Nonlinear Optics - uni-jena.de · Parametric vs non-parametric parametric process non-parametric...
Transcript of Nonlinear Optics - uni-jena.de · Parametric vs non-parametric parametric process non-parametric...
Single-Molecule Microscopy Group
Nonlinear Optics
Physical Optics
21-06-2017
Maria Dienerowitz
www.single-molecule-microscopy.uniklinikum-jena.de
Introduction
• light matter interactions
• nonlinear optical susceptibility
• phase matching
Nonlinear optics in microscopy
• parametric vs non-parametric
• 2nd harmonic generation (SHG)
• two photon excitation fluorescence (TPEF)
• osteoarthritic cartilage imaging
• 3rd harmonic generation (THG)
• cell lineage reconstruction of early zebrafish embryos
• stimulated Raman scattering (SRS)
• coherent anti-Stokes Raman scattering (CARS)
Contents
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Nonlinear Optics
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nonlinear response
linear response
Hooke’s law
cell division
Classification of optical media
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refractive index
metals n>0 dielectric
n<0 negative-index materials ?
optical frequencies
Speed of light in matter
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speed of light in matter
refractive index
vacuum speed of light
vacuum permeability
vacuum permittivity
relative permittivity relative permeability
Material parameters
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relative permittivity electric susceptibility
permittivity
Constitutive equations
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response of bound charge and current to applied field
displacement field
polarisation density
Nonlinear material response
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polarisation density
electric dipole moment
linear polarisation
2nd order nonlinear polarisation
3rd order nonlinear polarisation
Nonlinear material response
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2nd order nonlinear optical susceptibility
3rd order nonlinear optical susceptibility
atomic fields
laser light
-> 1mW – 1020W
sun light
linear optical susceptibility
Nonlinear material response
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Inversion symmetry
inversion symmetry = centrosymmetric no inversion symmetry = non-centrosymmetric
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Nonlinear material response
inversion-symmetric materials (gases, glasses, fluids, benzene (C6H6),…)
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)
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Time-varying polarisation
wave equation in nonlinear optical media
source term
example: second harmonic generation
electric field
2nd order polarisation
optical rectification
2nd harmonic frequency radiation 13
2nd order interactions
2nd order polarisation
electric field
sum frequency generation (SFG)
difference frequency generation (DFG)
optical rectification (OR)
second harmonic generation (SHG)
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3rd order interactions
3rd order polarisation
electric field
contains 22 different frequency components (and the negative of each):
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Phase matching
frequency matching condition SHG
SFG
phase matching condition SHG
SFG
temporal phase matching
spatial phase matching
destructive interference
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Phase matching
SHG
same material different frequency
dispersion
normal dispersive material birefringent uniaxial crystal
© A
drian
Pin
gston
e
wavenumber
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Birefringent phase matching
momentum mismatch
coherence length
non critical phase matching temperature matching
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Birefringent phase matching
critical phase matching angle matching
extraordinary rays: -> beam walkoff
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Summary theoretical foundation
source term
frequency matching
phase matching
birefringence
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Introduction
• light matter interactions
• nonlinear optical susceptibility
• phase matching
Nonlinear optics in microscopy
• parametric vs non-parametric
• 2nd harmonic generation (SHG)
• two photon excitation fluorescence (TPEF)
• osteoarthritic cartilage imaging
• 3rd harmonic generation (THG)
• cell lineage reconstruction of early zebrafish embryos
• stimulated Raman scattering (SRS)
• coherent anti-Stokes Raman scattering (CARS)
Contents
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Quantitative cellular imaging
- site specificity
- altering physical or
physiological properties
fluorescent staining
© Justass, Wikipedia
bovine pulmonary artery endothelial cell
green: F-actin (Alexa Fluor 488)
red: mitochondria (MitoTrackerRed)
blue: nuclei (DAPI)
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Nonlinear optics in microscopy
- noninvasive
- intrinsic and often specific chemical contrast
second harmonic generation (SHG) -> non-centrosymmetric structures
microtubule in acute brain slice of 8-month-old wild-type mouse
A Kwan et al., PNAS 105, 11370 (2008), D Debarre et al., Optics Letters 29, 2881 (2004)
third harmonic generation (THG) -> optical heterogeneities (interface)
Drosophila embryo, lipid droplets imaged by THG
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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
absorption complex index of refraction
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Parametric vs non-parametric
parametric process non-parametric process
two photon excitation fluorescence (TPEF)
second harmonic generation (SHG)
sum frequency generation (SFG)
third harmonic generation (THG)
coherent anti-Stokes Raman scattering (CARS)
stimulated Raman scattering (SRS)
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Second harmonic generation - SHG
process, non-centrosymmetric material
2nd harmonic generation
sum frequency generation - SFG
-> collagen
-> microtubules
-> muscle myosin
small excitation volume
decreased background
deeper imaging because of longer λexc
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Two photon excitation fluorescence - TPEF
arrive with 10-18s (1 attosecond) E < 2E1
less photobleaching
1 photon 2 photon
imaging depth: -> confocal laser scanning 50-80μm -> multi-photon imaging 200-1000μm
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SHG microscopy of early stage osteoarthritic cartilage
R Kumar et al., Biomedical Optics Express 6, 1895 (2015)
characterisation of extra-cellular cartilage matrix -> detection and understanding of progression of Osteoarthritis
SHG – 5μm SHG – 15μm SHG – 30μm TPEF – 30μm
TPEF – 3D SHG – 3D
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Third harmonic generation - THG
interface -> cell surface, intercellular lipid droplets
no molecular asymmetry required
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Cell Lineage Reconstruction of Early Zebrafish Embryos
N Olivier et al., Science 329, 967 (2010)
TPEF + SHG + THG
spherical sample
-> deep imaging: 400μm penetration depth
observe development in real time
-> minute-temporal resolution
high resolution microscopy
-> 1μm spatial resolution
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Structural imaging with THG
N Olivier et al., Science 329, 967 (2010) zebrafish embryo from the one-cell stage
λexc=1200nm
animal pole
vegetal pole
100μm
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Comparison structural detail imaged by THG and TPEF
THG signal TPEF – membrane staining
100μm
N Olivier et al., Science 329, 967 (2010) 32
Nuclear envelope fragmentation revealed by THG
N Olivier et al., Science 329, 967 (2010)
50μm
nuclear envelope dynamics during mitosis
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Mitotic spindle imaging with SHG
N Olivier et al., Science 329, 967 (2010)
SHG mitotic spindle -> microtubules present during mitosis
TPEF H2B/mCherry fusion protein -> chromatin condensation, chromosome formation and displacements
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Mitosis steps revealed by TPEF THG SHG microscopy
N Olivier et al., Science 329, 967 (2010)
SHG mitotic spindle
THG cell contour
TPEF β-actin
overlay
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Raman Scattering – linear optics
non-parameteric process parameteric process
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Raman Scattering – nonlinear optics
non-parameteric process
Coherent Anti-Stokes Raman scattering (CARS)
Stimulated Raman scattering (SRS)
parameteric process
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