multimodal optical platform for condensed matter...
TRANSCRIPT
Dr Grigory ARZUMANYAN
On behalf of the team,
Department of Raman spectroscopy,
Centre “Nanobiophotonics”, JINR, Dubna, Russia.
RAMAN SPECTROSCOPY SEMINAR
26-27 November 2015, Minsk, Belarus
Multimodal Optical Platform
for Condensed Matter Studies
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Moscow
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Basic Scientific Directions at JINR
High Energy Physics
Nuclear Physics
Condensed Matter Physics
JINR’s staff members ~ 4500
Main Supporting Activities
Theory
Networking and computing
Physics instruments and methods
Training of young staff
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Laser scanning confocal luminescente “CARS” microscope General developer: “SOL instruments” Ltd., Minsk, Belarus
Raman and nonlinear optical spectroscopy
and microscopy (CARS, SONICC) at JINR
Raman
Multimodal optical platform at JINR
CARS
Up-conversion
luminescence Transmitted and
reflected signals
channels
6
ССD, PMT: spectra, images,
E-CARS, F-CARS, P-CARS
200 400 600 800 1000 1200 1400 1600 1800
0
1000
2000
3000
4000
5000
6000
7000
632nm_grat-600_pin-100_1s_40x
Inte
nsity (
a.u
.)
Raman shift (cm-1)
3.1
3.2
3.3
3.4
1730
1
16162
14613
14184
1295
5
11876
1117
1096
7
8
10019
860
10
79711
63213
70512283
14 435358301
129
174229
2015
SONICC Second harmonic & sum frequency
generation
SERS
CARS history in brief The first recordings of Coherent Anti-Stokes
Raman Scattering go back to the 60s of the last
century, when two researchers of the Scientific
Laboratory at the “Ford Motor Company”,
P. D. Maker and R. W. Terhune, published an
article about their experiments (they simply
called their work “three wave mixing
experiments”). “Study of Optical Effects Due to an Induced
Polarization Third Order in the Electric Field
Strength”, Phys. Rev. 148, 990 (1966)
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Coherent Anti-Stokes Raman Scattering (CARS) Nonlinear Laser Microscopy
CARS derives its name from the fact that it uses two coherent
laser beams and the resulting signal has Anti-Stokes
(blue-shifted) frequency.
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ωvib = ωpump - ωStokes
CARS is a third-order nonlinear process that involves a pump beam at
a frequency ωp and a Stokes beam at a frequency of ωs. The signal at
the anti-Stokes frequency of ωas = 2ωp- ωs is generated in the phase-
matching direction.
The sample is stimulated through a four-wave mixing parametric process.
The vibrational contrast in CARS is created when the pump-Stokes frequency
difference matches molecular vibration of a particular chemical bond = ωvib
and the oscillations of molecules with that bond are driven coherently.
Thus, CARS provides a chemically specific signature of various molecules.
Physical background
ICARS(ω) ~ |CARS(3)|2 x Ip
2 IS x N2 (3) – third-order nonlinear susceptibility
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The CARS process in detail
CARS energy diagram
a b c d
Non-resonant component arises from
the electronic contributions to (3) :
b) off-resonance transitions, c) two-photon
enhanced non-resonant contribution.
ωAS = 2ωp- ωSt
CARS geometry varieties and detection schemes
Forward CARS (F-CARS) The signal is detected in the forward phase-matched direction and the signal is selected by a set of
spectral filters. The F-CARS signals are generally very strong (about 1% of the pump beam intensity)
and can sometimes be observed by the naked eye. The forward CARS signal is accompanied by a
strong nonresonant background which may overshadow weak signals that are of interest. Epi CARS (E-CARS) When detected in the backward direction, the nonresonant signal from the solvent (water) is completely
eliminated. E-CARS is particularly sensitive to objects in focus that are smaller than the optical
wavelength. When the sample is highly scattering, the forward propagating CARS signal can be
backscattered, giving rise to a strong epi-signal. Polarization sensitive CARS (P-CARS) By taking advantage of the Raman depolarization ratio of certain modes, the resonant signal can be
separated from the nonresonant background when polarization sensitive detection is employed. Multiplex CARS (M-CARS) A picosecond laser is combined with a femtosecond laser to cover a broad range of vibrational frequencies.
Time-resolved CARS By employing a femtosecond time-scale delay between the excitation and probe pulses, time-resolved CARS
allows for the almost complete suppression of nonresonant signals. 10
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JINR’s setup
General view of the
multimodal optical platform at JINR, Dubna
Pneumatic Vibration Isolation Workstation
STANDA, (Lithuania)
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Front view Front view Back view
Picosecond laser “EKSPLA” (Lithuania)
1 – picosecond Nd:YVO4 laser; 2 – picosecond SOPO (LBO); 3 – module for formation
and combination of the pump (SOPO) and Stokes (Nd:YVO4) beams; 4 – HeNe laser
1 2
4
3
Nd:YVO4 laser parameters:
Master oscillator – diode pumped mode-locked ND:YVO4 laser: - wavelength – 1064 nm - pulse duration – 7 ps - output power – 5 W @ 1064 nm and 2 W @ 532 nm - repetition rate – 85 MHz Synchronously pumped optical parametric oscillator (SOPO) - tuning range: (690 – 990) nm - pulse duration – 6 ps - output power (70-150) mW - spatial mode – TEM00
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Resonance selectivity of CARS-imaging (polysterene beads)
λ (nm) 804 803 802 801 798 799 800 797
CARS
resonance
Transmitted light
F-CARS channel
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λOPO = (690-990)nm λStokes = 1.06µm
CARS, λAntiStokes
CARS stringent requirement:
spatial and temporal overlap of two ps laser beams
6 ps 7 ps
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Sum Frequency Generation (SFG) in KTP: ƛSt. + ƛp.
ƛsum = 490.2 nm
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Transmitted light
F-CARS channel
Sensitivity and selectivity of CARS-imaging vs
temporal overlap of laser beams
Delay line (ps) 0 1 3 2 4 5
Two ps pulses
are superimposed
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Short summary
CARS microscopy is an efficient
technique for imaging with stronger
signal and chemical contrast
without using tags.
Raman
Multimodal optical platform at JINR
CARS
Up-conversion
luminescence Transmitted and
reflected signals
channels
19
ССD, PMT: spectra, images,
E-CARS, F-CARS, P-CARS
200 400 600 800 1000 1200 1400 1600 1800
0
1000
2000
3000
4000
5000
6000
7000
632nm_grat-600_pin-100_1s_40x
Inte
nsity (
a.u
.)
Raman shift (cm-1)
3.1
3.2
3.3
3.4
1730
1
16162
14613
14184
1295
5
11876
1117
1096
7
8
10019
860
10
79711
63213
70512283
14 435358301
129
174229
2015
SONICC Second harmonic & sum frequency
generation
SERS
SONICC – Second Order Nonlinear Imaging of Chiral Crystals
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SONICC relies on two-photon scattering (actually
second harmonic generation) which eliminates all
background from randomly oriented molecules, but
produces a strong signal from chiral
(noncentrosymmetric) molecules arranged in a
crystal, resulting in high contrast images.
crystals are clearly
visible in SONICC
Raman, CARS, P-CARS and SONICC images of
bacteriorhodopsin (BR) crystals as a model
of membrane proteins (MP)
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Micrograph of BR crystals
24 µ
Membrane proteins (MPs) are responsible for vital functions of the
cells and their studies are of great importance for both
science and practical medicine.
Structural study of MPs is a major challenge due to dramatic difficulties
with growing, detection and imaging of the crystals suitable
for X-ray crystallography.
900 1050 1200 1350 1500 16500
800
1600
2400
3200
4000
4800
5600 1538
1191
Inte
ns
ity
(a
.u.)
Raman shift (cm-1)
1018
Raman spectrum and images of BR excitation – 785nm, scan area: (24 x 24)µ, resolution – 48x48pel (1pel-0.5µ)
Raman spectrum of crystal BR
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Visualization of BR crystals
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F-CARS vs P-CARS
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Comparison of F-CARS and P-CARS contrasts
in resonant and nonresonant conditions
P-CARS at 1529 cm-1
Scan area 225x225um
Resolution: 2pl/1um
Data accumulation time ~2s
Pump =915.4nm, Stokes = 1.06um
Image at ~ 802nm
Scan area 225x225um
Resolution: 2pl/1um
Data accumulation time ~2s
Pump (Stokes) = 1.06um
Image at 532nm
SONICC at pump 1064nm
Polarized P-CARS image versus SONICC
50 мкм
Transmission image
50 мкм
SONICC image at 532nm (pump 1064nm)
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Polarized P-CARS image versus SONICC (cell C-9)
25 мкм
P-CARS (48x48um)
Scan area 48x48um
Resolution: 2pl/1um
Data accumulation time ~ 2s
Pump = 915.5nm, Stokes = 1.06um
Image at ~ 802nm
25 мкм
SONICC (48x48um)
Scan area 48x48um
Resolution: 2pl/1um
Data accumulation time ~ 2s
Pump (Stokes wave ) = 1.06um
Image at 532nm
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25 мкм
P-CARS (48x48um), 1529сm-1
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7000
Inte
nsity, a.u
.
Raman shift, cm-1
Raman spectra of BR crystall in center area
10s_pin 100
1529cm-1
600 800 1000 1200 1400 1600 1800
0
1000
2000
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4000
1529cm-1
Raman spectra of BR crystall in "obodok"
10s_pin 100
Inte
nsity, a.u
.
Raman shift, cm-1
Raman spectra in the central part
of the BR crystal is similar to that of
in the boundary rim.
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3D image (P-CARS and SONICC) of the same crystal
3D
P-CARS
3D
SONICC
Z=30um, step 1um
Z=30um, step 1um
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More stronger signals than spontaneous Raman CARS signal is at high frequency (lower
wavelength) – minimal fluorescence interference Microscopy – faster, more efficient imaging for
real-time analysis
Contrast signal based on vibrational
characteristics, no need of fluorescent tagging Higher spatial resolution
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Why develop CARS?
Main advantages:
Drawback – nonresonant background; can be
minimized by the polarization sensitive detection, etc.
To achieve an efficient upconversion luminescence emission
several requirements (prerequisites) are imposed on the host
matrices and dopants of UC phosphors. 30
The characterization of UC phosphors typically involves: Structure analysis (XRD, SEM, TEM, SANS, RAMAN, …)
Measurement of absorption and UC luminescent spectra
Kinetics of luminescence (excited state lifetimes)
Photon upconversion – sequential absorption of two or more
photons leads to the emission of light at shorter wavelength
than the excitation wavelength.
It is an anti-Stokes type emission.
Dopants for upconversion luminescence
Well defined discrete energy levels with the potential for the
UC process.
Lanthanide (rare earth) ions are a proper choice as they have rich
energy level structure of luminescence active transitions in the NIR,
VIS and UV spectral range. With two or more metastable, intermediate excited states to store
population during the upconversion process.
Matrix can be single ion doped or a combination of various
different ions. 31
Upconversion (UC) luminescence studies
Samples: oxyfluoride glasses provided by Belarusian
State Technological University, Minsk
(1) SiO2 – PbO – PbF2 – Er2O3
(2) SiO2 – GeO2 – PbО – PbF2– Er2O3
(3) SiO2 –Al2O3 – Y2O3 – Na2O – NaF – LiF – Er2O3 –YbF3
precursors and heat-treated (t = 350oC) samples (glass ceramics)
were available for those samples.
1.0 mol % Er3+
0.3 mol % Er3+
4.3 mol % Yb3+
Photograph of samples
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Structural studies: XRD, SANS and SEM
Comparison of the XRD pattern
of the crystalline precipitate (PbF2)
with that of the β-PbF2 crystal
SANS curves of precursor (black) and
glass ceramics (red).
3D model of the shape of PbF2 nanocrystals
structure (ab initio modeling ATSAS)
XRD
SANS
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CuKα radiation, λ = 1.540 Å
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SEM images showing dendritic shape of PbF2
nanocrystals embedded in the glassy matrix
Scanning Electron Microscopy (SEM)
precursor glass-ceramics
glass-ceramics
2µ
10µ
Energy Dispersive Spectrum (EDS)
of 1.0 mol% Er3+ doped glass and glass-ceramics
precursor glass-ceramics
keV keV
UCL spectra of precursors with various
Er3+/Yb3+ doping levels
500 550 600 650 700 750
0
5000
10000
15000
20000
25000
30000
35000
Inte
ns
ity
(a
.u.)
Wavelegth (nm)
Er, Yb,
mol% mol%
0,1 0
0,1 0,1
0,1 0,5
0,1 0,7
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0
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400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
Inte
ns
ity
(a
.u.)
Wavelegth (nm)
Er Yb
mol% mol%
0,5 0
0,5 4,0
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UCL emission significantly increases by factors
of ~ 25 (green) and ~ 150 (red) upon heat treatment
of precursor samples (glass-ceramics).
0 2 4 6 8 10 12 14
0,0
0,5
1,0
Inte
nsi
ty, a.u
Time, µs
precursor
glass-ceramics
Comparison of the UCL emission spectra
of precursor sample (top) with that of the glass ceramics (bottom).
UCL kinetics excited by 10ns pulses: black – precursor red – glass-ceramics
Energy diagram of Er3+ ions with
possible mechanisms of UCL radiation
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Low phonon energy of the medium: the energy level structure of Er ions is independent of the host materials,
however the phonon energy influences the non-radiative transitions rate.
Raman spectra of PbF2
~ 230cm-1
UCL efficiency in glass ceramics
The energy gap between the two
metastable levels 4I11/2 and 4I13/2
is about 3700 cm-1, and the
vibration stretching energy of
Si-O bond is about 1100 cm-1.
Interionic interactions of Er ions in PbF2 lattice followed by
cross-relaxation process: Er3+- Er3+ (4F7/2, 4I15/2 → 4F9/2,
4I13/2).
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UCL for bioimaging
UCL NPs are promising alternative to
traditional organic dyes.
а) imaged with a blue light filter
b) upconversion image with excitation at 980 nm
c) fluorescence image of the carbocyanine dye with excitation at 737 nm
d) merged image of the upconversion and fluorescence signals * Royal Society of Chemistry, http://dx.doi.org/10.1039/b905927j
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Optical imaging of blood vessels in the mouse ear *
Upconversion for solar technologies application
Schematic of upconversion layer application in silicon solar cell.
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Biomedical applications of CARS microscopy
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Ji-Xin Cheng, Y. Kevin Jia, X. Sunney Xie, et all, Harvard University
A.Pliss, A.Kuzmin, A.Kachynski, Paras N. Prasad, University of Buffalo
Biophotonic probing of macromolecular
transformations during apoptosis
The distribution of proteins in dividing and apoptotic HeLa cells
visualized by CARS imaging at 2928 cm−1.
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Biomedical applications of CARS microscopy
CARS microscopy for tissue imaging
Cancerous tissue recorded at
2817 cm-1 (nonresonant condition)
and 2850 cm-1 (resonance)
Healthy tissue recorded at
2849 cm-1 and 2881 cm-1
N. Vogler, T. Bocklitz, D. Akimov, A. Ramoji, Ch. Krafft, M. Schmitt, B. Dietzek, J. Popp
CARS images of pathological and physiological colon tissue
2015: Analysis of the State of the Art –
Raman Spectroscopy
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(a panel of experts in honor of Spectroscopy’s celebration of 30 years
covering the latest developments in material analysis)
DuPont representative: “Developments in coherent Raman
scattering microscopy approaches such as CARS and SRS
microscopy offer exciting potential in biological imaging applications”.
Juergen Popp, a professor at Friedrich-Schiller University Jena: “The most important recent advances have been developments in
instrumentation (SERS, CARS, TERS) that have pushed Raman
spectroscopy further into life sciences and biomedicine”.
Z.D. Schultz, an associate professor at the University of Notre Dame: “Certainly, we see the increased interest in SERS for applications and the
emergence of TERS for sub-diffraction imaging. It is really becoming
possible to talk about using Raman to investigate individual molecules”.
Thank You!
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