Aquaphotomics & NIR hyperspectral imaging

tools for understanding the role of water in foods

aoife.gowen@ucd.ie

School of Biosystems Engineering, University College Dublin, Ireland.

Key Questions

• What does water look like in the NIR?

• What can hyperspectral imaging show us?

• How can we use this to understand the role of water in foods?

H2O molecule

H2O molecule

• Nonlinear molecule: 3N-6 vibrational modes

• 3 vibrational degrees of freedom

Band locations from Chs 1 & 9 in Siesler , Ozaki, Kawata and Heise, 2002, Near-Infrared Spectroscopy: Principles, Instruments and Applications, Wiley GIFs from http://chemwiki.ucdavis.edu/Physical_Chemistry/Spectroscopy/Vibrational_Spectroscopy

4

νs

3756 cm-1

2662 nm

νas

3657 cm-1

2735 nm

δs

1595 cm-1

6270 nm

NIR spectrum of Water

1000 1500 2000 25000

0.5

1

1.5

2

2.5

3

3.5

4

Wavelength (nm)

Absorb

ance (

log (

1/T

))

1200 1300 1400 1500 1600 17000

0.5

1

1.5

Wavelength (nm)

Absorb

ance (

log (

1/T

))

NIR spectrum of Water

1200 1300 1400 1500 1600 1700-3

-2

-1

0

1

2

3x 10

-3

Wavelength (nm)

Absorb

ance (

log (

1/T

))

λ(nm) Tentative assignment [expected λ]

1343 2vs [1331-1371]

1412 2vas [1367-1408]

1431 2vδs+ vs [1440-1502]

1463 vas+ 2vδs [1460-1525]

1647 vas+ 2vδs [1460-1525]

Siesler , Ozaki, Kawata and Heise, 2002, Near-Infrared Spectroscopy: Principles, Instruments and Applications, Wiley

2n

d d

eri

vati

ve

NIR spectra of Water: temperature

1300 1350 1400 1450 1500 1550 1600 16500

0.5

1

1.5

Wavelength (nm)

Absorb

ance (

log (

1/T

))

1425 1430 1435 1440 1445 145030

35

40

45

50

55

60

65

70

Peak Wavelength (nm)T

em

pera

ture

“Blue Shift”: as T λ

Hydrogen Bonding

Proton Donor

Proton Acceptor

• Normal modes are perturbed when the vibrating O-H bond ‘senses’ another water molecule oriented so that its lone e’s face H of the vibrating bond.

• Frequency of vs decreases from 3707 cm-1 in the isolated molecule to 3628 cm-1 in liquid water and to 3277 cm-1 in ice.

*Adapted from Ch 2 (p. 14) in Franks, 2000, Water: A matrix of life 8

Suggested Assignments of Water Bands

*Observed in solid N2 matrix. Adapted from Luck, W.A.P. Infrared studies of Hydrogen Bonding in Pure Liquids and Solutions, in Franks, 1973, Water: A comprehensive treatise (Ch 4 p. 276-9) 9

Indicates blue shift of bands when less H bonding

between H2O molecules

Assignment 2vs 2vas

Free OH in free water molecule 1342-1383 1373-1415 Free (“Dangling”) OH 1351-1392 1381-1423 H bond in dimer 1410-1453 1455-1499 H bond in trimer 1424-1467 - H bond in tetramer 1475-1519 1533-1580

Water bands in NIR

Assignment Min Max 3vs 887 1021 vas+2vs 895 913 2vas+vs 903 921 3vas 911 1048

Assignment Min Max 2vds+vs 1440 1502 2 vs H bond in dimer 1456 1499 vas+2vds 1460 1525

Assignment Min Max vds+2vs 1098 1125 vas+vds+vs 1110 2vas+vds 1122 1150

Assignment Min Max 2vs 1331 1371 2Vas Free OH 1343 1383 vas+vs 1349 2vas Dangling OH 1352 1392 2vas 1367 1408 2vs Free OH 1374 1415 2vsDangling OH 1381 1423 2vas H bond in dimer 1411 1453 2vas H bond in trimer 1425 1467

Assignment Min Max 2vas H bond in tetramer 1475 1519 2vas H bond in polymer 1500 1544 2 vs H bond in tetramer 1534 1580 2 vs H bond in polymer 1553 1599

887-1048 nm

1098-1150 nm

1331-1467 nm

1440-1525 nm

1475-1599 nm

λ

Prof. Roumiana Tsenkova

Kobe University, Japan

Aquaphotomics

11

www.aquaphotomics.com

Aims to extract water absorbance patterns to describe biological systems e.g. animals, plants…

How to extract water absorbance pattern (WAP) from NIR Spectrum?

Spectra of water over 1 hour at “constant” temperature of 28 oC

1300 1350 1400 1450 1500 1550 16000

0.2

0.4

0.6

0.8

1

1.2

1.4

Wavelength (nm)

Lo

g (

1/T

)

12

PC1 of water over 1 hour at “constant” temperature of 28 oC

1300 1350 1400 1450 1500 1550 1600-0.15

-0.1

-0.05

0

0.05

0.1

0.15

0.2

Wavelength (nm)

PC

1

92.7 % Explained Variance

13

Expt. 1: Salts in Water

• 4 salts:

NaCl, KCl, MgCl2 or AlCl3

• Concentration range:

0.002 – 0.1 Mol.L-1

• Control: Millipore water

x 3

Gowen et al, Talanta, 2014

Experimental Design

A. Gowen (Dublin)

NIR Systems 6500 Y. Tsuchisaka (Kobe)

NIR Systems 6500

S. De Luca (Rome)

Nicolet 6700 FT-NIR

Water spectra

PC 1 Water only

PC 1 Salts

PC 2 Salts

Question

• How to remove effect of temperature in spectra?

• Extended Multiplicative Signal Correction!

...231210 IbIbXbbX

1

23

1

12

1

0ˆb

Ib

b

Ib

b

bXX

Interference Spectra

Corrected spectrum

EMSC Model

21

1300 1350 1400 1450 1500 1550 1600-0.15

-0.1

-0.05

0

0.05

0.1

0.15

0.2

Wavelength (nm)

PC

1

PC 1 EMSC Salts

LOD ~ 0.1 % (mass/mass)

How watery is food?

% water

90% 80% 70% 60% 50%

Hyperspectral Imaging

24/35

X

λ Y

Single wavelength image

0 20 40 60 80 100 120 140 160 180 2000

0.5

1

1.5

Pixel spectrum

Hyperspectral Imaging Equipment M

irror

Objective Slit Spectrograph Camera

Wavelength range (nm) 950–1650

Spectral resolution (nm) 7

Detector InGaAs

Expt. 2: Mushrooms

• 5 vibration times

• => induce surface damage

• x 24 mushrooms

• x 3 reps

Mushroom Curvature

EMSC on Mushrooms

1000 1100 1200 1300 1400 1500 16000

0.2

0.4

0.6

0.8

1

1.2

Wavelength (nm)

Re

fle

cta

nce

1000 1100 1200 1300 1400 1500 16000

0.2

0.4

0.6

0.8

1

1.2

Wavelength (nm)

EM

SC

(Re

fle

cta

nce

)

Raw Spectra: 1 mushroom EMSC Spectra: 1 mushroom

EMSC on Mushrooms

1000 1100 1200 1300 1400 1500 16000

0.5

1

1.5

2

2.5

3

Wavelength (nm)

2n

d d

eriva

tive

(E

MS

C)

1000 1100 1200 1300 1400 1500 16000.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Wavelength (nm)

2n

d d

eriva

tive

(E

MS

C)

Mean Spectra: 120 mushrooms Mean EMSC Spectra: 120 mushrooms

1000 1100 1200 1300 1400 1500 1600-0.06

-0.04

-0.02

0

0.02

0.04

0.06

0.08

Wavelength (nm)

Diffe

rence E

MS

C (

log(1

/R))

60s 120s 300s 600s

EMSC Mushrooms: difference spectra

Blue shift indicating more free water as damage proceeds

1000 1100 1200 1300 1400 1500 1600-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0.1

0.15

0.2

Wavelength (nm)

PC

1 E

MS

C (

log(1

/R))

1000 1100 1200 1300 1400 1500 1600-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0.1

0.15

0.2

Wavelength (nm)

PC

1 E

MS

C (

log(1

/R))

PC1 EMSC Mushrooms

λ (nm) 2D Tentative assignment

1055 -I 3vas

1384 -I 2vas dangling OH or 2vs Free OH in free water molecule

1454 -I 2vds+vs

or vas in dimer

Abs Peaks Mushroom

0 100 200 300 400 500 6000.175

0.18

0.185

0.19

0.195

0.2

0.205

Vibration time

Abs 1

05

5 n

m

0 100 200 300 400 500 6000.49

0.5

0.51

0.52

0.53

0.54

0.55

0.56

0.57

Vibration time

Abs 1

38

4 n

m

0 100 200 300 400 500 6000.76

0.77

0.78

0.79

0.8

0.81

0.82

0.83

0.84

0.85

Vibration time

Abs 1

45

4 n

m

3vas 2vas dangling OH 2vds+vs or vas in dimer

Undamaged Damaged

EMSC corrected images at 1384 nm

Free v’s Bound water

• Free water: can be extracted easily from foods by squeezing or cutting or pressing

• Bound water: cannot be extracted easily

• Bound water molecules can’t escape as vapor

=> Even upon dehydration food contains bound water.

Water activity

• W J Scott (1952) established that it was not water content that correlated with bacterial growth in foods, but

• Water activity: ratio of the water vapor pressure of the food to the water vapor pressure of pure water under the same conditions

Moisture Content Vs Water Activity

http://vssweb1.landfood.ubc.ca/courses/fnh/301/water/wprin.htm

Expt 3. Aqueous solutions

• Transflectance Cell

• 300 μL

• Aqueous solutions:

Salt Aw

LiCl 0.25

LiCl 0.5

NaCl 0.76

NaCl 0.92

KCl 0.98

W 1

Mean Sample Spectrum

1000 1100 1200 1300 1400 1500 16002.8

3

3.2

3.4

3.6

3.8

4

4.2

Wavelength (nm)

Log(1

/Reflecta

nce)

0.25 0.5 0.76 0.92 0.98 1

1000 1100 1200 1300 1400 1500 16000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Wavelength (nm)

Log(1

/Reflecta

nce)

0.25 0.5 0.76 0.92 0.98 1

No pretreatment EMSC

Difference Spectrum (EMSC (Water -))

900 1000 1100 1200 1300 1400 1500 1600 1700-0.02

-0.015

-0.01

-0.005

0

0.005

0.01

Wavelength (nm)

EM

SC

(W)

- E

MS

C(A

W)

0.98

0.92

0.76

900 1000 1100 1200 1300 1400 1500 1600 1700-0.02

-0.01

0

0.01

0.02

0.03

0.04

0.05

Wavelength (nm)

EM

SC

(W)

- E

MS

C(A

W)

0.5

0.25

900 1000 1100 1200 1300 1400 1500 1600 1700-0.02

-0.015

-0.01

-0.005

0

0.005

0.01

Wavelength (nm)

EM

SC

(W)

- E

MS

C(A

W)

0.98

0.92

0.76

Difference Spectrum EMSC Aw > 0.5

2D λ (nm) Tentative assignment

I+ 1104 vδs+2vs

- 1167 2vas+ vδs

+ 1384 2vas or 2vs dangling OH

M- 1433 2vas in dimer or trimer

I+ 1517 vas+ 2vδs or 2vas in tetramer

900 1000 1100 1200 1300 1400 1500 1600 1700-0.02

-0.01

0

0.01

0.02

0.03

0.04

0.05

Wavelength (nm)

EM

SC

(W)

- E

MS

C(A

W)

0.5

0.25

Difference Spectrum EMSC Aw<=0.5

2D λ (nm) Tentative assignment

-I 1405 2vas or 2vs dangling OH

-I 1489 2vds+vs or 2 vs H bond in dimer or vas+2vds

Expt. 4: Water temperature

• 1ml water pipetted on ceramic tile

• heating wire placed through centre of drop

• Glass slide placed on top =>thin layer of water

• Heating wire switched on

• Images obtained every minute for a total of 15 mins.

White tile

Glass slide

Heating wire

Expt. 2: Water temperature

1000 1100 1200 1300 1400 1500 1600-0.3

-0.2

-0.1

0

0.1

0.2

Wavelength (nm)P

C1

EM

SC

(lo

g(1

/R))

1000 1100 1200 1300 1400 1500 16000

0.5

1

1.5

2

2.5

3

3.5

4

Wavelength (nm)

EM

SC

(lo

g(1

/R))

Classic “effect of temperature”

1000 1100 1200 1300 1400 1500 1600-0.3

-0.2

-0.1

0

0.1

0.2

Wavelength (nm)

PC

1 E

MS

C (

log

(1/R

))

PC 1 EMSC

λ (nm) 2D Tentative assignment

1405 I- 2vas dangling OH

1475 I- vas+2vds

Or 2vas in tetramer

Absorbance at PC1 peaks

0 2 4 6 8 10 12 14 162.62

2.64

2.66

2.68

2.7

2.72

2.74

2.76

2.78

2.8

2.82

Time (min)

Abs 1

40

5 n

m

0 2 4 6 8 10 12 14 163

3.05

3.1

3.15

Time (min)

Abs 1

47

5 n

m

2vas dangling OH vas+2vds

or 2vas in tetramer

0 2 4 6 8 10 12 14 162.62

2.64

2.66

2.68

2.7

2.72

2.74

2.76

2.78

2.8

2.82

Time (min)

Abs 1

40

5 n

m

0 2 4 6 8 10 12 14 163

3.05

3.1

3.15

Time (min)

Abs 1

47

5 n

m

PC1 Score images

Expt. 5: Hydration of Dry foods

Wafer Soybeans

Coffee

Sample # Sample size

Coffee 33 2g

Wafer 21 0.6g

Soybeans 42 20 kernels

Hydrate to different MC & AW

Measure NIR HSI, MC, AW

AW/MC Wafer

0.2 0.3 0.4 0.5 0.6 0.72

4

6

8

10

12

14M

C

AW

AW/MC Coffee

0.2 0.3 0.4 0.5 0.6 0.74.5

5

5.5

6

6.5

7

7.5

8

8.5M

C

AW

Soybean

0.65 0.7 0.75 0.8 0.85 0.9 0.9511

12

13

14

15

16

17

18M

C

AW

Correlation between Aw, MC: wafer

1000 1100 1200 1300 1400 1500 1600-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

Wavelength (nm)

Corr

ela

tion C

oeff

icie

nt

MC

Aw

Correlation between Aw, MC: coffee

1000 1100 1200 1300 1400 1500 1600-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

Wavelength (nm)

Co

rre

latio

n C

oe

ffic

ien

t

Correlation between Aw, MC: soybean

1000 1100 1200 1300 1400 1500 1600-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

Wavelength (nm)

Corr

ela

tion C

oeff

icie

nt

MC

Aw

PC 1 EMSC Spectra

1000 1100 1200 1300 1400 1500 1600-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

Wavelength (nm)

PC

1 E

MS

C (

log

(1/R

))

Coffee

Wafer

Soybean

Difference EMSC (subtracting T0) Wafer

900 1000 1100 1200 1300 1400 1500 1600 1700-0.03

-0.02

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

Wavelength (nm)

Diffe

ren

ce

Sp

ectr

um

7.9%

10.1%

11.2%

11.3%

11.8%

11.9%

Wafer: PC1 EMSC loading

1000 1100 1200 1300 1400 1500 1600-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

Wavelength (nm)

PC

1 E

MS

C (

log

(1/R

))

1000 1100 1200 1300 1400 1500 1600-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

Wavelength (nm)

PC

1 E

MS

C (

log

(1/R

))

PC1 EMSC Wafer

2D λ (nm) Tentative assignment

-M 1097 vδs+2vs

+I 1335 2vs

-I 1412 2vs Free OH in free water molecule or 2vs Dangling OH

-M 1454 2vs in dimer or 2vas in dimer

-M 1573 2vδs in tetramer

Abs Peaks Wafer

2 4 6 8 10 12 140.66

0.665

0.67

0.675

0.68

0.685

0.69

0.695

MC

Abs 1

097 n

m

2 4 6 8 10 12 140.698

0.7

0.702

0.704

0.706

0.708

0.71

0.712

0.714

0.716

0.718

MC

Abs 1

335 n

m

2 4 6 8 10 12 140.86

0.87

0.88

0.89

0.9

0.91

0.92

0.93

MC

Abs 1

412 n

m

2 4 6 8 10 12 141.1

1.105

1.11

1.115

1.12

1.125

MC

Abs 1

454 n

m

2 4 6 8 10 12 141.012

1.014

1.016

1.018

1.02

1.022

1.024

1.026

1.028

MC

Abs 1

573 n

m

0.2 0.3 0.4 0.5 0.6 0.70.66

0.665

0.67

0.675

0.68

0.685

0.69

0.695

AW

Abs 1

097 n

m

0.2 0.3 0.4 0.5 0.6 0.70.698

0.7

0.702

0.704

0.706

0.708

0.71

0.712

0.714

0.716

0.718

AW

Abs 1

335 n

m

0.2 0.3 0.4 0.5 0.6 0.70.86

0.87

0.88

0.89

0.9

0.91

0.92

0.93

AW

Abs 1

412 n

m

0.2 0.3 0.4 0.5 0.6 0.71.1

1.105

1.11

1.115

1.12

1.125

AW

Abs 1

454 n

m

0.2 0.3 0.4 0.5 0.6 0.71.012

1.014

1.016

1.018

1.02

1.022

1.024

1.026

1.028

AW

Abs 1

573 n

m

vδs+2vs 2vs 2vs Free OH in free water molecule or 2vs Dangling OH

2vs in dimer or

2vas in dimer

2vδs in tetramer

Difference EMSC (subtracting T0) Coffee

900 1000 1100 1200 1300 1400 1500 1600 1700-0.03

-0.02

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

Wavelength (nm)

Diffe

ren

ce

Sp

ectr

um

5.1%

5.6%

5.8%

6.5%

6.6%

7.0%

Coffee – PC1 EMSC loading

1000 1100 1200 1300 1400 1500 1600-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

Wavelength (nm)

PC

1 E

MS

C (

log

(1/R

))

1000 1100 1200 1300 1400 1500 1600-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

Wavelength (nm)

PC

1 E

MS

C (

log

(1/R

))

PC1: EMSC Coffee

D2 λ (nm) Tentative assignment

M- 1097 vδs+2vs

I+ 1342 2vs or 2vas Free OH in free water molecule

I- 1419 2vs dangling OH or 2vas H bond in dimer

Abs Peaks Coffee

4.5 5 5.5 6 6.5 7 7.5 8 8.50.47

0.475

0.48

0.485

0.49

0.495

MC

Ab

s 1

09

7 n

m

4.5 5 5.5 6 6.5 7 7.5 8 8.50.392

0.394

0.396

0.398

0.4

0.402

0.404

0.406

0.408

0.41

0.412

MC

Ab

s 1

34

9 n

m

4.5 5 5.5 6 6.5 7 7.5 8 8.50.68

0.69

0.7

0.71

0.72

0.73

0.74

0.75

0.76

0.77

MC

Ab

s 1

41

9 n

m

0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.60.47

0.475

0.48

0.485

0.49

0.495

AW

Ab

s 1

09

7 n

m

0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.60.392

0.394

0.396

0.398

0.4

0.402

0.404

0.406

0.408

0.41

0.412

AW

Ab

s 1

34

9 n

m

0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.60.68

0.69

0.7

0.71

0.72

0.73

0.74

0.75

0.76

0.77

AW

Ab

s 1

41

9 n

m

2vs or 2vas Free OH in

free water molecule

2vs dangling OH or 2vas H bond in dimer

vδs+2vs

Difference EMSC (subtracting T0) Soybeans

900 1000 1100 1200 1300 1400 1500 1600 1700-0.02

-0.01

0

0.01

0.02

0.03

0.04

Wavelength (nm)

Diffe

ren

ce

Sp

ectr

um

12.6%

15.3%

14.9%

17.4%

14.6%

Soybean: PC1 EMSC loading

1000 1100 1200 1300 1400 1500 1600-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

Wavelength (nm)

PC

1 E

MS

C (

log

(1/R

))

1000 1100 1200 1300 1400 1500 1600-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

Wavelength (nm)

PC

1 E

MS

C (

log

(1/R

))

PC1 EMSC Soybean

2D λ (nm) Tentative assignment

I+ 1335 2vs

-I 1412 2vs Free OH in free water molecule or 2vs Dangling OH

-M 1587 2vδs in tetramer

Abs Peaks Soybean

11 12 13 14 15 16 17 180.875

0.88

0.885

0.89

MC

Ab

s 1

34

9 n

m

11 12 13 14 15 16 17 181.155

1.16

1.165

1.17

1.175

1.18

1.185

1.19

1.195

1.2

MC

Ab

s 1

40

5 n

m11 12 13 14 15 16 17 18

1.325

1.33

1.335

1.34

1.345

1.35

MC

Ab

s 1

58

7 n

m0.65 0.7 0.75 0.8 0.85 0.9 0.95 1

0.875

0.88

0.885

0.89

AW

Ab

s 1

34

9 n

m

0.65 0.7 0.75 0.8 0.85 0.9 0.95 11.155

1.16

1.165

1.17

1.175

1.18

1.185

1.19

1.195

1.2

AW

Ab

s 1

40

5 n

m

0.65 0.7 0.75 0.8 0.85 0.9 0.95 11.325

1.33

1.335

1.34

1.345

1.35

AWA

bs 1

58

7 n

m

2vs 2vs Free OH in free water molecule or 2vs Dangling OH

2vδs in tetramer

Key Questions

• What does water look like in the NIR?

• What can hyperspectral imaging show us?

• How can we use this to understand the role of water in foods?

Acknowledgements

67