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

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ССD, PMT: spectra, images,

E-CARS, F-CARS, P-CARS

200 400 600 800 1000 1200 1400 1600 1800

0

1000

2000

3000

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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.

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4800

5600 1538

1191

Inte

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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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nsity, a.u

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Raman shift, cm-1

Raman spectra of BR crystall in center area

10s_pin 100

1529cm-1

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1529cm-1

Raman spectra of BR crystall in "obodok"

10s_pin 100

Inte

nsity, a.u

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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

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(a

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Wavelegth (nm)

Er, Yb,

mol% mol%

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0,1 0,7

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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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