1. IPC Friedrich-Schiller-Universität Jena 1 ASP_MP_S2j Biophotonics Prof. Dr. Rainer Heintzmann Institut für Physikalische Chemie Friedrich-Schiller-Universität Jena Lecture 1 http://www.nanoimaging.de/Lectures/Biophotonics2010/ http://www.nanoimaging.de/Lectures/Biophotonics2011/

2. IPC Friedrich-Schiller-Universität Jena 2 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

3. IPC Friedrich-Schiller-Universität Jena 3 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

4. IPC Friedrich-Schiller-Universität Jena 4 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

5. IPC Friedrich-Schiller-Universität Jena 5 1. Introduction Engineering Optical Engineering Medical Engineering Sciences Biology Physics Chemistry Medicine (wealth of disciplines) Bio- photonics Biophotonics a highly interdisciplinar approach

6. IPC Friedrich-Schiller-Universität Jena 6 Light-Matter Interactions as the basis for Biophotonics 1. Introduction

7. IPC Friedrich-Schiller-Universität Jena 7 Light-Matter Interactions Absorption Scattering RefractionReflection  ( ) =absorption cross-section  S = scattering cross-section I(z) = intensity in depth z I 0 = incident intensity I( ) = transmitted intensity incident light reflected light scattered light transmitted light tissue 1. Introduction: Light-Matter Interactions

8. IPC Friedrich-Schiller-Universität Jena 8 water epidermis skin aorta blood melanosom 1. Introduction: Light-Matter Interactions

9. IPC Friedrich-Schiller-Universität Jena 9 1. Introduction: Light-Matter Interactions + - E + Polarisation P : Dipole moment per unit volume

10. IPC Friedrich-Schiller-Universität Jena 10 Linear Polarisation 1. Introduction: Light-Matter Interactions

11. IPC Friedrich-Schiller-Universität Jena 11 Nonlinear Polarisation 1. Introduction: Light-Matter Interactions for convergence:

12. IPC Friedrich-Schiller-Universität Jena 12 Nonlinear Polarisation 1. Introduction: Light-Matter Interactions yields:

13. IPC Friedrich-Schiller-Universität Jena 13 Example 1. Introduction: Light-Matter Interactions Terms in P: Frequency Name DC polarizability optical polarizability (refractive index) DC hyperpolarizability linear electrooptic effect (Pockels Effect) DC hyperpolarizability second harmonic generation third harmonic generation Kerr effect (n=n 0 +n 2 I)

14. IPC Friedrich-Schiller-Universität Jena 14 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  Two-photon absorption (TPA)  (3)  Third harmonic generation (THG)  CARS (Coherent Anti-Stokes- Raman-Scattering) 1. Introduction: Light-Matter Interactions

15. Light as waves: Refraction What is the reason for refraction of light? Atom in Glass Scattered Wave Direction of Light

16. Interference incoming wave scattered wave total outgoing wave

17. Interference incoming wave scattered wave total outgoing wave Phase shift of resulting wave!  Shorter wavelength in medium

18. Interference incoming wave scattered wave total outgoing wave Phase shift of resulting wave!  Shorter wavelength in medium

19. Light as waves: Refractive index n What is the reason for refraction of light? Atoms in Glass 1 2 = 1 /n 1

20. IPC Friedrich-Schiller-Universität Jena 20 AbsorptionDispersion  Bright field  Dark field  Phase contrast  Differential phase contrast 2. Contrast modes in light microscopy Amplitude difference Wavelength Phase difference Refractive indices n R : real part of refractive index n I : imaginary part of refractive index : 1D monochr. wave

21. IPC Friedrich-Schiller-Universität Jena 21 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 A 0 –A 1,2  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  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 imageBright field image of Moss reeds