Endoscope non linéaire pour l'imagerie en profondeur dans les tissus

Hervé Rigneault - Institut Fresnel Marseille - INSIS

Alexandre Kudlinsky – PhLAM Lille - INP Olivier Piot – URCA Reims - INSB

Didier Marguet – CIML Marseille - INSB

NLO Endoscope - DEFI 2013

TPEF SHG THG Raman CARS SRS ωP – ωS = ΩR

Nonlinear optical contrast mechanisms: label free imaging

CARS

ωP ωP

ωS ωS

SRS

Molecular Imaging

Collagen Cells (NADH)

Immuno-stained cryo section of melanoma

1mm 100µm

Image stitching: 70x70 images - size 100 µm x 100 µm

Collagen structure modification in the tumor

Two-Photon / Second harmonic image of tissues

85µm 85µm 55µm

30µm 55µm

30µm 0µm 0µm

Epi CARS Fwd CARS

Ear mouse (2845cm-1 CH aliphatic)

Coherent Anti-Stokes Raman Scattering (CARS) Imaging

2705cm-1 - NR- CARS

2845cm-1 - R-CARS TPEF SHG

Multimodal nonlinear imaging of living tissues

Composite CARS (R-NR) SHG TPEF

Cortex

Hippocampus

1 mm

6

Linear and nonlinear microscopy are limited to few 100µm

Can we bring nonlinear imaging in endoscopy?

« Noise » resulting from light propagation in conventional fiber

- Group velocity dispersion (GVD) - Material GVD - Waveguide dispersion

- Self Phase Modulation - Transient raise the local refractive index, phase shift and a corresponding spectral distortion and temporal chirp

- Raman - Inelastic scattering produces wavelength generation and gain

- Four wave mixing - Phase matched generation of new wavelengths

- Soliton - Solutions of nonlinear propagation equation –NLO Schrödinger equation (Anomalous dispersion regime)

- Autofluorescence - defects - unoxydized metal ions - color centers…

CW light

Ultra-short pulses

- Raman - Inelastic scattering produces wavelength generation and gain

NLO effects in SMF and Solid Core PCF in the ps regime

Single Mode Fiber (SMF)

with a single pump beam

Solid Core : Nonlinear

effects

Solid core PCF

0

0,25

0,5

0,75

1

720 724 728 732 736

WithoutWith bundle

Wavelength / nm

I

0

0,25

0,5

0,75

1

720 724 728 732 736

WithoutWith bundle

Wavelength / nm

I

650 700 750 800 850 900 950 1000 1050

Wavelength / nm

763 nm

756 nm

663 nm

990 cm-1

1855 cm-1

Stokes : 1064 nm

OPO1 : 816.8 nm

OPO2 : 888.7 nm

2845 cm-1

I

650 700 750 800 850 900 950 1000 1050

Wavelength / nm

763 nm

756 nm

663 nm

990 cm-1

1855 cm-1

Stokes : 1064 nm

OPO1 : 816.8 nm

OPO2 : 888.7 nm

2845 cm-1

I

SPM effect FWM effects

817nm 889nm 1064nm

1855cm-1 991cm-1

2845cm-1

Fiber

Hollow core (HC) Photonic crystal fiber (PCF)

G. Humbert, et al, Opt Express 12(8), 1477-1484 (2004).

R. F. Cregan, et al., Science 285, 1537-1539 (1999).

Suitable for ultra-fast beam delivery

NO SPM and FWM in hollow core (HC) PCF

0

0,25

0,5

0,75

1

727 728 729 730 731

Wavelength pump (nm)

Inte

nsity

(a.u

)

With HCWithout

In the time domain: - Pulse duration before HC PCH: 6 ps - Pulse duration after HC PCH: ~6 ps In the spectral domain:

@ 728.7 nm

FWHM before HC PCF : 0.8 nm FWHM after HC PCF : 0.8 nm

Pump beam

NO SPM, NO FWM (NR CARS) in Hollow Core fiber

Φ ~ 20 µm

Commercial HC fiber: NA~0.02

40x 1.2

0.5 50x

AP

D

x

z

filter

Anti-Stokes

fiber

fiber

Hollow Core

1.5 m

0%

10%

20%

30%

40%

50%

60%

720 750 780 810 840

Wavelength (nm)

Tran

smis

sion

(%)

Transmission

Forward

mirror Pompe

Stokes

spectrometer

P

L M

λ/2

Y X

Ti:Saph wavelength

790 nm

or 1080 nm

D

M

1m long fiber APD TPEF

560nm SHG

540 nm

De-scanned detection using Kagome for pump delivery and

signal collection

Ti:Saph fs Chameleon laser

‘Endoscope like’ proof of principle experiment

2-Photon imaging in ‘endoscope like’ scheme using Kagome fiber

Microscopy Endoscope

Microscopy Endoscope

TTB molecular crystals

Drosophila embryo