http://www.iap.uni-jena.de/multiphoton Nanomaterials and their Optical Applications Winter Semester 2012 -2013 Lecture 11 [email protected]
[email protected] Lecture 11 http://www.iap.uni-jena.de/multiphoton
Nanomaterials and their Optical Applications
Winter Semester 2012 -2013
Lecture 11
[email protected] Lecture 11
Schedule until the end of the semester 2
11 14.01.2013 Nanomarkers12 21.01.2013 Seminars presentations by students (2)13 28.01.2013 Seminars presentations by students (2)x 04.02.2013 no lecture
Lecture, Mondays 16-17.30
Turn in HW 4 on Tuesday 20.01.2013
Seminar, Tuesdays
p / / Q 7 22.01.2013
Examination : February 14th Beutenberg campus, IAP, 14.30-16.30
[email protected] Lecture 11
Choose your slot 3
Lecture : Nanomaterials and their optical applications
Date Room Time Speaker Title of the talk
21.01 IAP 16.00 Yera Ussembayev Plasmonic nanoparticles for biomedicine
16.30 Sebastian Unger Nanodiamonds
17.00 Zhi Upconversion nanoparticles
28.01 IAP 16.00 Pavlo Kliniev Nanowires as biosensors
16.30 Can Boran Akdal SPASERs
17.00 Wondimu Alemu High resolution microscopy
[email protected] Lecture 11
Location Institute of Applied Physics 4
[email protected] Lecture 11
Outline: Nanomarkers 5
• Contrast in imaging modalities
• Static versus dynamic probes
• Dynamic markers
• Multiphoton nanomarkers : SHRIMPs
www.spps.fi/
[email protected] Lecture 11
Possible contrast mechanism for imaging 6
[email protected] Lecture 11
Possible contrast mechanism for imaging 7
[email protected] Lecture 11
Contrast agents 8
Tissue itself Melanin , collagen
Dyes Nanoparticles Quantum dots
[email protected] Lecture 11
Contrast agents 9
Goal of biomedical imaging: structural and functional information
[email protected] Lecture 11
Contrast agents 10
[email protected] Lecture 11
Contrast agents: dynamic probes 11
Scattering probes: static, thus background noise from scattering of the tissues
Dynamic probes: modulated externally by slight modulation, then the unmodulated background can be filtered out efficiently
Contrast agents: paramagnetic particles, 20-30 nm with a magnetic susceptibility of χ=1 In tissue χ< 10-5
Contrast agents: iron oxide like magnetite, already used in MRI
Under a high magnetic field gradient… What happens ?
[email protected] Lecture 11
Contrast agents: dynamic probes 12
Optical coherence tomography (OCT) to detect the changes: The optical setup typically consists of an interferometer with a low coherence, broad bandwidth light source. Light is split into and recombined from reference and sample arm, respectively.
optical ultrasound !
The key benefits of OCT are: • Live sub-surface images at
near-microscopic resolution • Instant, direct imaging of tissue
morphology • No preparation of the sample or
subject • No ionizing radiation
[email protected] Lecture 11
Contrast agents: dynamic probes 13
[email protected] Lecture 11
Contrast agents: dynamic probes 14
[email protected] Lecture 11
Contrast agents: dynamic probes 15
[email protected] Lecture 11
Imaging applications: Two-photon confocal 16
16
The Journal of Cell Biology, Volume 105, 1987
Fertilized egg of a sea urchin stained with antitubulin. Scale bar, 50 um.
Conventional vs Confocal microscopy
Single-photon vs two-photon confocal
• Small excitation volume • Longer wavelength
[email protected] Lecture 11
Some history 17
17
1986 First biological samples
1974 First SHG microscopy
Freund et al.. Biophys. J. 50, 693 1986
1999 High resolution SHG
Campagnola et al Biophys. J. 77, 3341 (1999).
Hellwarth et al, Opt Comm, 12, 3, 1974
rat tail tendon
1978 First scanning image
Gannaway et al., OQE, 10, 435, 1978
[email protected] Lecture 11
What is the Second Harmonic Generation (SHG) ? 18
18
Take the simplest atom: hydrogen Put it into an electric field
where α is the answer of the atom to electric field
Microscopic view: 1 atom
Macroscopic view: N atoms
p Eα=
You end up with a dipole moment
the macroscopic dipole moment (per unit volume) is called the POLARIZATION :
Term responsible of second harmonic generation Only present in special material that do not have a center of symmetry
2 30 1 0 2 0 3 ...P E E Eε χ ε χ ε χ= + + +
• 3rd-order : multiphoton absorption, third harmonic generation, coherent anti-Stokes Raman scattering
• 1st-order : absorption and reflection
• 2nd-order: SHG, sum and difference frequency generation, hyper-Rayleigh
[email protected] Lecture 11
19
19
Energy diagram
ω
ω 2ω
ω
ω
Real state
< 2ω
Ground state
Non-radiating relaxation
2-photon fluorescence
Ground state
Virtual state
SHG
Purely scattering mechanism
ω 2ω
Excitation and emission spectra Laser pump
(> 450 nm) 800 850 900 400 425 450
SHG 2-photon fluorescence
directional Non directional
What is the Second Harmonic Generation (SHG) ?
[email protected] Lecture 11
Endogenous SHG : no staining 20
C. elegans
Green SHG from collagen
Red is autofluorescence
Green: SHG (collagen) Red: endogenous 2PEF Pink: THG
C. elegans
Human colon adenocarcinoma
Rat lung tissue
Edward Brown and Rakesh Jain Massachusetts General Hospital
Nature Methods 3, 47 (2006)
50 μm.
@ BIOP
@ BIOP
[email protected] Lecture 11
Endogenous SHG : no staining 21
C. elegans
C. elegans
50 μm.
@ BIOP
@ BIOP
Microscope settings: Pump at 812 nm Detection in reflection with filter set
Cut the pump
Narrow band pass at 406 nm
[email protected] Lecture 11
Endogenous SHG vs SH nanoprobes 22
C. elegans
C. elegans
50 μm.
@ BIOP
@ BIOP
Label with SHRIMPs
Second Harmonic Radiation IMaging Probes
SHRIMPs
• Flexibility to target any type of cells or tissue • No phase matching issue due to the particle
size smaller than the wavelength
HeLa cells
[email protected] Lecture 11
Who are SHRIMPs ? 23
23
Crystalline organic-inorganic hybrid particles
Bonacina et al. Appl. Phys. B 2007
Delahaye, et al. Chem. Phys. Lett. 2006
Nakayama et al., Nature 2007
KNbO3
Fe(IO3)3
100 nm
BaTiO3
Hsieh et al, Opt. Exp. 2009
Zielinski et al. Small 2009
CdTe/CdS BaTiO3 /Au
Pu et al, PRL 2010
Le Xuan APL 2006
KTP
Kachynski et al. J. Phys. Chem. C 2008
ZnO
Non centrosymmetric material = space group lacking an inversion center
[email protected] Lecture 11
Challenges in Biomedical Imaging 24
24
Life processes are … • Extremely complex • Highly dynamic • KEY: imaging probes
The wish list for “ideal” probes … • Small (the smaller the better) • Sensitive (easily detected) • Sustainable (no bleaching) • Stable (no blinking) • Fast (quick response) • Scalable (no signal saturation) • Biocompatible (no toxicity) • …
GFPs
www.Invitrogen.com
QDs Dr. Kalju Kahn at UCSB
SHRIMPs
[email protected] Lecture 11
Advantages of SHRIMPs 25
25
Long-term observation: Due to the SHG physical mechanism over virtual energy state
Flexibility in excitation wavelength: SHG is a nonresonant process
Coherent signals: interferometric detection techniques possible, such as holography
Narrow signal bandwidth: more effectively suppress the background.
Ultrafast response time: in the subfemtosecond range
Biocompatibility: little toxicity and excellent biocompatibility to cells and tissues.
Laser pump
(> 450 nm) 800 850 900 400 425 450
SHG 2-photon fluorescence
[email protected] Lecture 11
BaTiO3 nanoparticles characterization 26
100x NA 1.4
Ti:sapphire oscillator 800nm, 76MHz, 150fs
Peak intensity 1 GW/cm2 100x less than cell damage threshold
SHG: Quadratic power dependency
σ 2p : Two-photon cross section
1GM (Goeppert-Mayer) = 10-50 ⋅ cm4 ⋅ sec / photon)
W2p = σ2p ⋅ (Iincident)2
C. Hsieh, R. Grange, Y. Pu, D. Psaltis, Opt. Express. 17, 2880 (2009)
Total radiated power
[email protected] Lecture 11
BaTiO3 nanoparticles characterization 27
100x NA 1.4
Ti:sapphire oscillator 800nm, 76MHz, 150fs
Peak intensity 1 GW/cm2 100x less than cell damage threshold
Comparison with fluorescent and SH markers
Type Size (nm) σ 2p (GM) GFP < 5 6 QD 10 2’000 - 47’000 BaTiO3 30 8.5 BaTiO3 90 6’500
[email protected] Lecture 11
Plasmonics to engineer a resonance 28
28
SHG enhancement > 1000 x Tunability with thickness
Noble metal nanoparticles or shells: oscillation of conduction electrons at the surfaces generating a strong resonance at optical frequencies
Scientific American 2007
[email protected] Lecture 11
Plasmonics to engineer a resonance 29
Y. Pu, R. Grange, Ch.-L. Hsieh, and D. Psaltis, Physical Review Letters, 104, 2010.
Amine Au seeds Shell Bare SHRIMPs
SEM pictures at different stages
Chemical synthesis of the nanoshell
[email protected] Lecture 11
Superresolution microscopy 30
http://zeiss-campus.magnet.fsu.edu/articles/superresolution/introduction.html
[email protected] Lecture 11
Superresolution microscopy 31
stimulated emission depletion (STED)
http://zeiss-campus.magnet.fsu.edu/tutorials/superresolution/stedconcept/index.html
[email protected] Lecture 11
Superresolution microscopy 32
Single-Molecule Localization Microscopy
http://zeiss-campus.magnet.fsu.edu/tutorials/superresolution/stedconcept/index.html