Nonlinear Optics. Single-Molecule Microscopy Group. Physical Optics Maria Dienerowitz.

Transcription

1 Single-Molecule Microscopy Group Nonlinear Optics Physical Optics Maria Dienerowitz

2 Contents Introduction light matter interactions nonlinear optical susceptibility phase matching Nonlinear optics in microscopy parametric vs non-parametric 2 nd harmonic generation (SHG) two photon excitation fluorescence (TPEF) osteoarthritic cartilage imaging 3 rd harmonic generation (THG) cell lineage reconstruction of early zebrafish embryos stimulated Raman scattering (SRS) coherent anti-stokes Raman scattering (CARS) 2

3 Nonlinear Optics nonlinear response cell division linear response Hooke s law 3

4 Classification of optical media metals n>0 dielectric optical frequencies n<0 negative-index materials? refractive index 4

5 Speed of light in matter vacuum speed of light vacuum permittivity vacuum permeability speed of light in matter refractive index relative permittivity relative permeability 5

6 Material parameters permittivity relative permittivity electric susceptibility 6

7 Constitutive equations displacement field polarisation density response of bound charge and current to applied field 7

8 Nonlinear material response electric dipole moment polarisation density linear polarisation 2 nd order nonlinear polarisation 3 rd order nonlinear polarisation 8

9 Nonlinear material response linear optical susceptibility 2 nd order nonlinear optical susceptibility 3 rd order nonlinear optical susceptibility atomic fields laser light -> 1mW W sun light 9

10 Nonlinear material response 10

11 Nonlinear material response inversion-symmetric materials (gases, glasses, fluids, benzene (C 6 H 6 ), ) materials with and without inversion symmetry second-harmonic generation sum-frequency generation difference-frequency generation optical parametric oscillation third-harmonic generation self-focussing saturable absorption two-photon absorption stimulated Raman scattering 4-wave mixing (CARS) 12

12 Inversion symmetry inversion symmetry = centrosymmetric no inversion symmetry = non-centrosymmetric 11

13 Time-varying polarisation source term wave equation in nonlinear optical media example: second harmonic generation electric field 2 nd order polarisation optical rectification 2 nd harmonic frequency radiation 13

14 2 nd order interactions electric field 2 nd order polarisation second harmonic generation (SHG) sum frequency generation (SFG) difference frequency generation (DFG) optical rectification (OR) 14

15 3 rd order interactions electric field 3 rd order polarisation contains 22 different frequency components (and the negative of each): 15

16 Phase matching frequency matching condition phase matching condition SHG SFG SHG SFG spatial phase matching temporal phase matching destructive interference 16

17 Phase matching SHG wavenumber dispersion same material different frequency normal dispersive material birefringent uniaxial crystal Adrian Pingstone 17

18 Birefringent phase matching non critical phase matching temperature matching coherence length momentum mismatch 18

19 Birefringent phase matching critical phase matching angle matching Adrian Pingstone extraordinary rays: -> beam walkoff 19

20 Summary theoretical foundation source term frequency matching phase matching birefringence 20

21 Contents Introduction light matter interactions nonlinear optical susceptibility phase matching Nonlinear optics in microscopy parametric vs non-parametric 2 nd harmonic generation (SHG) two photon excitation fluorescence (TPEF) osteoarthritic cartilage imaging 3 rd harmonic generation (THG) cell lineage reconstruction of early zebrafish embryos stimulated Raman scattering (SRS) coherent anti-stokes Raman scattering (CARS) 21

22 Quantitative cellular imaging fluorescent staining - site specificity - altering physical or physiological properties bovine pulmonary artery endothelial cell green: F-actin (Alexa Fluor 488) red: blue: mitochondria (MitoTrackerRed) nuclei (DAPI) Justass, Wikipedia 22

23 Nonlinear optics in microscopy - noninvasive - intrinsic and often specific chemical contrast second harmonic generation (SHG) -> non-centrosymmetric structures third harmonic generation (THG) -> optical heterogeneities (interface) 15μm microtubule in acute brain slice of 8-month-old wild-type mouse Drosophila embryo, lipid droplets imaged by THG A Kwan et al., PNAS 105, (2008), D Debarre et al., Optics Letters 29, 2881 (2004) 23

24 Parametric vs non-parametric parametric process non-parametric process -> no net energy transfer -> involves energy loss (or gain) duration of virtual state real susceptibility complex susceptibility complex index of refraction absorption 24

25 Parametric vs non-parametric parametric process non-parametric process second harmonic generation (SHG) two photon excitation fluorescence (TPEF) sum frequency generation (SFG) third harmonic generation (THG) stimulated Raman scattering (SRS) coherent anti-stokes Raman scattering (CARS) 25

26 Second harmonic generation - SHG process, non-centrosymmetric material -> collagen -> microtubules -> muscle myosin 2 nd harmonic generation sum frequency generation - SFG small excitation volume decreased background deeper imaging because of longer λ exc 26

27 Two photon excitation fluorescence - TPEF 1 photon 2 photon less photobleaching arrive with s (1 attosecond) E < 2E 1 imaging depth: -> confocal laser scanning 50-80μm -> multi-photon imaging μm 27

28 SHG microscopy of early stage osteoarthritic cartilage characterisation of extra-cellular cartilage matrix -> detection and understanding of progression of Osteoarthritis SHG 5μm SHG 15μm SHG 30μm TPEF 30μm SHG 3D TPEF 3D R Kumar et al., Biomedical Optics Express 6, 1895 (2015) 28

29 Third harmonic generation - THG interface -> cell surface, intercellular lipid droplets no molecular asymmetry required 29

30 Cell Lineage Reconstruction of Early Zebrafish Embryos spherical sample -> deep imaging: 400μm penetration depth observe development in real time -> minute-temporal resolution high resolution microscopy -> 1μm spatial resolution TPEF + SHG + THG N Olivier et al., Science 329, 967 (2010) 30

31 Structural imaging with THG animal pole λ exc =1200nm vegetal pole 100μm N Olivier et al., Science 329, 967 (2010) zebrafish embryo from the one-cell stage 31

32 Comparison structural detail imaged by THG and TPEF 100μm THG signal TPEF membrane staining N Olivier et al., Science 329, 967 (2010) 32

33 Nuclear envelope fragmentation revealed by THG 50μm nuclear envelope dynamics during mitosis N Olivier et al., Science 329, 967 (2010) 33

34 Mitotic spindle imaging with SHG SHG mitotic spindle -> microtubules present during mitosis TPEF H2B/mCherry fusion protein -> chromatin condensation, chromosome formation and displacements N Olivier et al., Science 329, 967 (2010) 34

35 Mitosis steps revealed by TPEF THG SHG microscopy TPEF β-actin THG cell contour SHG mitotic spindle overlay N Olivier et al., Science 329, 967 (2010) 35

36 Raman Scattering linear optics non-parametric process parametric process 36

37 Raman Scattering nonlinear optics non-parametric process parametric process Stimulated Raman scattering (SRS) Coherent Anti-Stokes Raman scattering (CARS) 37

38 Live-cell stimulated Raman imaging (SRS) alkynes metabolically incorporated into replicating DNA two-colour SRS imaging HeLa cells alkyne tagged nucleic acid intrinsic lipid signal overlay S Hong et al., Angew. Chem., Int. Ed. 53, 5827 (2014)

39 Imaging myelin sheath with CARS healthy spinal cords injured spinal cords treated spinal cords intraaxonal free Ca 2+ myelin sheath: CARS (coherent anti-stokes Raman scattering) calcium indicator Oregon Green 488: TPEF (two photon excited fluorescence) Y Shi et al., Nat. Nanotechnol. 5, 80 (2010)

40 Porcine tissue imaging with CARS and SHG lipocytes: CARS (coherent anti-stokes Raman scattering) muscle fibre and collagen: SHG (second harmonic generation) Biomedizinische Optik, Laser Zentrum Hannover e.v.