ASPMPS 2 j Biophotonics Prof Dr Rainer Heintzmann

ASP_MP_S 2 j Biophotonics Prof. Dr. Rainer Heintzmann http: //www. nanoimaging. de/Lectures/Biophotonics 2010/ http: //www. nanoimaging. de/Lectures/Biophotonics 2011/ Institut für Physikalische Chemie Friedrich-Schiller-Universität Jena Lecture 1 1 IPC Friedrich-Schiller-Universität Jena

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 2 IPC Friedrich-Schiller-Universität Jena

Content 7. Fluorescence microscopy 7. 1 Fluorochromes 7. 2 Confocal fluorescence microscopy 7. 3 FRET 7. 4 FRAP, i. FRAP, FLIP 7. 5 Ultramicroscopy / SPIM / HILO 7. 6 Multi-photon microscopy 7. 7 4 Pi 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 3 IPC Friedrich-Schiller-Universität Jena

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 4 IPC Friedrich-Schiller-Universität Jena

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

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

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

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

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

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

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

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

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=n 0+n 2 I) 13 IPC Friedrich-Schiller-Universität Jena

1. Introduction: Light-Matter Interactions Process c(1) § Linear absorption § Spontaneous emission (Fluorescence) § Reflection § Elastic scattering § Inelastic scattering: Ramanscattering § Diffraction 14 c(2) c(3) § 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) IPC Friedrich-Schiller-Universität Jena

$Light as waves: Refraction What is the reason for refraction of light? Direction of$

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

Interference incoming wave scattered wave total outgoing wave

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

$Light as waves: Refractive index n What is the reason for refraction of light?$

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

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

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 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 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 21 Bright field image of Moss reeds IPC Friedrich-Schiller-Universität Jena