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

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

3. Advantages Disadvantages Minimal sample prep.Minimal sample prep. Very generalVery general Rich in informationRich in information Aqueous samplesAqueous samples “Special” techniques“Special” techniques Good spatial resolutionGood spatial resolution Simple operationSimple operation ExpensiveExpensive Fluorescence interferesFluorescence interferes Time consumingTime consuming Weak effectWeak effect Underdeveloped processing toolsUnderdeveloped processing tools

4. Raman Intensity / Arbitrary 750500100012501750300027501500 Raman Shift / cm -1 CH 3 CH 3 CH 3 O H O CH 3 CH 3CH3 CH 3 CH 3  -tocopherol  tocopherol  -tocopherol 3 C H 3 C H OH O CH 3 CH 3 CH 3 CH 3 CH 3 Raman spectroscopy can distinguish tocopherol homologues CH 3CH3 CH 3 CH 3 CH3 CH 3 CH 3 CH 3CH3

5. Multivariate Analysis Unlocks the Raman Spectrum xy00 10 11 01 MultivatiateTransform(spectrum*loading) xy00 10 11 01score 1 1 ReconstructImage 0 01 1 x y10 MultivatiateAnalysisLoading 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. Intensity of tocopherol signal vs protein or fat is proportional to its relative concentration. R2 = 0.99 = 0.99 0 20 40 60 80 100 020406080100 measured wt % aT predicted wt % aT weight % of aT in PAME 0 5 051015202530 Predicted aT [nmol/mg] R 2 = 0.95 R 2 0 5 10 15 20 25 30 051015202530 Measured aT [nmol/mg] HPLC measured aT in A549 cells 10 25 50 aT/prot[nmol/mg] 0 10 20 30 0 Supplemented aT /  M A549 cells supplemented with aT

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

8. 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

9. CH3 CH 3 CH 3 OH O CH 3CH3 CH 3 CH 3 OH O CH 3 COOH Raman spectroscopy can distinguish tocopherol metabolites and oxidation products O O CH 3 CH 3 CH 3 CH 3CH3 OH CH 3 CH 3 Raman Intensity / Arbitrary  -tocopherol quinone Raman Shift / cm -1  - carboxyethyl hydrochroman 50010001500200025003000OO CH 3CH3 CH 3 CH 3 COOH 5-nitro- γ-tocopherol O CH 3 CH 3 OH CH 3 CH 3NO2 CH 3 CH 3 CH 3 CH 3CH3 CH 3 CH 3 NO2

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

11. 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

12. 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 CH 3CH3 CH 3 CH 3 CH 3 CH 3 CH 3CH3 O OHCH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 β-tocotrienol - γ-tocotrienol Raman Intensity / Arbitrary

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

14. Raman microscopy is capable of: DetectingIdentifyingDistinguishingQuantifyingMapping Tocopherol homologues Tocopherol Oxidation products Tocopherol Metabolites Tocotrienols Raman microscopy simultaneously provides information on: Oxidative enzymes (anything with porphyrin group) Fatty acids ProteinsDNA Summary

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