionselectric fields

viscosity

polaritypH

temperature

Fluorescence Probes are essentially molecular stopwatches which monitor dynamic events which occur during the excited state lifetime – such as movements of proteins or protein domains

• it provides information on the molecular environment

• it provides information on dynamic processes on the nanosecond timescale

Also fluorescence is very, very, very sensitive!

Work with subnanomolar concentrations is routine while femtomolar

and even SINGLE MOLECULE studies are possible with some effort

Experimental Systems

ActinMitochondria

Nucleus

Cell organization and function

Multicellular organisms

Molecular structure and dynamics

GFP in a mouse

Biological membrane

Instrumentation

Microscopes Fluorimeters

High throughput platereaders

2. Christian Huygens 1692: Developed a wave theory of light

A very brief history of the study of light

Showed that the component colors of the visible portion of white light can be separated through a prism, which acts to bend the light (refraction) in differing degrees according to the wavelength. Developed a “corpuscular” theory of light .

1. Sir Isaac Newton 1672:

3. Hans Christian Oersted 1820Showed that there is a magnetic field associated with the flow of electric current

4. Michael Faraday 1831Showed the converse i.e. that there is an electric current associated with a change of magnetic field

5. James Clark Maxwell: 1865Published his “Dynamical theory of the electromagnetic field” which combined the discoveries of Newton, Young, Foucault, Oersted and Faraday into a unified theory of electromagnetic radiation

Light consists of electromagnetic transverse waves of frequency and wavelength related by = nc where n is the index of refraction of the medium and c is the speed of the light in vacuum c = 3x1010 cm/s

E

B

Ecn

B

we are interested in interactions of the electric field with the matter

0

BD

t

DjH

t

BE

6. Max Karl Ernst Ludwig Planck: 1900Explained the laws of black body radiation by postulating that electromagnetic radiation is emitted at discrete energetic quanta E = h , where Planck constant h = 6.6256 *10-34 Js.

7. Albert Einstein: 1905Explained the explained the photoelectric effect by assuming that light is adsorbed at discrete energetic quanta E = h , photons.

8. Louis de Broglie: 1924Introduced properties of electromagnetic waves to all particles – the wave-corpuscular dualism of quantum physics. A freely moving particle of momentum p has wavelength =h/p.

Wavelength and energy scale, appropriate units

X-ray

UV

Visible light

IR

Microwave

Radio

Wavelength nm10-4 10-2 100 104102 106 108 1010

Frequency Hz 1021 1019 1017 1015 1013 1011 109 107

Wavenumber cm-1 1011 109 107 105 103 101 10-1 10-3

Energy Kcal108 106 104 102 100 10-2 10-4 10-6

Energy eV107 105 103 101 10-1 10-3 10-5 10-7

The optical region of the electromagnetic spectrum

UV

Visible light

IR

nm10-4 10-2 100 104102 106 108 1010

Wavelength nm

wavelength << optical elementsmolecules << wavelength

vs. microwave or r.f. techniquesthe whole molecules sense the same phase of light (vs. X-ray diffraction)

Interaction of electromagnetic waves with matter

• Atoms and molecules described as electric multipoles, first approximation: electric dipole

• Classical electrodynamics: dipoles oscillate at the frequency of the external electromagnetic field

+

-

Elastic scattering of light

Interaction of molecules with photons - quantum description

• Light exists in form of discrete quanta – photons E = h • Atoms and molecules occupy discrete energetic states,

which can be found as the solution of Schroedinger’s equation.

electronic states

vibrational states

rotational states

J = 1

microwave region

N = 1

IR – VIS region

UV – VIS regionE

• Exchange of energy with photons is accompanied by transitions between those states.

Interaction of light with matter – overview of processes

• elastic scattering – no exchange of energy between the molecule and the photon

• inelastic (Raman) scattering – the photon either gives a part of its energy to the molecule or vice versa

• absorption or emission of photons by the molecule

1

2absorption

spontaneous emission

induced emission

•induced emission is coherent with incident light

•spontaneous emission by individual molecules is incoherent

•scattering is coherent and instantaneous

Elastic scattering of light• Rayleigh scattering – small molecules (x<0.3) as a “point

dipole”, Isc ≈ 4 blue sky, red sunset

• Larger scatterers – macromolecules, cells, Mie theory for spherically symmetrical scatterers

x = 0.07

x = 7

an

x

http://omlc.ogi.edu/calc/mie_calc.html

Raman scattering

C.V. Raman

(1888-1970)

•1923 theoretically predicted by Adolf Smekal using classical physics

•1928 observed by C. V. Raman

Stokeselastic anti-Stokes

branch of Raman spectrum

the photon and the molecule exchange energy

the photon is not absorbed:

scattering is an instantaneous and coherentv1

v2

0

0-

h

+

Raman spectrum

intensity of Stokes branch is higher by a factor

Tk2h

exp22

4

0

0

Stokesanti-Stokes

Absorption of light

wFt

P)(cos22

1212 D

d

dI0dx I

1

2

E = hv0

Nf1 molecules

Nf2 molecules

angle between polarization and D12, for random

orientation of molecules31cos2

F shape of the spectral line – conservation of energy

S

small energy approximation – assumes that absorption does not change f2/f1=exp(E/kT)=(T)

electromagnetic energy density

xSM

wdh

M – number of photons

tnx dcd

N – number of molecules

N = c NAS dx

Absorption of light

wFt

P)(cos22

1212 D

d

dI0

dx I

1

2

E = hv0

Nf1 molecules

Nf2 molecules

S

xcx

MhTF

tP

ft

PfN

tM

dNd

)()(cosd

dd

ddd

A22

1221

212

1

D

McTFxM

)()(cosConst.dd 22

12 D

cxbMxM )(e)0()( cxbII )(0 e

lcII

)(log0

the molar extinction coefficient (molar absorptivity)

the Lambert Beer law

Absorption: measurement

The Beer Lambert Law

Absorption (Optical Density) = log Io / I = c l

l is the path length of the sample (1 cm)

Deuterium/TungstenLamp

PMT sample

PMT reference

I

Io

Mono-chromator

sample

blank

Detector

•a typical sample: a solution in a cuvette

•the solvent and the reflection from the cuvette walls contribute to the

extinction of light

•relative measurement of absorption

Probability HIGH

HIGH

MEDIUM

LOW

Energy

Inter-nuclear distance

G

S1

v 0

v 1

v 2

v 3

v1 0

v 11

v 12

v1 3

Electronic transitions from the ground state to the excited state

Probability

Wavelength nm

Inter-nuclear distance

G

S1

v 0

v 1

v 2

v 3

v1 0

v 11

v 12

v1 3

Electronic transitions from the ground state to the excited state

Shaded areas reflects the probability of where the electron would be if it were in that vibrational band

Most favored transitions occur From the

maximum shaded areas of the ground state

To the maximum shaded areas of the excited state

Electronic – vibrational spectrum

other transitions (other vibrational modes, non-fundamental transitions,…)

effect of room temperature effect of molecular surroundings

The wavelength value of the absorption maximum

and the molar absorptivity

are determined by the degree of Conjugatation of -bonds

Absorption maxima : The importance of conjugation

Increasing the number of double bonds shifts the absorption to lower energy

mola

r ab

sorb

tivity

Wavelength nm

N=55 pi-bonds, 10 electrons

N=44 pi-bonds, 8 electrons

N=33 pi-bonds, 6 electrons

As the degree of conjugation increases (i.e the number of electrons involved in the delocalized -orbitals)

the absorption energy decreases (> , the energy between the ground and excited state decreases)

the absorption becomes more intense (>, increased probability of absorption)

Benzene < Naphthalene < Anthracene < naphthacene < pentacene Abs. Max 262nm 275 nm 375 nm 475 nm 580 nm Log 3.84 3.75 3.90 4.05 4.20 (Extinction)

Log

Exti

ncti

on

Coeffi

cie

nt

275 nm 375 nm 475nm absorption wavelength

Increasing the number of aromatic rings increases the absorption maximum

Emission of light - Luminescence

Luminescence – the excess of light emitted above thermal radiation. The emission follows after the molecule has resided for some time in the excited state.

according to excitation mechanism:

photoluminescence – absorption of light

chemiluminescence – chemical reaction

thermoluminescence – heat

electroluminescence – electric current

…

fluorescence

phosphorescencephotoluminescence – absorption of light

Typical sources of luminescence

• organic molecules (usually with conjugated -bonds) – synthetic fluorophores (fluorescein, rhodamine, …), biological molecules (aromatic amino acids – Trp, Tyr, chlorophyll, …)

• inorganic crystals (diamond, Si, GaAs, … ) – the spectra depend on the bandgap size, which depends on the size of the crystal (nanocrystals emit in VIS – quantum dots), extreme photostability

• small inorganic molecules – noble gases (in discharge lamps), N2 (in lasers, responsible for bluish colour of spark discharges), …

quantum dots – same material, different sizes

Acknowledgement

The course was inspired by courses of:

Prof. David M. Jameson, Ph.D.

Prof. RNDr. Jaromír Plášek, Csc.

Prof. William Reusch

Financial support from the grant:

FRVŠ 33/119970