Raman Spectroscopy and imaging to explore skin and hairC3$A... · Raman Spectroscopy and imaging to...

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Pr. Igor Chourpa, Université de Tours, EA4244

1

Raman Spectroscopy and imaging to explore skin and hair

Sir C.V. Raman – Nobel price inphysics in 1930

10 µm

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What is the Raman scattering effect?Incident mono-chromatic beam

Scattered light is polychromatic

Échantillon opaque

Échantillon transparent

ν0 = 1/λ0

ν0 + νvib; ν0 ; ν0 - νvib+ νvib; 0 ; - νvib

anti-StokesStokes

λν

νvib

3500

cm

-1

O-H

élon

g

3500

cm

-1

O-H

élon

g

1700

cm

-1

C=O

élon

g

1700

cm

-1

C=O

élon

g

Spectrum of bands shifted compared to theexcitation position, cm-1

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Molecule-photon energy exchange

v2v1v0

E1

E0

États virtuels

Raman Stokesscattering

hνdif = hν0 − hνvib

Raman anti-Stokesscattering

hνdif = hν0 + hνvib

Rayleigh scattering

hνdif = hν0

UV-visibleabsorption

v2v1v0

Fluorescenceemission

absorption IR

non-radiative relaxation

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How to record a Raman spectrum?

More or less simple intruments

- sample, no need of preparation

- distant analysis is possible

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Advantages in Raman :- no sample preparation (non destructive analysis, in situ)- distant analysis and in vivo analysis is allowed (optical fiber)- better spatial resolution (mixtures) and spatial resol-n (microscopy)- low frequencies are analysable (inorganic substances)

Raman spectroscopy is complementary to IR, both beingstructure-specific methods

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Difficulties in Raman

Low signal from low density samples like cells (progres of instrumentation goes on)

2800

2600

2400

2200

2000

1800

1600

Inte

nsity

(a.u

.)

400 600 800 1000 1200 1400 1600

Wavenumber (cm-1)

protein solution ~5g/L

protein powder

- Poor sensitivity: 10-1 M (RR : 10-4 M) - interference with emissions

Use of powerful lasersemitting in red and near-IR

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Raman spectrum from tissueComposed of the bands of:

– proteins– lipids– water– DNA

A. Whitley and F. Adar. Cytometry Part A (2006) 9A:880–887

lipid

protein

lipid+protein

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Raman spectrum of a proteina) Primary structure: aromatic AA content

155

4

140

2 1

414

115

6

745

107

8

165

3

411

563

667

894

941

105

0

110

7

117

3

127

0 131

3 1

336

161

5 1

585

144

8

120

6

112

9 103

1 1

004

853

8

29

641

6

20

509

1200

1000

800

600

400

200

0

Inte

nsity

(a.u

.)

400 600 800 1000 1200 1400 1600

Wavenumber (cm-1)

1563

883

756

TyrFermi doublet:853/829 increases upon Tyr exposure

Tyr

Phe

Phe

Cys

•Quantitation of the band area in the spectrum normalised at ~1450 cm-1

Trp

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Raman spectrum of a protein (BSA)a) Secondary structure (conformations)

155

4

140

2 1

414

115

6

745

107

8

411

563

667

894

941

105

0

110

7

117

3

127

0 131

3 1

336

161

5 1

585

144

8

120

6

112

9 103

1 1

004

853

8

29

641

6

20

509

1200

1000

800

600

400

200

0

Inte

nsity

(a.u

.)

400 600 800 1000 1200 1400 1600

Wavenumber (cm-1)

1563

883

756

525 55

0

1669

168316

33

Peptide bonds: Amide I

Ran

dom

coi

lsh

eetβυ(C-C)

(α) Peptide bonds:Amide III

1240

C-H

2sc

isso

ring

Bridges S-S (C-C-S-S-C-C)

568

543

521

505

25

20

15

10

5

0

450 500 550 600

g-g-gg-g-t

t-g-t

165

3 hé

lix α

124

1

131

5

134

0

165

4

hélic

e α hé

lice α

1654

hé

lix α

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Quantitative structure description

% of conformations estimated by deconvolution of the Amide I band(Ducel et al., 2006).

Inte

nsity

(a.u

.)

110

0

124

1

595

6

20

640

742

7

56 829

8

52

883

9

39

100

2

103

3

112

8

120

7

131

5

134

0

139

6

141

8

144

8

156

3

158

4

161

7

166

6

620

6

40

742

7

56

829

8

52

883

9

22

100

2

103

3

110

0

112

8

120

7

141

8

144

8

155

3

158

4 1

617

600 800 1000 1200 1400 1600Wavenumber (cm -1)

507

5

07

globulin gliadin

rand

om

rand

om

pea

alpha 1654 : 32 % 1669 : 10 % 1683 : 15 %

155

2

158

2 1

602

161

6

163

3

165

4

166

9

168

3

170

5

1550 1600 1650 1700

1654: 24 %1669: 15 %1683: 20 %

155

4

158

4

160

2

161

6

163

3

166

9

168

3

170

5

1550 1600 1650 1700 Wavenumber (cm-1)

Trp

rand

omsh

eetβ

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Raman measurements of skin in vivo

Laser 633,785 or 830 nm, 5-10 mW; obj 20X LWD or 50x LWD;

Resolution in Z (Si): 6 to 30 µmDepth max ~300 µm

Inversed microscope (immobilisation against a quartzwindow d.~2 mm)

Otherwise: optical fiber with focus/collect headEx: Skrebova N. et al., J. Biomed. Opt. 2005: 10, 014013LabRam INV, Horiba JY

Application note : horiba.com

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Raman spectra of skin in vivo

633 nm

830 nm785 nm

ν(O-H)ν(C-H)

Application note : horiba.com

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Hydration profiles

non-hydrated hydrated

After superficial application of a hydrating agent (cosmetology)pMPC (2-methacryloyloxethylphosphorylcholine polymer) with hyaluronic acid. L. Chrit et al. Biopolymers, 2006, 85: 359-369

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FT-Raman of lipids in human stratumcorneum (HSC)

FT-Raman spectra of HSC:temperature: 25- 100°C.

25°C

HSC wholeHSC no lipids

A: Band separation (cm-1) ν(CH2)s and ν(CH2)as2847 and 2931 cm-1

of HSC for the temperatures69 -75°C

B: Thermal transition diagram

B

Lawson et al., Spectrochimica Acta Part A 54 (1998) 543–558

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Carotenoids in human skin

Lutein, zeaxanthin, lycopene and its Z-isomers, and - α, -β, -γ, and ξ-carotene conc. quantitated by HPLC, compared with the 1524 cm-1 Raman intensity (counts).

Carotenoid concentration in the skincorrelates with the presence or absence of skin cancer and precancerous lesions

Exc. 488 nm: strong fluorescence: correction

Hata TR et al. J Invest. Dermatol. (2000) 115, 441–448

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Human tissue : pathologic or not?

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Raman spectroscopy of human hair:effect of chemicals (1) or of aging (2)

(1) Kuzuhara A., Biopolymers, Vol. 77, (2005) 335-344

(2) Kuzuhara et al., BiopolymersVolume 87, (2007) 134-140

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18

Molecular imaging by Raman spectral mapping

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Spectra acquisition : ~5-10 sec per spectrum in Raman

2 µm

multi-spectralmap

~900-2500spectra

Principle of spectral mapping

treatments :1) Spectrals : baseline correction

mapping of intensity / form2) Statistics

Sample video caption

Statistics >10 maps

Co-localisation with TV image

1500

1000

500

0

1000 1200 1400 1600

Wavenumber (cm-1)

Mean=62.6 Error= 5.4

10

8

6

4

2

0

Inte

nsit

y (a

.u.)

1000 1200 1400 1600Wavenumber (cm-1)

10 µm

0

10

20

30

40

50

60

70

80

naturels c12 à10%

Con

trib

utio

n R

aman

, %

Parametric mapsoverlay

Mean=37.4

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Spatial resolution of a confocal Raman micro-spectrometer

the Raman intensity follows the cuticle layer

1.0

0.5

0.0

400 600 800 1000 1200 1400 1600Wavenumber (cm-1)

-Cuticle

-Cortex

-Medulla

Spectra from different layers

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Hair X-Y mapping algorithms : decomposition intospectral models

2.0

1.5

1.0

0.5

0.0

400 600 800 1000 1200 1400 1600

40

60

40 60

60

40

20

0

40

60

40 60

200

150

100

50

0

40

60

40 60

100

50

0

5 µm

40

45

50

55

60

30 40 50 60 70 80

Region-specificspectral models

for decomposition

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Profiles of the hair composition andstructure: fitting with gaussian profiles

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Coloured gray hair

Dye penetration profiles

0

20

40

60

80

100

120

-10 0 10 20 30 40

depth, µm

Ram

an c

ontr

ibut

ion,

dye

/pro

tein

, %

10

20

30

40

50

Length Y (µm)

0 20 40 60Length X (µm)

167

4

157

9 1

525 1

476

130

6 1

279

124

7

100

8

852

731

351

420

523

5

57

616

7

00

758

8

23

911

103

8

114

7 1

193

123

1

133

6 1

380

145

4 1

541

160

5

15

10

5

0

Inte

nsit

y (a

.u.)

400 600 800 1000 1200 1400 1600

Wavenumber (cm-1)

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Raman maps by spectra deconvolutionTissue after the sub-cutaneous implant of alginates in rat

10 µm

Mean=41.8 lipidm= 6.1

Error= 3.2

800

600

400

200

0

1000 1200 1400 1600Wavenumber (cm-1)

0

10

20

30

40

50

60

70

80

naturels c12 à10%

Con

trib

utio

n R

aman

, %

Spectra fitted with 3 models « DNA » (cells), « protein » (serum),« lipid »

Mean=52.1

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Parametric maps of the same sample,corresponding to different Raman bands

2 µm

0.7

0.6

0.5

2 µm

0.5

0.4

0.3

2 µm

0.4

0.3

0.2

2 µm

0.5

0.4

2 µm

0.6

0.5

0.4

2 µm

0.5

0.4

2 µm

0.8

0.7

0.6

2 µm

0.2

0.1

2 µm

0.6

0.4

2 µm

0.6

0.5

0.4

2 µm

0.4

0.3

0.2

2 µm

0.6

0.4

2 µm

0.4

0.3

0.2

2 µm

0.2

0.1

2 µm

1.2 1 .0 0 .8 0 .6 0 .4 0 .2 0 .0

1000 1200 1400 1600

Models may bemultiple, includinginflammation,pathology, etc.

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Acknowledgments

Dr. Simone Cohen-Jonathan, Pr. Pierre Dubois, UFR de Pharmacie de ToursPhilippe Maingault, Laboratoires BrothierRégion Centre

Raman Spectroscopy and imaging to explore skin and hairC3$A... · Raman Spectroscopy and imaging to...

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