ASP_MP_S2j Biophotonics Prof. Dr. Rainer Heintzmann

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

2. Content • Introduction • 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 • Optical coherent tomography • Molecular many electron systems:electronic and nuclear movement • UV-Vis absorption • 5.1 Franck-Condon principle • 5.2 Electronic chromophores • 5.3 Polarimetry & circular dichroism • 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. Content • 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 • 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. Content • Non-linear Raman microspectroscopy • 9.1 Hyper Raman • 9.2 Coherent anti-Stokes Raman scattering (CARS) • 9.3 Stimulated Raman microscopy • Future trends in non-linear microscopy

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

6. 1. Introduction Light-Matter Interactions as the basis for Biophotonics

7. Absorption Scattering Reflection Refraction 1. Introduction: Light-Matter Interactions Light-Matter Interactions incident light reflected light tissue transmitted light scattered light a(n) =absorption cross-section aS = scattering cross-section I(z) = intensity in depth z I0 = incident intensity I(n) = transmitted intensity

8. 1. Introduction: Light-Matter Interactions blood melanosom aorta water skin epidermis

9. - + E 1. Introduction: Light-Matter Interactions + Polarisation P : Dipole moment per unit volume

10. 1. Introduction: Light-Matter Interactions Linear Polarisation

11. 1. Introduction: Light-Matter Interactions Nonlinear Polarisation for convergence:

12. 1. Introduction: Light-Matter Interactions Nonlinear Polarisation yields:

13. 1. Introduction: Light-Matter Interactions Example 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=n0+n2I)

14. c(1) c(2) c(3) • Linear absorption • Spontaneous emission (Fluorescence) • Reflection • Elastic scattering • Inelastic scattering: Raman-scattering • Diffraction • Second harmonic generation (SHG) • Sum-frequency generation (SFG) • Difference-frequency generation (DFG) • Optical parametric amplification • Two-photon absorption (TPA) • Third harmonic generation (THG) • CARS (Coherent Anti-Stokes-Raman-Scattering) 1. Introduction: Light-Matter Interactions Process

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

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 l1 l2= l1/n l1

20. 2. Contrast modes in light microscopy : 1D monochr. wave Absorption Dispersion nR : real part of refractive index nI : imaginary part of refractive index Phase difference Amplitude difference Refractive indices Wavelength l • Dark field • Phase contrast • Differential phase contrast • Bright field

21. 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 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 Amplitude difference Wavelength l Uniform bright field image Bright field image of Moss reeds