1. Fluorescence spectroscopy and microscopy for biology and medicine
CZECH TECHNICAL UNIVERSITY IN PRAGUE
FACULTY OF BIOMEDICAL ENGINEERING
Fluorescence spectroscopy and microscopy for biology and medicine
Martin Hof, Radek Macháň

2. Fluorescence spectroscopy and microscopy for biology and medicine
CZECH TECHNICAL UNIVERSITY IN PRAGUE
FACULTY OF BIOMEDICAL ENGINEERING
Fluorescence spectroscopy and microscopy for biology and medicine
Martin Hof, Radek Macháň
Absorption of light and electronic transitions
Basic principles of fluorescence, fluorescence spectra
Lifetime of fluorescence and its measurement
Quenching of fluorescence and its biological applications
Anisotropy of fluorescence and its biological applications
Influence of solvent on fluorescence spectra
Foerster resonance energy transfer and excimer fluorescence
Fluorescent proteins
Fluorescence microscopy, confocal and 2-photon microscopy
Resolution of fluorescence microscope and its enhancement
Fluorescence correlation spectroscopy
Photodynamic Therapy

3. Basic literature:
Lakowicz J.R.: Principles of Fluorescence Spectroscopy, 3rd edn. Springer 2006 cfs.umbi.umd.edu/
Hof M., Hutterer R., Fidler V.: Fluorescence Spectroscopy in Biology. Springer Verlag
Gauglitz G., Vo-Dinh T.: Handbook of Spectroscopy. Wiley VCH Verlag, Weinheim 2003
Prosser V. a kol.: Experimentální metody biofyziky. Academia,Praha 1989
Invitrogen Tutorials
Becker W.: The bh TCSPC Handbook

4. Also fluorescence is very, very, very sensitive!
Why fluorescence?
Fluorescent
Probe
ions
electric fields
viscosity
polarity pH
temperature
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
it provides information on the molecular environment
it provides information on dynamic processes on the nanosecond timescale
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

5. Experimental Systems Molecular structure and dynamics
Cell organization and function
Actin
Mitochondria
Nucleus
Biological membrane
Multicellular organisms
GFP in a mouse

6. Instrumentation
Microscopes
Fluorimeters
High throughput platereaders

7. 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:
2. Christian Huygens 1692:
Developed a wave theory of light
3. Hans Christian Oersted 1820
Showed that there is a magnetic field associated with the flow of electric current
4. Michael Faraday 1831
Showed the converse i.e. that there is an electric current associated with a change of magnetic field

8. 5. James Clark Maxwell: 1865
Published 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
we are interested in interactions of the electric field with the matter

9. 6. Max Karl Ernst Ludwig Planck: 1900
Explained the laws of black body radiation by postulating that electromagnetic radiation is emitted at discrete energetic quanta E = hn , where Planck constant h = *10-34 Js.
7. Albert Einstein: 1905
Explained the explained the photoelectric effect by assuming that light is adsorbed at discrete energetic quanta E = hn , photons.
8. Louis de Broglie: 1924
Introduced properties of electromagnetic waves to all particles – the wave-corpuscular dualism of quantum physics. A freely moving particle of momentum p has wavelength l=h/p.

10. Wavelength and energy scale, appropriate units
X-ray UV
Visible light IR
Microwave
Radio
Wavelength nm
10-4
10-2
100
104
102
106
108
1010
Frequency Hz
1021
1019
1017
1015
1013
1011
109
107
Wavenumber
cm-1
105
103
101
10-1
10-3
Energy Kcal
10-6
Energy eV
10-5
10-7

11. The optical region of the electromagnetic spectrumUV
Visible light IR nm
10-4
10-2
100
104
102
106
108
1010
Wavelength nm
wavelength << optical elements
molecules << wavelength
the whole molecules sense the same phase of light (vs. X-ray diffraction)
vs. microwave or r.f. techniques

12. 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

13. Interaction of molecules with photons - quantum description
Light exists in form of discrete quanta – photons E = hn
Atoms and molecules occupy discrete energetic states, which can be found as the solution of Schroedinger’s equation.
Exchange of energy with photons is accompanied by transitions between those states.
rotational states
DJ =  1
microwave region
vibrational states
DN =  1
IR – VIS region E
electronic states
UV – VIS region

14. 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 2
absorption
spontaneous emission
induced emission
induced emission is coherent with incident light
spontaneous emission by individual molecules is incoherent
scattering is coherent and instantaneous

15. Elastic scattering of light
Rayleigh scattering – small molecules (x<0.3) as a “point dipole”, Isc ≈ n4 blue sky, red sunset
x = 0.07
Larger scatterers – macromolecules, cells, Mie theory for spherically symmetrical scatterers
x = 7

16. Raman scattering
1923 theoretically predicted by Adolf Smekal using classical physics
1928 observed by C. V. Raman
C.V. Raman
( )
the photon and the molecule exchange energy
n0-Dn
n0+Dn n0
the photon is not absorbed:
scattering is an instantaneous and coherent v2
hDn v1
elastic
Stokes
anti-Stokes
branch of Raman spectrum

17. Raman spectrum intensity of Stokes branch is higher by a factor
anti-Stokes
Stokes

18. Absorption of light N = c NAS dx DE = hv0 I I02
Nf2 molecules
DE = hv0 1
Nf1 molecules I0 dx I
 angle between polarization and D12, for random orientation of molecules
electromagnetic energy density
F shape of the spectral line – conservation of energy
M – number of photons
small energy approximation – assumes that absorption does not change f2/f1=exp(DE/kT)=f(T)
N – number of molecules
N = c NAS dx

19. Absorption of light the Lambert Beer law DE = hv0 I I02
Nf2 molecules
DE = hv0 1
Nf1 molecules I0 dx I
the Lambert Beer law
e the molar extinction coefficient (molar absorptivity)

20. Absorption: measurement
The Beer Lambert Law
Absorption (Optical Density) = log Io / I =  c l
l is the path length of the sample (1 cm)
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
Deuterium/
Tungsten
Lamp
PMT sample
PMT reference I Io
Mono-
chromator
sample
blank
Detector

21. Electronic transitions from the ground state to the excited state
Energy S1
Probability
HIGH
MEDIUM
LOW
v1 3
v 12
v 11
v1 0 G
Probability
v 3
v 2
v 1
v 0
Inter-nuclear distance
Wavelength nm

22. Electronic transitions from the ground state to the excited state
Inter-nuclear distance G S1
v 0
v 1
v 2
v 3
v1 0
v 11
v 12
v1 3
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

23. Electronic – vibrational spectrum
other transitions (other vibrational modes, non-fundamental transitions,…)
effect of room temperature
effect of molecular surroundings

24. Absorption maxima : The importance of conjugation
The wavelength value of the absorption maximum
and the molar absorptivity
are determined by the degree of Conjugatation of p-bonds
Increasing the number of double bonds shifts the absorption to lower energy
molar absorbtivity
Wavelength nm
N=5
5 pi-bonds, 10 electrons
N=4
4 pi-bonds, 8 electrons
N=3
3 pi-bonds, 6 electrons

25. Increasing the number of aromatic rings increases the absorption maximum
Benzene < Naphthalene < Anthracene < naphthacene < pentacene
Abs. Max nm nm nm nm nm
Log e
(Extinction)
Extinction
Coefficient
Log
275 nm nm nm absorption wavelength
As the degree of conjugation increases
(i.e the number of electrons involved in the delocalized p-orbitals)
the absorption energy decreases (> l, the energy between the ground and
excited state decreases)
the absorption becomes more intense (>e, increased probability of absorption)

26. 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
phosphorescence
photoluminescence – absorption of light

27. Typical sources of luminescence
organic molecules (usually with conjugated p-bonds) – synthetic fluorophores (fluorescein, rhodamine, …), biological molecules (aromatic amino acids – Trp, Tyr, chlorophyll, …)
small inorganic molecules – noble gases (in discharge lamps), N2 (in lasers, responsible for bluish colour of spark discharges), …
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
quantum dots – same material, different sizes

28. 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