ASP_MP_S2j Biophotonics Prof. Dr. Rainer Heintzmann

IPC Friedrich-Schiller-Universität Jena1

ASP_MP_S2jBiophotonics

Prof. Dr. Rainer Heintzmann

Institut für Physikalische ChemieFriedrich-Schiller-Universität Jena

Lecture 1


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


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


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


1. Introduction

Engineering

Optical EngineeringMedical Engineering

Sciences

Biology Physics Chemistry

Medicine

(wealth of disciplines)

Bio-photonics

Biophotonics a highly interdisciplinar approach


Light-Matter Interactions as the basis for Biophotonics

1. Introduction


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


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




+ -

E

+

Polarisation P : Dipole moment per unit volume


Linear Polarisation


litysusceptibilinear :

space free ofty permittivi :)1(

0

)1(0

EP

12)1( n


Nonlinear Polarisation


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:


Nonlinear Polarisation


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


Example


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)


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)



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


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

ASP_MP_S2j Biophotonics Prof. Dr. Rainer Heintzmann

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