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IPC Friedrich-Schiller-Universität Jena1
ASP_MP_S2jBiophotonics
Prof. Dr. Rainer Heintzmann
Institut für Physikalische ChemieFriedrich-Schiller-Universität Jena
Lecture 1
IPC Friedrich-Schiller-Universität Jena2
Content
1. Introduction2. Contrast modes in light microscopy
2.1 Bright field microscopy2.2 Dark field microscopy2.3 Phase contrast microscopy2.4 Polarisation microscopy2.5 Differential interference contrast
3. Optical coherent tomography4. Molecular many electron systems:
electronic and nuclear movement5. UV-Vis absorption
5.1 Franck-Condon principle5.2 Electronic chromophores5.3 Polarimetry & circular dichroism
6. Fluorescence spectroscopy6.1 Stokes shift6.2 Fluorescence life time6.3 Fluorescence quantum yield6.4 Steady state fluorescence emission6.5 Fluorescence excitation spectroscopy
IPC Friedrich-Schiller-Universität Jena3
Content
7. Fluorescence microscopy7.1 Fluorochromes7.2 Confocal fluorescence microscopy7.3 FRET7.4 FRAP, iFRAP, FLIP7.5 Ultramicroscopy / SPIM / HILO7.6 Multi-photon microscopy7.7 4Pi microscopy7.8 STED microscopy7.9 linear and nonlinear structured illumination 7.9 PALM/STORM
8. Vibrational microspectroscopy8.1 Normal modes8.2 IT-absorption microspectroscopy8.3 Raman microspectroscopy8.4 Protein structure determination8.5 Biomedical diagnostics8.6 Resonance Raman spectroscopy8.7 SERS
IPC Friedrich-Schiller-Universität Jena4
Content
9. Non-linear Raman microspectroscopy9.1 Hyper Raman9.2 Coherent anti-Stokes Raman scattering (CARS)9.3 Stimulated Raman microscopy
10. Future trends in non-linear microscopy
IPC Friedrich-Schiller-Universität Jena5
1. Introduction
Engineering
Optical EngineeringMedical Engineering
Sciences
Biology Physics Chemistry
Medicine
(wealth of disciplines)
Bio-photonics
Biophotonics a highly interdisciplinar approach
IPC Friedrich-Schiller-Universität Jena6
Light-Matter Interactions as the basis for Biophotonics
1. Introduction
IPC Friedrich-Schiller-Universität Jena7
Light-Matter InteractionsAbsorption
Scattering
RefractionReflection () =absorption cross-sectionS = scattering cross-sectionI(z) = intensity in depth zI0 = incident intensityI() = transmitted intensity
Gewebe
Anregungslicht
transmittiertes Licht
gestreutes Licht
reflektiertes Licht
Gewebe
Anregungslicht
transmittiertes Licht
gestreutes Licht
reflektiertes Licht
incident light reflected light
scattered light
transmitted light
tissue
1. Introduction: Light-Matter Interactions
IPC Friedrich-Schiller-Universität Jena8
100 1000 10000
Wellenlänge / nm
Abs
orp
tions
koef
fizie
nt /
cm
-1
100
10
1
1000
10000
UV Vis IR
Aorta
Haut
gesamtes Blut
Melanosom
Epidermis
Wasser
100 1000 10000
Wellenlänge / nm
Abs
orp
tions
koef
fizie
nt /
cm
-1
100
10
1
1000
10000
UV Vis IR
Aorta
Haut
gesamtes Blut
Melanosom
Epidermis
Wasser water
epidermis skin
aorta
blood
melanosom
1. Introduction: Light-Matter Interactions
IPC Friedrich-Schiller-Universität Jena9
1. Introduction: Light-Matter Interactions
+ -
E
+
Polarisation P : Dipole moment per unit volume
IPC Friedrich-Schiller-Universität Jena10
Linear Polarisation
1. Introduction: Light-Matter Interactions
litysusceptibilinear :
space free ofty permittivi :)1(
0
)1(0
EP
12)1( n
IPC Friedrich-Schiller-Universität Jena11
Nonlinear Polarisation
1. Introduction: Light-Matter Interactions
litiessusceptibinonlinear order thirdand second :,
space free ofty permittivi :
...
)3()2(
0
3)3(2)2()1(0
EEEP
EEE )1(2)2(3)3( for convergence:
IPC Friedrich-Schiller-Universität Jena12
Nonlinear Polarisation
1. Introduction: Light-Matter Interactions
frequency :2
amplitude :
)cos(
E
tEE
yields:
...Re
...)3cos()2cos()cos(3
32
210
2312010
tititi ePePePP
tPtPtPPP
...3)3(2)2()1(0 EEEP
IPC Friedrich-Schiller-Universität Jena13
Example
1. Introduction: Light-Matter Interactions
frequency :2
amplitude :
field DC :
)cos(
0
0
E
E
tEEE
0)1( E
Terms in P:
)cos()1( tE
Frequency Name
DC DC polarizability
optical polarizability (refractive index)
)cos(0)2( tEE
20
)2( E DC hyperpolarizabilityDC
linear electrooptic effect(Pockels Effect)
2)2(wE DC hyperpolarizability
)2cos(2)2( tE second harmonic generation
)3cos(3)3( tE third harmonic generation
2
3
DC
)cos(2)3( tE Kerr effect (n=n0+n2I)
IPC Friedrich-Schiller-Universität Jena14
Process
(1)
Linear absorption Spontaneous emission
(Fluorescence) Reflection Elastic scattering Inelastic scattering: Raman-
scattering Diffraction
(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-Stokes-
Raman-Scattering)
1. Introduction: Light-Matter Interactions
IPC Friedrich-Schiller-Universität Jena15
Absorption Dispersion
Bright field
Dark field Phase contrast Differential phase contrast
2. Contrast modes in light microscopy
Amplitude difference
Wavelength
Phase difference
Refractive indices
nR : real part of refractive indexnI : imaginary part of refractive index
: 1D monochr. wavec
nnkn
kvac
0
22
IPC Friedrich-Schiller-Universität Jena16
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 is called amplitude object. Contrast is A0 –A1,2
Bright filed microscopy is the most simpleand basic light microscopy method
Sample is illuminated from belowby a light cone
In case there is no sample in the opticalpath a uniform bright image is generated
An amplitude object absorbs light at certain wavelengths and therefore reduces the amplitude of the light passing through the object
2. Contrast modes in light microscopy: Bright field
Amplitude difference
Wavelength
Uniform bright field image Bright field image of Moss reeds