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Raman microscopy can localize vitamin E and its metabolic/oxidation products in biological samples

C. C. R. M. Raman microscopy can localize vitamin E and its metabolic/oxidation products in biological samples. Workshop ISH-Themennetzwerks Biowirkstoffe. Rene Beattie. Centre for Clinical Raman Microscopy Queen’s University of Belfast. 16 th November, 2007. Raman Microscopy.

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Raman microscopy can localize vitamin E and its metabolic/oxidation products in biological samples

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  1. C C R M Raman microscopy can localize vitamin E and its metabolic/oxidation products in biological samples Workshop ISH-Themennetzwerks Biowirkstoffe Rene Beattie Centre for Clinical Raman Microscopy Queen’s University of Belfast 16th November, 2007

  2. Raman Microscopy • Irradiate sample with monochromatic radiation • Most scattered light unchanged (Rayleigh scattering) • For some molecules vibrate, removing energy from radiation before scattering • Frequency difference gives vibrational spectrum hn Rayleigh hn Intensity hn hn’ hn’ hn 0 n-n’ hn • High magnification objectives allows micron spatial resolution

  3. Advantages Disadvantages • Weak effect • Minimal sample prep. • Expensive • Simple operation • Time consuming • Very general • Fluorescence interferes • Rich in information • Good spatial resolution • Underdeveloped processing tools • Aqueous samples • “Special” techniques

  4. C H 3 C H C H C H 3 3 3 C H C H C H 3 3 3 C H 3 C H 3 H O g-tocopherol O C H C C H H 3 C H C C H H C C H H 3 3 3 3 3 3 3 H C 3 C C H H 3 3 a-gtocopherol Raman spectroscopy can distinguish tocopherol homologues C H 3 O H O H C 3 C H a-tocopherol 3 C H 3 Raman Intensity / Arbitrary 500 750 1000 1250 1500 1750 2750 3000 Raman Shift / cm-1

  5. x y x y score 1 1 0 0 1 1 0 0 1 0 0 -1 1 0 1 1 Multivatiate Analysis y 0 1 0 1 0 1 1 -1 -1 0 1 x Multivatiate Transform (spectrum*loading) Reconstruct Image Multivariate Analysis Unlocks the Raman Spectrum Loading PCA – Prinicpal Component Analysis, analyses variation within spectra only PLS – Projection to Latent Structures, regression method that analyses covariation between spectra and reference parameters

  6. HPLC measured aT in A549 cells weight % of aT in PAME 30 25 2 2 R R = 0.95 = 0.95 30 20 Predicted aT [nmol/mg] 15 20 10 10 5 5 0 0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Measured aT [nmol/mg] A549 cells supplemented with aT 100 R 2 = 0.99 aT/prot [nmol/mg] 0 25 80 60 predicted wt % aT 40 Supplemented aT / mM 20 10 50 0 0 20 40 60 80 100 measured wt % aT Intensity of tocopherol signal vs protein or fat is proportional to its relative concentration.

  7. x100 x20 Absolute Signal intensity aT,Porphyrin, Nuclear Protein aT,gT aT / PAME 5 mm Raman spectroscopy can map tocopherol distribution in biological tissues Mouse Lung, 10 mm section Relative Signal Intensity Brightest spot c.a. 1 pg

  8. Raman spectroscopy can map tocopherol distribution in biological tissues 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

  9. C H C H C H O O H 3 3 3 H C C H C C C C C C C C H H H H H H H H 3 3 3 3 3 3 3 3 3 3 H C C H C H C H C H 3 3 3 3 3 O C H 3 O O C C H H 3 3 H H O O COOH O O H C H C 3 3 C C H H 3 3 C H C H 3 3 N O N O 2 2 H O O H C C H 3 3 C H 3 Raman spectroscopy can distinguish tocopherol metabolites and oxidation products a-tocopherol quinone a- carboxyethyl hydrochroman Raman Intensity / Arbitrary COOH 5-nitro-γ-tocopherol 500 1000 1500 2000 2500 3000 Raman Shift / cm-1

  10. x100 x20 Porphyrin, aTQ aT,aCEHCQ aT,aTQ 5 mm 5 mm Raman spectroscopy can map tocopherol metabolism and oxidation in biological tissues Mouse Lung, 10 mm section

  11. Raman spectroscopy can map tocopherol metabolite distribution in biological tissues 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

  12. H O O C C C C C C C C C C C C C C H H H H H H H H H H H H H H 3 3 3 3 3 3 3 3 3 3 3 3 3 3 H O H C O 3 Raman spectroscopy can distinguish tocotrienol homologues β-tocotrienol Raman Intensity / Arbitrary γ-tocotrienol β-tocotrienol - γ-tocotrienol 500 1 000 1 500 2 000 2 500 3 000 Raman Shift / cm-1

  13. Raman spectroscopy can map tocotrienol distribution in tobacco seeds 100x 5 mm fatty acids carbohydrates tocotrienol unknown substance

  14. Summary Raman microscopy is capable of: Detecting Identifying Distinguishing Quantifying Mapping Tocopherol homologues Tocopherol Oxidation products Tocopherol Metabolites Tocotrienols Raman microscopy simultaneously provides information on: Oxidative enzymes (anything with porphyrin group) Fatty acids Proteins DNA

  15. Acknowledgements Centre for Clinical Raman Microscopy Prof John McGarvey Prof Madeleine Ennis Prof Alan Stitt Prof Peter Hamilton Prof Stuart Elborn Dr Bettina Schock Dr Vicky Kett Dr Lindsay Barrett Mr Ciaran Maguire Collaborators Dr Christine Desel (trienols) Dr Fransesco Galli (metabolites) The Audience

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