Nonlinear Optical Methods for Biophotonics Applications

Valdas Pasiskevicius vp@laserphysics.kth.se

Applied Physics

School of Engineering Sciences Royal Institute of Technology (KTH)

Stockholm, Sweden

ADOPT Winter school 2016 1

Outline

Introduction Nonlinear material response Applications: SFG, SHG, MPEF SRS, CARS, SERS Tissue ablation

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Outline

Introduction: the classics Nonlinear material response Applications: SFG, SHG, MPEF SRS, CARS, SERS Tissue ablation

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Applications: Clinical diagnostics, Cell functional imaging

Picture by Dr. Sonja Pyott University of North Carolina, Wilmington Wilmington, NC, USA

Fluorescent dye Stained biopsy

“Gold standard” for diagnostics

Imaging Spectroscopy

Cochlea and Hair Cells

• Depth resolution • Functional imaging

Exogenous Fluoresece Imaging - Confocal scanning laser microscopy (CSLM)

• Tissue specific • ”Molecular” resolution

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CSLM principle

Lichtman, J. W. and J.-A. Conchello (2005). "Fluorescence microscopy." 2(12): 910-919 5

CSLM resolution: point spread function (PSF)

T. Wilson, Journal of Microscopy, Vol. 244, 2011, pp. 113–121

PSFConfocal ~(PSFwide field)2

𝐿𝐹𝑊𝐻𝑀𝑐𝑜𝑛𝑓 = 0.37𝜆/𝑁𝐴

𝐿𝐹𝑊𝐻𝑀𝑤𝑖𝑑𝑒 = 0.51𝜆/𝑁𝐴

𝐴𝐹𝑊𝐻𝑀𝑐𝑜𝑛𝑓 = 0.64𝜆

𝑛 − 𝑛2 − 𝑁𝐴2

𝐴𝐹𝑊𝐻𝑀𝑤𝑖𝑑𝑒 = 0.89𝜆

𝑛 − 𝑛2 − 𝑁𝐴2

Lateral resolution Axial resolution

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Field shaping with opposing lenses and Superresolution

S. W. Hell, R. Schmidt, A. Egner, Nat. Phot. , 3, 381 (2009)

STED is also a nonlinear optical method regardless of claims to opposite!

STED – simulated emission depletion PALM – photo-activation localization iPALM – interferometric PALM STORM – stochastic optical reconstruction

sm IInz

/

2ln,

STED:

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Sensitivity to surroundings Bleaching (excitation triplet states)

Some aspects to take into account in fluorescence methods

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Excitation Fluorescence

Endogenous fluorophores in tissue

A. Wagnieres, et al, Photochem. Photobiol. 68, 603 (1998)

Some aspects to take into account in fluorescence methods

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Outline

Introduction Nonlinear material response Applications: SFG, SHG, MPEF SRS, CARS, SERS Tissue ablation

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Material response to EMG fields

• Strongest response form bond-forming (valence) electrons • Delocalised

Electron density: peptide bond in anifreeze protein

Generated by MoPro package

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Some definitions

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• Constitutive relations:

;

;0

t

DEJ

MHB

;0 PED

Usual simplifications: nonconductive (=0), nonmagnetic (M =0), no space charge (=0).

EP :0 EPolarization vector:

• Polarization P and magnetization vectors fully describe material response to external

electromagnetic wave.

Magnetization vector: HM :0 M

• Material parameters: dielectric susceptibility E and magnetic susceptibility M

;D

Polarization = dipole moment per unit volume

)1(

0

)1(

00 PPEPP

Linear response

Harmonic oscillator model:

22222

22

0

2)1(

4)()Re(

m

Ne22222

0

2)1(

4)(

2)Im(

m

Ne

- damping of material resonance at .

n

w1 w2 w3

RF-FIR Visible UV

w Acoustic )1Re(,1 )1()1( n

Dielectric tensor, refractive index:

)1Im(2 )1(

c

Power absorption coefficient:

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Electronic Vibrational Rotational

Nonlinear response to EMG fields

P

E

Polarization due to anharmonic oscillator driven by external field E:

...... )3()2()1(

0

)3()2()1(

00 PPPPEEEEEEPP

inter-atomic electric field ~ ]/[1010 1110

2

0

mVr

e

n

nnnEEχP n

nn

...

1

)(

...0

)(

1

111)())(,...,;()(

Anharmonicity results in nonlinear polarisation at combination frequencies

Summations: n ...1

over Cartesian coordinates (x,y,z)

over different sets of frequencies

n

n

...

,...,,

21

21

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Microscopic viewpoint Alternative description, nonlinear response of a single dipole (molecular bond):

)()(:),...,;( 11

)()(

, nmmn

nn

m EEe γr

),,;(),,;(

),;(),;(

);();(

321

)3(

321

21

)2(

21

1

)1(

1

γγ

γ

γ

Common notation in literature:

polarizability

hyperpolarizability

)()1()0( ... n

mmmm eeee rrrr

Nonlinear response of a dipole characterized by polarizability tensor:

Local fields

;3/))(2()()( jExternaljLocaljm EE

For isotropic spherical molecules:

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2. Resonant

1. Perturbative

...... )3()2()1(

0

)3()2()1(

00 PPPPEEEEEEPP

• Photon far from electron transition resonances • Optical electric field complies :

km

mkaE

)(

km - dipole moment for k <->m

b

aea

E 0

No bound-bound multiphoton transitions

No bound-free multiphoton transitions

ba - Bohr radius, e – electron charge

Typically intensity < 1013 W/cm2

• P Series diverges if

• Nonlinearities for specific processes can be resonantly enhanced

0)( nmk

Regimes of nonlinear optics

Bound-bound multiphoton transitions

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3. Strong-field

4. Relativistic

b

aea

E 0

• Bound-continuum transitions: atom and molecule ionization with optical field

• High-harmonic generation in plasma

• as-pulse generation and detection

• Electron acceleration in optical field close to

• High-coherence -ray generation by laser Compton scattering

• Electron-positron pair generation

• Laser proton sources

• Thermonuclear fusion

2mc

1.03pm2MeV2.1 20 cme

218W/cm10I

Regimes of nonlinear optics

Bound-free multiphoton transitions

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Outline

Introduction Nonlinear material response Applications: SFG, SHG, MPEF: imaging SRS, CARS, SERS Tissue ablation

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)()(:),;()( 32321)2(

1 EEχP NL

Sum-frequency mixing (SFM) Second harmonic generation (SHG)

32321 ,

32321 ,

Second-order nonlinearity

Processes useful for imaging:

32 1

Forward and backward processes

LkkkkL )( 321Momentum conservation requires:

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Second-order nonlinearity

In materials with inversion symmetry 2nd order nonlinear processes are

absent: 0),;( 321

)2( ijkχ

Inversion symmetry exists in:

• Crystal symmetry classes (432),(622) ,(422).

• Isotropic solids.

• Atomic gasses.

• Molecular gasses.

• Liquids.

Inversion symmetry absent in:

• Remaining 29 symmetry classes.

• Surfaces of isotropic media.

• Chiral media

• Aligned polymers

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SHG imaging microscopy (SHIM), beginnings: I. M. Freund, et al, ”Connective tissue polarity,” Biophys. J. 50, 693 (1986)

Second Harmonic generation imaging

SHIM Features: • No fluorescent labels • No toxicity • No photobleaching • High contrast 3D imaging • Spatial resolution • Polarized SH signals: information about structural anisotropy

Sources of SHG in tissue: • Membranes • Colagen (connective tissue fibrils) • Chiral protein structures (e.g. Tubulin structures , myelin sheaths)

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SHG signal (for interaction length < Lc): • Inversely propotional to beam area and pulse length • Depends quadraticaly on molecule concentration

Second Harmonic generation imaging

Beam area

power Pulse length

SHG microscopy image 250 µm deep in muscle tissue. Scale bar – 50 µm

P. J. Campagnola, et al, Biophys. J., 81, 493 (2002) 22

Sum-frequency generation imaging

• Conceptually similar to SHG • Except for the cases where one wave is on

molecular resonance • Typically NIR (~1 µm) and resonant MIR (~3

µm) • Linear SFM power dependence on the

powers of exciting waves

32321 ,

Picture source: V. Raghunathan et al Optics Lett. , 36, 3891 (2011)

Picosecond OPO

Resonant Off-Resonant

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Nonresonant SFM can make interpretation complicated

Sum-frequency generation imaging

V. Raghunathan et al Optics Lett. , 36, 3891 (2011)

Colagen SFM imaging on methylene mode 2959 cm-1

MIR wave off resonance MIR wave on resonance

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Two-photon excitation of fluorescence TPEF relies on two-photon absorption (TPA) process

• Third-order resonant nonlinearity • Absorption coefficient depends on intensity

R. W. Boyd, Nonlinear optics, 3rd ed. 2007

In dielectrics

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Predicted by Maria Goeppert-Mayer in 1931 in her doctoral dissertation (Göttingen)

Picture source: UCF, College of Sciences

Two-photon excitation of fluorescence W. Denk, J. H. Strickler, W. W. Webb, Science, 248, 73 (1990)

M. Drobyzhev, et al, Nature Methods, 8, 393 (2011)

...32 IIInPA

Two-photon excitation of fluorescence

TPA cross-section

M. Drobyzhev, et al, Nature Methods, 8, 393 (2011)

Goeppert-Mayer units, 1 GM = 10−50 cm4 s photon-1

Sensitivity to local electric field )()( 1

2

10 gA

)()( 2

2

10

2

102 gB E)(5.0 21

0

1010

Linear extinction

• Spatial resolution

• Sensitivity to surounding fields

• NIR excitation: less scattering

• Lower infuence of auofluorescence

Two-photon excitation of fluorescence

V. E. Centonze et al, Biophys. J. 75, 2015 (1998)

Confocal vs TPEF

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Multiphoton excitation ”guide star”

R. Aviles-Espinosa et al, Biomed.Opt.Exp. 2, 3125 (2011) X. Tao, et al ,SPIE Proc. 8978, 89780D (2014)

TPFE imaging with NADH autofluorescence guide star: • In vivo • 30 ms/WF measurement • Depth 51 µm

C Elegans in vivo

Combined SHG, TPEF (autofluorescence)

Zipfel W R et al. PNAS 2003;100:7075-7080

©2003 by National Academy of Sciences 30

Upconversion fluorescence in nanoparticles

H. Liu, C. T. Xu, S. Andersson-Engels, Opt. Express, 22, 17782 (2014)

2PE

3PE

Core-shell NaYF4 : Yb3+, Tm3+ @NaYF4

Increasing imaging resolution through scattering medium 31

Outline

Introduction Nonlinear material response Applications: SFG, SHG, MPEF: imaging SRS, CARS, SERS: imaging, sensing Tissue ablation

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.:))((

:))(( )0(

)0(EE

αEα

rr

X

XX

X

XeXeH m

mm

mI

Orientation energy Rayleigh scattering Raman scattering IR absorption

Electric dipole energy:

0))(( )0(

X

Xe mr

0))((

X

Xmα

If molecular vibration mode is called IR-active.

If differential polarizability tensor molecular vibration is Raman-active.

Symmetric stretching mode Asymmetric stretching mode Bending mode

)(Xmα X X X

X )()0( Xe mr X X

Raman activity

IR activity

yes

yes yes no

no no

Raman Scattering, basics

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Vibrational RS

E

X

Stokes

Anti-Stokes

|n2m2l2>

|n1m1l1>

E

X

|n1m1l1>

<n2m2l2|

Electronic resonant RS

,...2,1,

,

mm

m

pa

ps

Raman Scattering, basics

Stokes

Antistokes

• Spontaneous Stokes efficiency is higher • Ratio is temperature dependent

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spsppsRs EEP2

)3(

0 ),,;(

SRS: Stimulated Raman Scattering

Stimulated Raman Scattering

Combined EDFA+Raman Amplifier

),,;()3(

sppsR

affects amplitude (gain) and phase (refractive index) at

• Vibrational and rotational (molecular gasses) Raman scatering • SRS automatically phase-matched • Efficiency (contrast in microscopy) depends on intensity of the pump

s

Raman susceptibility: a complex quantity

))0(exp()0()( LIgILI pRss

Nnnc

TNg

sp

RsR

220

2

2

4

Stimulated Raman gain:

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sprpasprpsprpaRa EEEP ;),,;( *)3(0

CARS Coherent Antistokes Raman Scattering:

• Four wave mixing process: phase sensitive • Epr should be applied before vibrational (rotational) coherence relaxation • Phase-matching is required

kp

ks

ka

kpr ks

kpr

ka

kp

0 asprp kkkkk

Two-beam scheme BOXCARS scheme

• In CARS nonresonant background from FWM process: • Bacground suppression: polarization and time gating, heterdyne CARS

),,;()3(

sprpa

p s pr a

v=0 v=1

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CARS imaging

Antistokes scattering reported: P. D. Maker and R. W. Terhune, Phys. Rev. 137, A801(1965). CARS name invented: R. F. Begley,et al, Appl. Phys. Lett. 25, 387 (1974). First CARS microscope: M. Duncan, et al, Opt. Lett. 7, 350 (1982).

• Stimulated Raman scattering is automatically phase matched

• CARS is not • In CARS microscopy: large NA

collinear excitation works fine

NR )3(

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CARS imaging

Typical numbers: • Picosecond pulses t>1/Raman spectral width • Pulse energy <1 nJ: avoid damage • Average powers ~10 mW: avoid heating • Easy region 2500-3500 cm-1, • More difficult but more important 800-1800 cm-1

Example: 0.1 nJ, 5ps, 1.2NA, gives 0.2 TW/cm2 in focus, and 500 antistokes photons per pulse at OH 3300cm-1 mode

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CARS imaging with chirped pulses

Th. Hellerer, et al, Appl. Phys. Lett. 85, 25 (2004)

C-N

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Vibrational spectra in tissue (H-C-H, O-H stretching)

Cholesterol Lipid tristearin

HOH

• Multiple overlapping vibrational bands • Work in sweet-spot or ”quiet region” in vibrational spectrum (good for some molecules) • In CARS – phase sensitivity adds more complexity • Advanced algorithms required for extraction of vibrational spectrum

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Heterodyne CARS

E. Potma, et al, Opt. Lett. 31, 241 (2006)

sinImcosRe2 )3()3()3(22

RRNRpLOaLO EEEES

1. Add ELO field at a and use interferometer to control phases 2. Modulate phase of ELO and use heterdyne detection

d-DMSO

• =90 gives background-free Raman response • Amplification by increasing and not pump ELO

• Imaging 103x faster than with spontaneous Raman

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Th. Hellerer et al PNAS, 104, 14658 (2007)

CARS @2845 cm-1 (CH2) TPEF: Nile red

CARS imaging: comparison with TPEF

• Sensitivity ~105 lipid molecules in focus volume (sub mM concentrations) • Somewhat better with heterodyne CARS

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M. Lee, et al, IntraVital, 4, e1055430 (2015)

CARS multispecies imaging

CH2 2845 cm-1 CH3 2930 cm-1 OH 3030 cm-1

Lipid (b)-(f) Amino (d)-(b)-(f) HOH (f)-(d)

Overlay

Chemically selective imaging of tumor histopathology in MMTV-PyMT mammary tumors

• Stokes: 7 ps, 1064 nm, 80 MHz. Pump(probe): 5ps, 750-920 nm SPOPO. • Combined power on sample: 30-60mW • Dwell time: 10 µs • Acquisition: 2.6 s/frame

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M. Lee, et al, IntraVital, 4, e1055430 (2015)

CARS combined multimodal imaging Red: SHG (colagen) Green: TPFE GFP expressing cancer cells Cyan: CARS CH3 2930 cm-1 host cells

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CARS (red)+SHG (green)

Hyperspectral CARS multimodal imaging

Lipids in mamary tumor tissue

CARS

Source of picture: Eric Potma, UC Irvine 45

SRS microscopy

• No nonresonant background • No labels • Linear dependence on concentration • Sensitivity similar and better than CARS (50 µM retinol, 5 mM methanol) • Spatial resolution similar to multiphoton imaging

Ch. W. Freudiger, et al, Science, 322, 1857 (2008)

7 ps Nd:YVO4 + OPO. <40 mW, 30 MWcm-2

Efficiency ~ 4 , but avoid short due to autofluorescence background

• Stimulated Raman Gain (SRG) • Raman-stimulated pump loss (SRL)

46

SRS imaging Comparison with CARS

Ch. W. Freudiger, et al, Science, 322, 1857 (2008)

SRS Brain

SRS Skin

Through 1 mm

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Stimulated Raman spectroscopy

RIKE – Raman-induced Kerr effect dual comb spectroscopy

T. Ideguchi, et al, Optics Lett., 37, 4498 (2012)

• Measures amplitude and phase of vibrational modes • Single spectral measurement in ~3 µs

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Resonantly enhanced near-field

Surface enhanced Raman scattering (SERS) Albrecht MG, Creighton JA. 1977..J. Am. Chem. Soc. 99:5215–17

Nie S, Emory SR. 1997. Science 275:1102–6

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SRS and CARS: Relative (dis)advantages CARS microscopy • No labels required • Bond-specific imaging • Potentially background-free • Relaxed requirements for laser stability • Quadratic dependence on concentration • Phase matching required (propagation direction-sensitive) • Parasitic nonresonant FWM • CARS spectra need interpretation SRS microscopy • No labels required • Bond-specific imaging • Linear dependence on concentration • Free from parasitic FWM • Phase matching not required (insensitive to propagation direction) • Straighforward spectral information • Detection of small modulation on top of large signal • High demenads on laser stability

Outline

Introduction Nonlinear material response Applications: SFG, SHG, MPEF: imaging SRS, CARS, SERS: imaging, sensing Tissue ablation: minimally invasive surgery, nanosurgery

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Cell surgery

• TW/cm2 intensties: fs pulses • MPA: subdiffraction resulution • Independent on linear absorption properties of biological object • Low-energy breakdown threshold • Low heat deposition in surrounding tissue

Processes: • Optical breakdown : ionization, plasma formation, chemical dissociation • Material removal: pressure wave, cavitation

• Avoid nonlinear propagation, self focussing • Large NA optics (NA>0.9)

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Optical breakdown processes

• Multiphoton ionization, tunelling tunelling: ”instantaneus” • Impact ionization: requires scattering, ”slow”

A. Vogel, et al, Appl. Phys. B, 81, 1015 (2005) 53

Keldysh parameter: γ<1 tunelling (short pulses, low ω) γ>1 multiphoton ionization

Optical breakdown of water

Electron density dynamics

Electron density vs intensity

A. Vogel, et al, Appl. Phys. B, 81, 1015 (2005) Multiphoton ionization part 54

Optical breakdown thresholds

For fs pulses: • Higher intensity threshold • Lower energy fluence • Less colateral damage

Breakdown in water

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The breakdown process

Chemical changes: • Reactive oxygen species (ROS) • Dissociation by electron capture

Thermoelastic stress: • Pulse shorter than thermalisation time • Stress confinement • Shock wave formation • Nucleation of bubble in the focal region

Typical values for cavitation • λ = 800 nm, NA = 1.3, T=20 C, 100 fs • Required ΔT = 131.5 C for critical tensile stress p=−71.5MPa • Corresponding Energy density in the focus 551 J cm-3

• Electron density 0.23x1021 cm-3

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Time-dependence of the bubble radius

57

Cavitation @ tensile stress ~-70 MPa

Example for HOH breadown threshold: 800 nm, 100 fs, NA 0.65 0.1 µJ, 5.64 J/cm2, 56.4 TW/cm2

Cell surgery: intensity regimes

A. Vogel, et al, Appl. Phys. B, 81, 1015 (2005) 58

Cell optical trapping and surgery

Z. L. Shi, et al, Current Microscopy Contributions to Advances in Science and Technology (2012) 59

K. König, et al Optics Lett. 26, 819 (2001)

DNA ablation and drilling

• ~0.5 nJ, 80 MHz, 800 nm, 170 fs • 1.3 NA , 320 nm spot • Dwell time 3.2 ms (1.6 µJ cummulative) • 200 nm cuts

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NLO methods: some advantages and disadvantages

Optimist’s view: • Spatial resolution not limited by PSF • Staining is not required in most of the methods • Specificity to molecular bonds • Sensitivity to interfaces and types of tissue • Sensitivity to local electric fields (SFIM)

Pessimist’s view: • High intensities required • Might be difficult to apply in light-scattering surroundings • No clear standards yet (except for surgery) • Perceived as technically complicated

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