ASP_MP_S2j
Biophotonics
Prof. Dr. Rainer Heintzmann
Institut für Physikalische Chemie
Friedrich-Schiller-Universität Jena
Lecture 1
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Content
1. Introduction
2. Contrast modes in light microscopy
2.1 Bright field microscopy
2.2 Dark field microscopy
2.3 Phase contrast microscopy
2.4 Polarisation microscopy
2.5 Differential interference contrast
3. Optical coherent tomography
4. Molecular many electron systems:
electronic and nuclear movement
5. UV-Vis absorption
5.1 Franck-Condon principle
5.2 Electronic chromophores
5.3 Polarimetry & circular dichroism
6. Fluorescence spectroscopy
6.1 Stokes shift
6.2 Fluorescence life time
6.3 Fluorescence quantum yield
6.4 Steady state fluorescence emission
6.5 Fluorescence excitation spectroscopy
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Content
7. Fluorescence microscopy
7.1 Fluorochromes
7.2 Confocal fluorescence microscopy
7.3 FRET
7.4 FRAP, iFRAP, FLIP
7.5 Ultramicroscopy / SPIM / HILO
7.6 Multi-photon microscopy
7.7 4Pi microscopy
7.8 STED microscopy
7.9 linear and nonlinear structured illumination
7.9 PALM/STORM
8. Vibrational microspectroscopy
8.1 Normal modes
8.2 IT-absorption microspectroscopy
8.3 Raman microspectroscopy
8.4 Protein structure determination
8.5 Biomedical diagnostics
8.6 Resonance Raman spectroscopy
8.7 SERS
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Content
9. Non-linear Raman microspectroscopy
9.1 Hyper Raman
9.2 Coherent anti-Stokes Raman scattering (CARS)
9.3 Stimulated Raman microscopy
10. Future trends in non-linear microscopy
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1. Introduction
Biophotonics a highly interdisciplinar approach
Sciences
Medicine
Biology
Physics
(wealth of
disciplines)
Biophotonics
Chemistry
Engineering
Optical Engineering
Medical Engineering
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1. Introduction
Light-Matter Interactions as the
basis for Biophotonics
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1. Introduction: Light-Matter Interactions
Light-Matter Interactions
incident
light
Anregungslicht
Absorption
reflected
light
reflektiertes
Licht
tissue
Gewebe
Scattering
transmittierteslight
Licht
transmitted
gestreutes Licht
scattered
light
Reflection
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Refraction
a(n) =absorption cross-section
aS = scattering cross-section
I(z) = intensity in depth z
I0 = incident intensity
I(n) = transmitted intensity
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1. Introduction: Light-Matter Interactions
UV
IR
Vis
10000
Absorptionskoeffizient / cm-1
gesamtes
bloodBlut
melanosom
Melanosom
1000
100
Aorta
aorta
water
Wasser
10
1
skin
Haut
100
epidermis
Epidermis
1000
10000
Wellenlänge / nm
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1. Introduction: Light-Matter Interactions
+
+
-
E
Polarisation P : Dipole moment per unit volume
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1. Introduction: Light-Matter Interactions
Linear Polarisation
P  0 E
(1)
 0 : permittivi ty of free space
 : linear susceptibi lity
(1)
  n 1
(1)
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1. Introduction: Light-Matter Interactions
Nonlinear Polarisation


P   0  (1) E   ( 2) E 2   (3) E 3  ...
 0 : permittivi ty of free space
 ( 2) ,  (3) : second and third order nonlinear susceptibi lities
for convergence:
 E   E   E
( 3)
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3
( 2)
2
(1)
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1. Introduction: Light-Matter Interactions
Nonlinear Polarisation


P   0  (1) E   ( 2) E 2   (3) E 3  ...
E  E cos(t )
E : amplitude
  2n : frequency
yields:
P  P0  P1 cos(t  0 )  P2 cos( 2t  1 )  P3 cos(3t  2 )  ...


 Re P0  P1eit  P2ei 2t  P3ei 3t  ...
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1. Introduction: Light-Matter Interactions
Example
E  E0  E cos(t )
E0 : DC field
E : amplitude
  2n : frequency
Terms in P:
 (1) E0
 (1) E cos(t )
DC
Name
DC polarizability

optical polarizability (refractive index)
DC
DC hyperpolarizability

 ( 2) Ew 2
linear electrooptic effect
(Pockels Effect)
DC
DC hyperpolarizability
 ( 2) E 2 cos(2t )
2
second harmonic generation
 (3) E 3 cos(3t )
3
third harmonic generation

Kerr effect (n=n0+n2I)
 ( 2) E0 2
 ( 2) E0 E cos(t )
 (3) E 2 cos(t )
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Frequency
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1. Introduction: Light-Matter Interactions
Process
(1)
 Linear absorption
 Spontaneous emission
(Fluorescence)
 Reflection
 Elastic scattering
 Inelastic scattering: Ramanscattering
 Diffraction
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(2)
 Second harmonic generation
(SHG)
 Sum-frequency generation
(SFG)
 Difference-frequency
generation (DFG)
 Optical parametric
amplification
(3)
 Third harmonic generation
(THG)
 Two-photon absorption (TPA)
 CARS (Coherent Anti-StokesRaman-Scattering)
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2. Contrast modes in light microscopy
: 1D monochr. wave
Absorption
k
2
l

2n
lvac
 nk0  n
Dispersion
nR : real part of refractive index
nI : imaginary part of refractive index
Phase difference
Amplitude difference
Refractive
indices
Wavelength l
 Bright field
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 Dark field
 Phase contrast
 Differential phase contrast
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
c
2. Contrast modes in light microscopy: Bright field
2.1 Bright field transmission (absorption = imaginary part of refractive index)
An object, keeping the phase of an incoming wave constant and decreasing the
Amplitude difference
amplitude is called amplitude object.
Contrast is A0 –A1,2
Bright filed microscopy is the most simple
and basic light microscopy method
Sample is illuminated from below
by a light cone
In case there is no sample in the optical
path a uniform bright image is generated
Wavelength l
An amplitude object absorbs light at certain wavelengths and therefore reduces the
amplitude of the light passing through the object
Uniform bright field image
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Bright field image of Moss reeds
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