CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

9th French-Israeli Symposium on Non-linear & Quantum Optics

Coherent anti-Stokes Raman scattering (CARS) microscopy near boundaries David Gachet, Franck Billard, Nicolas Sandeau & Hervé Rigneault Mosaic group, Institut Fresnel – Marseille, France

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

Coherent anti-Stokes Raman scattering basics (1)

νs νp

νp

νs

νas

V=1

ΩR

V=0 Coherent anti-Stokes Raman scattering

CARS important features: 1. Resonant process (fluorescence) 2. Coherent process (SHG, THG)

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

Coherent anti-Stokes Raman scattering basics (2) Two contributions to CARS generation

ωp

ωs

ωas ωp ωs

ωp

ωas

ωp V=1 V=0

ΩR

V=1 V=0

ΩR

Resonant contribution (χR(3))

Nonresonant contribution (χNR(3))

(vibrational origin)

(electronic response of the medium)

Presence of molecules with oscillating vibrational mode ΩR Î Enhancement of the signal at angular frequency ωas

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

CARS microscopy: a stain free microscopy Forward detected signal (F-CARS) ωas z

35 30 25 20

y (µm)

y

Sample

10 5 0

Microscope objective

0

5

3

30 28 26 24

x10

x

15

10

15

20

25

30

35

x (µm)

Advantages: 1. Fluorescent staining useless. ωp

ωs ωas

Backward detected signal (E-CARS)

2. Chemical selectivity of the contrast. 3. Intrinsic 3D imaging.

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

CARS imaging of interfaces: motivations (1) From the literature… Polystyrene beads in agarose

Cheng et al., J. Opt. Soc. Am. B, 19, 1363 (2002)

Melanin beads in water Cheng et al., Biophys. J. 83, 502 (2002) Kano and Hamaguchi, Opt. Expr. 14, 2798 (2006)

Yeast cells in water

Interference or refractive effects ???

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

CARS imaging of interfaces: motivations (2) CARS as a third-order nonlinear process:

(

)

( ) ( ) ( )

Spring dephasing

P(3) r, −ωas = χ (3) (ωas ) : Ep r, ωp : Ep r, ωp : Es* r, ωs

Excitation volume

π/2

Excitation volume

π/4

Object 0 -4

-2

0

2

4

Normalized resonance detuning

Surrounding

Scanning direction

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

Spectral behavior of CARS signal (1) ( 3) χ (3) decomposit ion into two parts : χ ( 3) = χ R( 3) + χ NR

Oscillator

For an isolated Raman line :

χ R( 3)

a strength = (ω p − ω s − Ω R ) + iΓ

Electronic response spectrally independen t :

χ

I CARSα χ

( 3) R

I CARS α χ

( 3) 2 R

(3) NR

Raman line half-width

is real & constant

+χ

( 3) 2 NR

+χ

( 3) 2 NR

Homodyne terms

(

Vibrational frequency

+ 2 Re χ

( 3) R

Heterodyne term

CARS resonance lineshape Potma et al., J. Raman Spectrosc. 34, 642 (2003)

⋅χ

( 3) ∗ NR

)

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

Spectral behavior of CARS signal (2) Χ(3) polar form:

χ ( 3) (ζ ,η ) = ρ (ζ ,η ) ⋅ exp[iφ (ζ ,η )] 1/η − ζ ⎤ ⎡ ρ (ζ ,η ) = χ ⋅ ⎢1 + 4 2 η ( ζ + 1) ⎥⎦ ⎣ 2 tan[φ (ζ ,η )] = η (ζ 2 + 1) − 2ζ

1/ 2

( 3) NR

with

δω = ω p − ω s where

ζ = (δω − Ω R ) / Γ ( 3) η = −2Γ ⋅ χ NR /a

Laser detuning Normalized Raman resonance detuning Nonresonant part relative strength

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

Spectral behavior of CARS signal (3) 105 120

150

165

90

4

OR1 Bef. Res. P CARS Res. RP Raman Res. PM Phase max. D Spect. dip OR2 Aft. Res.

3

Nonresonant part relative strength ( 3) η = −2Γ ⋅ χ NR /a

P

45

RP

30

η=1 η=2

1

D

180

60

η=0.5

2

PM

75

5

135

χ(3) phase (°)

15

OR 0

1

2

3

4

5

χ(3)/χNR modulus

Representation of the resonance in the complex plane

0

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

CARS imaging of interfaces: 1D model (1) Excitation field

I CARS ( x) =

Object

∗

Surrounding

( ρ O , φO )

x

( ρ S ,0)

0

λ

1 0

λ⎞ ⎛ if x < ⎜ ⎟ 2⎠ ⎝

I CARS ( x) = ρ O2

[

]

2

⎛x⎞ I CARS ( x) = ρ O2 + ρ S2 − 2 ρ O ρ S ⋅ cos(φO ) ⋅ ⎜ ⎟ ⎝λ⎠ x 1 λ⎞ ⎛ + ρ O2 − ρ S2 ⋅ + ρ O2 + ρ S2 + 2 ρ O ρ S ⋅ cos(φO ) ⎜ if x ≤ ⎟ λ 4 2⎠ ⎝ λ⎞ ⎛ I CARS ( x) = ρ S2 ⎜ if x > ⎟ 2⎠ ⎝

(

)

[

]

ICARS admits a minimum on the [-λ/2; λ/2] range only if cos(ΦO) < min(ρO/ρS; ρS/ρO)

2

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

CARS imaging of interfaces: 1D model (2) Sample

I CARS ( x) =

Object

( ρ O , φO )

CARS intensity intensity (AU) (AU) CARS

1.0 6

∗

Surrounding 0

(ρ S ,0)

x

1

Excitation field λ

2

0

Interface Interface

0.95

Obj. Obj. Peak Dip Phase max. Off resonance

4 0.83 2 0.7 1

Sur. Sur.

Off resonance Phase max.

0.60 -0.8

-0.4 -0.4 0.0 0.4 Scan Scanposition position(x/ (x/λ)λ)

0.8

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

x

z Scanning directions

y

Sample

Oil 6µm

NA=1.2 in water

Glass slide

Es(ωs) Ep(ωp) Backward detected signal (E-CARS)

Considered set-up

Normalized CARS intensity

Collection Forward detected signal NA=0.5 (F-CARS) ωas

Normalized CARS intensity

CARS imaging of interfaces: 3D model 6 5 4 3 2 1 0

Interface Resonance peak Dip Off resonance Phase maximum

Bead

1.0

1.0

Oil

1.5 2.0 2.5 3.0 Scan position (µm) Interface

0.9 0.8

Phase maximum Off resonance

0.7 0.6

Bead 1.0

Oil

1.5 2.0 2.5 3.0 Scan position (µm)

Theoretical 1D scans (3D model)

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

Experimental set-up λp = 730 nm Ps=500 μW Pulse rate=3.8 MHz Pulse width ~ 3 ps

ωp

BC: beam combiner BS: beam splitter C: condenser (NA=0.5) F: filter L: lens

Ep

ωs BC

Sample

F L E Epi CARS detector

Es

λs ~ 787 nm Ps=500 μW Pulse rate=3.8 MHz Pulse width ~ 3 ps

F

NA 1.2

ωas

ωas BS

Objective

C

LF Forward CARS detector

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

Experimental evidence (1)

Polystyrene bead (Φ=6 µm)

45 PM

0.5

P

30 15

D

Glass slide 0

OR 0.5 1 1.5 (3)

104 102

300

100 0 http://www.aist.go.jp 960Source:1000 1040 1080 Raman shift (cm-1)

100

96 96 98 100 102 104

2

50

X position (µm)

0

Bead 2D scans Off-resonance

1003cm-1 Polystyrene bead

200

150

98

χ /χ(3)NRmodulus

Immersion oil

200

100

500 400

250

70

104

65

102

60

100

55

98

50 45

96

40 96 98 100 102 104

X position (µm)

35

CARS intensity (kcps)

CARS intensity (kcps)

60

1.5 1

n~1.6@25°C, phenyl group

75

Y position (µm)

Scanning plane

2

90

CARS resonance

Phase (°)

CARS intensity (kcps)

Immersion oil

(3)

χ

Y position (µm)

n=1.556@25°C, phenyl free

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

400 300 200 100 960

980

1000 1020 1040 1060 1080

945cm-1 1002cm-1 1007cm-1 1007-1013cm-1 1013cm-1 1018cm-1 1018-1024cm-1 1024cm-1 1030cm-1 1035cm-1 1097cm-1

CARS intensity (AU)

6 5 4 3 2 1 96

100

104

Scan position (µm)

Bead experimental 1D scans

108

104

250

102

200

100 150

98

100

96 96 98 100 102 104

X position (µm)

50

CARS intensity (kcps)

Raman shift (cm-1)

Y position (µm)

CARS intensity (kcps)

Experimental evidence (2)

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

Y position (µm)

400 300 200 100 0 960

1000

1040

104

250

102

200

100 150

98

100

96

1080

Raman shift (cm-1)

96 98 100 102 104

50

CARS intensity (kcps)

CARS intensity (kcps)

Experimental evidence (3)

X position (µm)

CARS intensity (AU)

1.2

Scans features:

1.0

1. Influence of the bead/oil refraction index mismatch…

0.8 1007-1013cm-1 1024cm-1 1035cm-1 1097cm-1

0.6

96

100

104

Scan position (µm)

108

Bead experimental 1D scans (selected scans)

2. …but a spectral effect can be seen, conveying the interference variation between the bead and the oil.

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

Discussion (1)

Potma et al., J. Raman Spectr. 34, 642 (2003)

CARS spatial coherence

CARS spectral coherence

Oron et al., Phys. Rev. Lett. 89, 273001 (2002)

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

Discussion (2)

Path 1

Path 1 Giant

Path 2

Unilamellar

Glass slide

Vesicle Resonant

Non-

Path 2

Dephasing Variation of distance between the GUV and the slide Dephasing of the incident

Iin

resonant pulse spectral contribution contribution

Iout

1.2 CARS intensity (AU)

A fruitful analogy: the Michelson interferometer

components

1.0 0.8 1007-1013cm-1 1024cm-1 1035cm-1 1097cm-1

0.6

(e)

96

100

104

Scan position (µm)

Object

Surrounding

108

Experiment by Potma et al. Æ spatial interferences Experiment by Oron et al. Æ spectral interferences Our experiment Æ combined effect

Raman resonance detuning

CARS microscopy near boundaries

9th FrenchFrench-Israeli Symposium on NonNon-linear & Quantum Optics

Conclusion & perspectives

1. Theoretical & experimental study of CARS imaging of interfaces. 2. Strong analogy with previous works on spatial & spectral interferences in CARS Æ combined effect. 3. Experiment suitable for multiplex CARS microscopy (one shot experiment, phase-retrieval procedures in congested fingerprint region). 4. On small objects (biological membranes), the procedure seems interesting to improve image contrast (high non-resonant surrounding & reduced refraction-index mismatch)