raman microscopy can localize vitamin e and its metabolic/oxidation products in biological samples...
TRANSCRIPT
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’
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• 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
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hn
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• 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