Transcript Optical spectroscopy - UFCH JH

CZECH TECHNICAL UNIVERSITY IN PRAGUE
FACULTY OF BIOMEDICAL ENGINEERING
Fluorescence spectroscopy and microscopy
for biology and medicine
Martin Hof, Radek Macháň
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
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 Tutorials
www.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?
electric
fields
ions
• it provides information on
the molecular environment
Also fluorescence is very,
very,Fluorescent
very sensitive!
viscosity
temperature
• it provides information on
dynamic processes on the
nanosecond
timescale
Work with
subnanomolar
Probe
concentrations is
routine while femtomolar
polarity
pH
and even SINGLE MOLECULE studies are
possible with some effort
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
Experimental Systems
Molecular structure and dynamics
Cell organization and function
Actin
Mitochondria
Nucleus
Biological membrane
Multicellular organisms
GFP in a mouse
Instrumentation
Fluorimeters
High throughput platereaders
Microscopes
A very brief history of the study of light
1. Sir Isaac Newton 1672:
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 .
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
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





B
D
 E  
 H  j 
t
t


 D  
 B  0
B
E
n
B E
c
we are interested in interactions of the electric field with the matter
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 = h , where Planck constant h = 6.6256 *10-34 Js.
7. Albert Einstein: 1905
Explained the explained the photoelectric effect by assuming that light is adsorbed at
discrete energetic quanta E = h , 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
=h/p.
Wavelength and energy scale,
appropriate units
Visible light
X-ray
Microwave
UV
IR
Radio
Wavelength
nm
Frequency Hz
Wavenumber
cm-1
Energy Kcal
Energy eV
10-4
1021
1011
108
107
10-2
1019
109
106
105
100
1017
107
104
103
102
1015
105
102
101
104
1013
103
100
10-1
106
1011
101
10-2
10-3
108
109
10-1
10-4
10-5
1010
107
10-3
10-6
10-7
The optical region of the
electromagnetic spectrum
Visible light
UV
nm
Wavelength
10-4
nm
10-2
100
molecules << wavelength
the whole molecules sense the same
phase of light (vs. X-ray diffraction)
IR
102
104
106
108
wavelength << optical elements
vs. microwave or r.f. techniques
1010
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.
• 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
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
2
absorption
spontaneous
emission
induced emission
1
•
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
x
na

x = 0.07
• Larger scatterers – macromolecules, cells, Mie theory for
spherically symmetrical scatterers
x=7
http://omlc.ogi.edu/calc/mie_calc.html
Raman scattering
• 1923 theoretically predicted by Adolf Smekal using
classical physics
•
1928 observed by C. V. Raman
C.V. Raman
(1888-1970)
0-D
the photon and the molecule
exchange energy
0+D
0
the photon is not absorbed:
v2
hD
v1
elastic
Stokes
anti-Stokes
branch of Raman spectrum
scattering is an instantaneous and
coherent
Raman spectrum
4
 2 0  D 
 hD 


exp


intensity of Stokes branch is higher by a factor 

 2kT 
 2 0  D 
anti-Stokes
Stokes
Absorption of light
S
2
Nf2 molecules
DE = hv0
1
I0
dx
I
dx  c dt
n
 electromagnetic energy density
w
M h
S dx
 M – number of photons
 N – number of molecules
N = c NAS dx
Nf1 molecules
2
d P12
 D12 cos2  F ( )w
dt
  angle between polarization and D12,
cos2   1 3
for random orientation
of molecules
 F shape of the spectral line –
conservation of energy
 small energy approximation – assumes
that absorption does not change
f2/f1=exp(DE/kT)=f(T)
Absorption of light
S
2
Nf2 molecules
DE = hv0
1
I0
dx
I
Nf1 molecules
2
d P12
 D12 cos2  F ( )w
dt
2
dM
dP21 
Mh
 dP12
2
 N f1
 f2
cNAdx
   D12 cos  F ( )f(T )
dt
dt 
dx
 dt
2
dM
 C onst.D12 cos2  F ( )f(T )Mc
dx
M(x)  M(0) eb( )cx
I
log   () c l
I0
I  I0 eb( )cx
the Lambert Beer law
 the molar extinction coefficient (molar absorptivity)
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
sample
Deuterium/
Tungsten
Lamp
Detector
PMT sample
I
PMT reference
Io
Monochromator
blank
Electronic transitions from the ground state to the excited state
Energy
S1
Probability
v1 3
HIGH
v 12
HIGH
v 11
MEDIUM
v1 0
LOW
G
Probability
v3
v2
v1
Wavelength nm
v0
Inter-nuclear distance
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
S1
v1 3
v 12
v 11
v1 0
Most favored
transitions occur From
the
maximum shaded
areas of the ground
state
To the maximum
shaded areas of the
excited state
G
v3
v2
v1
v0
Inter-nuclear distance
Electronic – vibrational spectrum
other transitions (other
vibrational modes, nonfundamental transitions,…)
effect of room temperature
effect of molecular surroundings
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 -bonds
Increasing the number of double bonds shifts the absorption to lower energy
N=5
5 pi-bonds, 10 electrons
N=4
4 pi-bonds, 8 electrons
N=3
3 pi-bonds, 6 electrons
Wavelength nm
Increasing the number of aromatic rings increases the absorption maximum
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)
275 nm
375 nm
475nm
absorption wavelength
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)
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
Typical sources of luminescence
•
organic molecules (usually with conjugated -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
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