Imaging with Second-HarmonicGeneration Nanoparticles. Chia-LungHsieh, Rachel Grange, Ye Pu, Demetri Psaltis \

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1 Imaging with Second-HarmonicGeneration Nanoparticles Chia-LungHsieh, Rachel Grange, Ye Pu, Demetri Psaltis \ demetri.psaltis@epfl.ch 1

2 Outline Second-harmonic generation (SHG) in nanoparticles Cell imaging with SHG nanoparticles Holographic imaging Imaging through scattering media 2

3 Introduction Fluorescence bioimaging Aquired by T. Schallerat London s Global University Real state Non-radiating relaxation Fluorescence Ground state 3

4 Light-matter interaction: nonlinear polarization SHG from a SHRIMP P induced E scatt (2ω) E incident (ω) +Q E scatt (ω) E incident χ (2) bulk -Q E scatt P induced = χ (1) E (ω) Linear Scattering + χ (2) E (ω) E(ω) SHG Scattering (No phase matching) 4

5 Introduction Second harmonic generation (SHG) Virtual state Real state Non-radiating relaxation SHG Fluorescence Ground state Ground state SHG Excitation and emission spectra 2-photon fluorescence Laser pump (> 450 nm)

6 Introduction why SHG markers? Second Harmonic Radiation Imaging Probes (SHRIMPs) Long-term observation:due to the SHG physical mechanism over virtual energy state Flexibility in excitation wavelength:shg is a nonresonant process Coherent signals:complex field detection Narrow signal bandwidth:more effectively suppress the background Ultrafast response time:in the sub-femtosecond range Biocompatibility:excellent biocompatibility to cells and tissues 6

7 SHG nanomaterials KTiOPO 4 Crystalline organicinorganic hybrid particles KNbO 3 LiNbO 3 W = 50nm, L = 700nm 200 nm Le Xuan et al., Small Delahaye, et al., Chem. Phys. Lett Nakayama et al., Nature 2007 Grange et al, Appl. PhyLett Fe(IO 3 ) 3 ZnO CdTe/CdSQdot BaTiO 3 W = 50nm, L = 700nm Bonacina et al. Appl. Phys. B 2007 Opt. Exp Kachynskiet al. J. Phys. Chem. C 2008 Zeilinski et al. Small 2010 Hsieh et al, Opt. Express 2009 Biomaterials

8 Barium titanate (BaTiO 3 ) nanoparticles Noncentrosymmetric crystal structure: Tetragonal 90 nm BaTiO 3 SEM image 30 nm BaTiO 3 SEM image BaTiO 3 dry powder 8

9 SHG from single nanoparticles Ti:sapphire oscillator nm, 76MHz, 150fs 100x NA 1.4 Average intensity ~ 10 kw/cm 2 100x less than cell damage threshold λ/2 L1 Sample OBJ L2 Filter EMCCD 500 nm SEM image Corresponding SHG image 9

10 SHG from single nanoparticles Ti:sapphire oscillator nm, 76MHz, 150fs 100x NA 1.4 Average intensity ~ 10 kw/cm 2 100x less than cell damage threshold λ/2 L1 Sample OBJ L2 Filter EMCCD Power dependency Stability Tunability 10

11 Polarization-dependent SHG response (a) (b) (c) (d) 11

12 SHG efficiency +Q -Q ε p ε 0 E p 3ε 0 ( ω) = E0e ε + 2ε p 0 iω t P(ω,2ω, )=χ E (ω)+χ E (ω) E (ω)+ (1) (2) p p p W 2p = σ 2p (I incident ) 2 SHG Fluorescence x Biomarkers Size (nm) Brightness (GM) Photon budget BaTiO >10 9 BaTiO >10 9 GFP < Organic dyes < Qdots GM (Goeppert-Mayer) = cm 4 sec / photon 12

13 Plasmonic enhanced SHG nanoparticles 700 times SHG enhancement through engineered resonance Bare BaTiO 3 core Surface Seeding Complete Shell SEM pictures at different stages Phys. Rev. Lett. 104, (2010) 13

14 Nonspecific cell labeling with SHRIMPs Endocytosis SHG 2007 by Saunders, an imprint of Elsevier, Inc. Transmission 12 hours Myeloid cells Wait for16 hours before imaging 14

15 SHG and two-photon fluorescence imaging 9 µm 8 µm 7 µm 6 µm 5 µm 4 µm 3 µm 2 µm 1 µm 0 µm Green: SHG from SHRIMPs Red: fluorescent staining 15

16 SHRIMPs uptaken by HeLa cells via endocytosis Y Z X X 5 µm Y Z Acquisition time: 10 minutes 16

17 Dendritic cell imaging with SHRIMPs 1 hour video of dendriticcells in a collagen matrix y y x z x Start z 17

18 Specific labeling via surface functionalization 18

19 Specific labeling in protein microarrays Primary Ab 1 Secondary Ab Control Primary Ab 2 Primary Ab 3 O S BaTiO 3 NH O N O Antibody-SHRIMP conjugate 19

20 Specific labeling of cell surface proteins O S Target: cell membrane HLA class I antigen Secondary Ab BaTiO 3 O NH N O Primary Ab Transmission SHG Overlay Specific labeling Control (no primary Ab) 20

21 Harmonic holographic 3D imaging of nanoparticles Direct imaging Sample OBJ Lens Filter EMCCD Holographic imaging Reference 21

22 Dynamic harmonic holographic imaging of nanoparticles Frame rate: 100 Hz Motion speed: 1 mm/sec 22

23 Holographic 3D imaging of nanoparticles Beam propagation Reference R(x,y,z) h(x,y,z) S(x,y,z) Signal S(x,y,z) h(x,y,z) Complex field detection S(x,y,z) h(x,y,z) + R(x,y,z) 2 Digital back propagation 23

24 Harmonic holographic 3D imaging of nanoparticles 24

25 Harmonic holographic 3D imaging of nanoparticles µm z = 0 µm z = 3.12 µm z = 6.24 µm Distance (µm) 25

26 Imaging SHRIMPs in live animals Anesthetized mouse Y X X Dermal collagen Z Collaboration with Prof. Melody Swartz group at EPFL 26

27 Imaging SHRIMPs in live animals 27

28 Imaging SHRIMPs in scattering tissue 28

29 Undo the scattering by holography Scattering S(r) Record Holographic crystal Phase conjugation Scattering media S*(r) Holographic crystal Read-out 29

30 Imaging through tissue by phase conjugation Imaging through clear media Imaging through scattering media (0.5 mm chicken breast tissue) Imaging through scattering media by phase conjugation Read-out Z. Yaqoob, D. Psaltis, M. S. Feld, C.Yang Nature Photonics2, (2008) 30

31 Digital optical phase conjugation (DOPC) Digital camera Scattering < 1 sec Second harmonic nanoparticle Scattering media Phase conjugation < 1 sec Spatial light modulator (SLM) 31

32 Digital phase conjugation of SHRIMP beacon SHRIMP SLM Excitation Diffuser Reference BS3 M CCD1 DI OBJ1 OBJ2 L1BF S L2 BS2 BS1 BF CCD2 32

33 Focusing through random media Input phase Output intensity Linear Transformation 400 times stronger Bar = 5 µm 33

34 Imaging through turbidity by scanning phase conjugation SHRIMP Commercial diffuser (surface roughness standard deviation: 3 µm) 185 µm SHRIMP Optics Express, Vol. 18, Issue 20, pp (2010) 34

35 Imaging through turbidity by scanning phase conjugation SHRIMP Target Scanning image without DOPC Scanning image with DOPC Image size: 115 µm x 115 µm 35

36 Conclusion SHG in nanoparticles: Mechanism, efficiency, polar response Bioimaging: Bioconjugation, cell imaging, in vivo imaging Scan-free 3D imaging: Harmonic holographic microscope Imaging through turbidity: Digital phase conjugation using SHG beacon nanoparticles 36

37 Acknowledgements SHRIMP team: Chia_lung Hsieh Ye Pu Rachel Grange Thomas Lanvin Xin Yang Paul Bowen (Powder Technology Lab at EPFL) Marc Chambon (Biomolecular Screening Facility at EPFL) Thierry Laroche (Bioimaging and Optics Platform at EPFL) Keith Harshman (Protein and DNA Array Facility at Unil) Johann Weber Floriane Consales Marie-Agnes Doucey Funding: National Centre of Competence in Research Quantum Photonics 37

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