SHG Nanoprobes: Advancing harmonic imaging in biology. Periklis (Laki) Pantazis

Transcription

1 SHG Nanoprobes: Advancing harmonic imaging in biology Periklis (Laki) Pantazis

2 fluorescence microscopy in biomedical research: impact of fluorescent proteins 1960s and 1970s 1992 and wt-gfp purified along with luminsecent aequorin from Jellyfish Aequorea Victoria wt-gfp cloned and Expressed e-gfp introduced wt-gfp/e-gfp crystal structure reported relative Fluorescence E-GFP e-gfp excitation e-gfp emission wavelength [nm]

3 fluorescence microscopy in biomedical research: impact of fluorescent proteins dsred recovered in anthozoan corals photoconvertible fluorescent proteins photoactivatable fluorescent proteins 2004-present Numerous fluorescent variants by in vitro and in vivo directed evolution kaede pa-gfp san diego beach J Biomed Opt. 2007;12(4):044004(1-7).

4 fluorescence recovery after photobleaching (frap): kinetics of morphogen gradient formation frap of gfp-dpp FRAP recovery curve Tissue Autofluorescence Bleaching Bleaching normalized fluorescence effective diffusion coefficient production rate degradation rate immobile fraction time [min] dppgal4::uas-gfp-dpp/+ Science Jan 26;315(5811): Phys Rev E Jan;75(1 Pt 1): Phys Rev Lett Jan 14;94(1):

5 fluorescence microscopy in biomedical research: improving long-term photostability BRIGHT - PHOTOSTABLE - COLOR SELECTIVE - ph INSENSITIVE - WATER SOLUBLE Organic Dyes Alexa 488nm Molecular Probes

6 fluorescence microscopy in biomedical research: improving long-term photostability BRIGHT - PHOTOSTABLE - COLOR SELECTIVE - ph INSENSITIVE - WATER SOLUBLE Quantum Dots simultaneous excitation at 365nm core shell polymer coating size-dependent emission 10-20nm normalized fluorescence wavelength [nm]

7 fluorescence microscopy in biomedical research: limitations of quantum dots signal intensity [a.u.] blinking Signal Saturation Blinking Blinking signal intensity [a.u.] saturation time [s] no continuous tracking of individual molecule trajectories scan power [a.u.] time resolution of singlemolecule imaging is limited

8 single-photon excited fluorescence vs. two-photon excited fluorescence Excited state Excited state!i 2!i!<2!i ~10-9 s!i!<2!i ~10-9 s Ground state single-photon excited fluorescence Excitation not restricted to the focal point Limited penetration Increased phototoxicity Ground state two-photon excited fluorescence Excitation restricted to the focal point Deeper penetration Reduced phototoxicity

9 two-photon excited fluorescence vs. second harmonic generation (shg) Excited state Virtual state!i!i!=2!i!i!<2!i ~10-9 s!i ~10-15 s Ground state two-photon excited fluorescence Involves real transition Energy is partially lost Nanosecond response time Frequency lower than SHG Ground state second harmonic generation Involves virtual transition Energy is conserved Femtosecond response time Frequency exactly doubled

10 second harmonic generation (shg): phase-matching conditions for a strong signal oscillating electron nonlinear material constructive interference!i!=2!i } coherence length ordered, non-centrosymmetric structures at specific angles

11 shg microscopy in biomedical research: detecting endogenous shg SHG trans-detection condenser 2PEF specimen objective incident light epi-detection incident light

12 detecting endogenous shg: visualizing endogenous structures - zebrafish trunk muscles bodipy tr end. shg Proc. SPIE, 7183(1):

13 identifying shg nanoprobes: screening for a large second-order nonlinear susceptibility (" 2 ) optical response of a medium P(!) = " (1) E(!) + " (2) E(!) 2 + " (3) E(!) P(!) induced polarization E(!) optical field strength of incident light " (n) n th -order susceptibility of the material " (1) absorption or reflection " (2) second harmonic generation (shg) " (3) third harmonic generation (thg)

14 identifying shg nanoprobes: barium titanate (BaTiO3) nanoparticles BaTiO3 (~30nm) - 50 nm J. Appl. Phys. 98, (2005) cubic (>132 C) O2 hexagonal (>1432 C) - 25 nm - 0 nm ba tetragonal (<132 C) Proc Natl Acad Sci U S A. (2010); 107(33):

15 characterizing shg nanoprobes: barium titanate (BaTiO3) shg signal properties intensity [a.u.] 50 FWHM < 5nm wavelength [nm] Excitation 820nm Peak intensity ~10 9 Wcm -2 Proc Natl Acad Sci U S A. (2010); 107(33):

16 shg nanoprobes display a high signalto-noise ratio in optically challenging environments Phantom medium 2.5 % Intralipid/ 0.05 % Indian Ink / 20 % polyacrylamide Proc Natl Acad Sci U S A. (2010); 107(33):

17 shg nanoprobes display a high signalto-noise ratio in optically challenging environments QD BaTiO3 SNR [a.u.] broad Filter set narrow Proc Natl Acad Sci U S A. (2010); 107(33):

18 characterizing shg nanoprobes: barium titanate (BaTiO3) shg signal does not blink 5 5 BaTiO3 QD intensity [a.u.] intensity [a.u.] scans scans Proc Natl Acad Sci U S A. (2010); 107(33):

19 characterizing shg nanoprobes: barium titanate (BaTiO3) shg signal does not saturate BaTiO3 QD intensity [a.u.] QD intensity [a.u.] scan power [%] scan power [%] Proc Natl Acad Sci U S A. (2010); 107(33):

20 characterizing shg nanoprobes: barium titanate (BaTiO3) shg signal properties BaTiO3 intensity [a.u.] #SHG [nm] #Excitation [nm] Proc Natl Acad Sci U S A. (2010); 107(33):

21 characterizing shg nanoprobes: zinc oxide (ZnO) and silicon carbide (SiC) shg signal properties ZnO SiC intensity [a.u.] intensity [a.u.] #SHG [nm] #SHG [nm] #Excitation [nm] #Excitation [nm] Proc Natl Acad Sci U S A. (2010); 107(33):

22 multi-shg imaging modality: exploiting distinct shg signal profiles of BaTiO 3 and SiC BaTiO3 800nm intensity [a.u.] SiC 880nm 800 & 880 nm wavelength [nm] Proc Natl Acad Sci U S A. (2010); 107(33):

23 shg nanoprobes show low Toxicity in vivo Zebrafish survival rate after 72hpf [%] * 20 0 QD BaTiO3 PBS NI (n=120 each)

24 shg nanoprobes detectable in trans- and epi-direction trans-detection 410 nm Low power of 850nm trans High power of 850nm trans epi/trans condenser specimen epi epi merge objective epi-detection 410 nm incident light 820 nm Proc Natl Acad Sci U S A. (2010); 107(33):

25 shg nanoprobes readily detectable with increased imaging depth end. shg shg nanoprobes bodipy tr Proc Natl Acad Sci U S A. (2010); 107(33):

26 shg nanoprobes provide superior signal-to-noise ratio in Epi-Direction when in mammalian Tail Tendon specimen Low power of 850nm High power of 850nm objective epi-detection 410 nm incident light 820 nm Proc Natl Acad Sci U S A. (2010); 107(33):

27 shg nanoprobes compared to fluorescent labels Bioessays (2012); 34(5):

28 SHG Nanoprobes Functionalization: Hydroxylation and Silanization Nat Protoc. (2012); 7(9):

29 non-reactive coated shg nanoprobes disperse within zebrafish cells in vivo Nat Protoc. (2012); 7(9):

30 non-reactive coated shg nanoprobes disperse within zebrafish cells in vivo SHG memb-mdendra2 Nat Protoc. (2012); 7(9):

31 pegylated shg nanoprobes persist within cells of the developing zebrafish embryo SHG Bodipy CR Dextran 546 Bodipy TR Nat Protoc. (2012); 7(9):

32 antibody functionalization enables targeted linking of shg nanoprobes

33 targeting functionalized BaTiO3 - recognizing dystrophin Proc Natl Acad Sci U S A. (2010); 107(33):

34 SHG Nanoprobes Functionalization: Click Chemistry Linkage Nat Protoc. (2012); 7(9):

35 shg nanoprobes functionalization: click chemistry linkage - performed at room temperature, non-toxic, no byproducts Nat Protoc. (2012); 7(9):

36 shg nanoprobes functionalization: click chemistry linkage - performed at room temperature, non-toxic, no byproducts Control Functionalized SHG AF488 Colocalization Nat Protoc. (2012); 7(9):

37 shg nanoprobes functionalization: click chemistry linkage - performed at room temperature, non-toxic, no byproducts P<10-4 Nat Protoc. (2012); 7(9):

38 conclusion SHG Nanoprobes neither bleach nor blink superior single molecule detection probes analyzing molecular target dynamics with unmatched temporal resolution and precision SHG Nanoprobes do not saturate SHG offer very sensitive and rapid detection of cells/ molecules of interest without compromising on temporal resolution by simply increasing the illumination power SHG Nanoprobes signal is multidirectional allows imaging without any microscope modification, using settings very similar to those required for fluorescent probe imaging SHG Nanoprobes are not toxic and display a superb signal-to-noise ratio and virtually no signal background clinically valuable for diagnosis of disease conditions, progression and evaluation of potential treatments using minimally invasive optical microendoscopy

39 Thank Thank you! you! Scott Fraser William Dempsey Nicolas Plachta Ali Yasin Sonay Shirley Pease Jelena Čulić-Viskota Scott Fraser ETH Zurich ETH Zurich Caltech Caltech Postdoc and Phd positions available!