Raman microscopy can localize vitamin E and its

metabolic/oxidation products in biological samples

Workshop ISH-Themennetzwerks Biowirkstoffe

16th November, 2007

Centre for Clinical Raman MicroscopyQueen’s University of Belfast C

RM

C

Rene Beattie

Raman Microscopy• Irradiate sample with monochromatic radiation

hn

hn’

hn’

• For some molecules vibrate, removing energy from

radiation before scattering

Intensity

-n n’0

• Frequency difference gives vibrational spectrum

• High magnification objectives allows micron spatial

resolution

hn

hn

hn

hn

• Most scattered light unchanged (Rayleigh scattering)

Rayleigh

Advantages Disadvantages

• Minimal sample prep.

• Very general

• Rich in information

• Aqueous samples

• “Special” techniques

• Good spatial resolution

• Simple operation • Expensive

• Fluorescence interferes

• Time consuming

• Weak effect

• Underdeveloped processing tools

Ram

an I

nte

nsi

ty /

Arb

itra

ry

750500 1000 1250 1750 300027501500Raman Shift / cm-1

CH3

CH3

CH3

OH

OCH

3CH

3

CH3

CH3 CH

3

a-tocopherol

-a g tocopherol

g-tocopherol

3

CH3CH

OH

OCH

3CH

3

CH3 CH

3CH

3

Raman spectroscopy can distinguish tocopherol homologues

CH3

CH3

CH3 CH

3

CH3

CH3

CH3 CH

3CH

3

Multivariate Analysis Unlocks the Raman Spectrumx y

0 0

1 0

1 1

0 1

MultivatiateTransform

(spectrum*loading)

x y

0 0

1 0

1 1

0 1

score

-1

1

-1

1

Reconstruct

Image0

0 1

1

x

y

-1

1

0

Multivatiate

Analysis

Loading

PCA – Prinicpal Component Analysis, analyses variation within spectra onlyPLS – Projection to Latent Structures, regression method that analyses covariation between spectra and reference parameters

Intensity of tocopherol signal vs protein or fat is proportional to its relative concentration.

R2 = 0.99

0

20

40

60

80

100

0 20 40 60 80 100measured wt % aT

pre

dic

ted

wt

% a

T

weight % of aT in PAME

0

5

0 5 10 15 20 25 30

Pre

dic

ted

aT

[n

mo

l/mg

]R2 = 0.95R2 = 0.95

0

5

10

15

20

25

30

0 5 10 15 20 25 30

Measured aT [nmol/mg]

HPLC measured aT in A549 cells

10

25

50

aT/prot [nmol/mg]

0

10

20

30

0S

up

ple

men

ted

aT

/ m

M

A549 cells supplemented with aT

Raman spectroscopy can map tocopherol distribution in biological tissues

x20x100

5 mm

aT, gT aT / PAMEaT, Porphyrin, Nuclear Protein

Absolute Signal intensity Relative Signal Intensity

Brightest spot c.a. 1 pg

Mouse Lung,

10 mm section

aT concentrations were ca. 17.1 nmol/ng prot

aT was more concentrated than gT

highly localised tocopherol signals

aT was highly co-localised with saturated fatty acid

Fatty acid was close match for lung surfactant

Indicative of alveolar type II cells.

aT is relatively more concentrated in the lipids at exposed surfaces

Raman spectroscopy can map tocopherol distribution in biological tissues

CH3

CH3

CH3

OH

OCH

3

CH3

CH3

CH3

OH

OCH

3

COOH

Raman spectroscopy can distinguish tocopherol metabolites and oxidation products

O

O

CH3

CH3

CH3

CH3

CH3OH CH3 CH

3

Ram

an I

nten

sity

/ A

rbit

rary

a-tocopherol quinone

Raman Shift / cm-1

a- carboxyethyl hydrochroman

500 1000 1500 2000 2500 3000

O

O

CH3

CH3CH3 CH

3

COOH

5-nitro-γ-tocopherolO

CH3

CH3OH

CH3

CH3

NO2

CH3 CH3

CH3

CH3 CH3 CH3

CH3

NO2

Raman spectroscopy can map tocopherol metabolism and oxidation in biological tissues

x20x100

5 mm

Porphyrin, aTQ aT, aCEHCQ

5 mm

aT, aTQ

Mouse Lung,

10 mm section

aTQ concentrations were ca. 42 % that of aT

Hydroxychroman signal indicated a quinone form

Highly localised aTQ and aCEHCQ signals

Both highly co-localised with porphyrn (e.g. cytochrome)

Low co-localisation with lung surfactant and aT, but

close proximity

Raman spectroscopy can map tocopherol metabolite distribution in biological tissues

500 1 000 1 500 2 000 2 500 3 000

Raman Shift / cm-1

Raman spectroscopy can distinguish tocotrienol homologues

β-tocotrienol

γ-tocotrienol

O

OH

CH3

CH3

CH3CH3

CH3CH3CH3

CH3

O

OH

CH3CH3

CH3CH3

CH3 CH3 CH3

β-tocotrienol - γ-tocotrienol

Ram

an I

nte

nsi

ty /

Arb

itra

ry

100x

5 mm

tocotrienol unknown substance

fatty acids carbohydrates

Raman spectroscopy can map tocotrienol distribution in tobacco seeds

Raman microscopy is capable of:

Detecting IdentifyingDistinguishingQuantifyingMapping

Tocopherol homologuesTocopherol Oxidation productsTocopherol MetabolitesTocotrienols

Raman microscopy simultaneously provides information on: Oxidative enzymes (anything with porphyrin group)Fatty acidsProteinsDNA

Summary

Acknowledgements

Centre for Clinical Raman MicroscopyProf John McGarveyProf Madeleine EnnisProf Alan StittProf Peter HamiltonProf Stuart ElbornDr Bettina SchockDr Vicky KettDr Lindsay BarrettMr Ciaran Maguire

CollaboratorsDr Christine Desel (trienols)Dr Fransesco Galli (metabolites)

The Audience