1. Fund BioImag 2013 10-1 10: MR spectroscopy 1.How can the Bloch equations be used to describe the effect of T 1 on the magnetization ? 2.How can sensitivity be optimized ? 3.What nuclear property allows to distinguish the signal from different molecules ? 4.What is MR spectroscopy ? After this week you 1.can calculate the effect of multiple RF pulses on longitudinal magnetization 2.know the definition of Ernst angle 3.Understand the two basic mechanisms by which electrons influence the precession frequency of nuclear magnetization 4.Know the definition of chemical shift 5.Know how and under what molecular conditions NMR spectroscopy can provide non-invasive biochemical information

2. Fund BioImag 2013 10-2 Fat and water in MRI: Examples Retrobulbar fat Fat suppressed breast MRI (cancer does not contain fat) Fatty liver

3. Fund BioImag 2013 10-3 Why is NMR spectroscopy important for (bio)chemists ? Routine tool –Non-destructive analysis of samples » Wine in bottles » Synthesis outcome control Other applications –Structure determination in solution –Molecule dynamics –Chemical reactions in situ Kurt Wüthrich Biophysicist, 2002 Insert sample Analyze data Determine structure

4. Fund BioImag 2013 10-4 10-1. What is the effect of relaxation on M(t) ? Bloch equations revisited flip angle  =  B 1  : The effect of T 1 and T 2 on the signal : Effect of T 2 Effect of T 1 M z (0) TR, TI  T1T1 T2T2 M xy (0) Longitudinal coherence: Effect of T 1 on signal depends on prior RF manipulations Longitudinal coherence: Effect of T 1 on signal depends on prior RF manipulations Longitudinal coherence

5. Fund BioImag 2013 10-5 What are the optimal conditions to measure T 1 ? Inversion recovery Measured signal TI Optimal choice of  for measuring T 1 ? Use noise error propagation calculation (Lesson 1) sin  =0   =  Converts M z to M xy (signal) TI = T 1 Optimal t=TI to detect changes in T 1 ? Inversion-Recovery Multipulse experiment with two RF pulses Usual experiment to measure T 1 (  =  )   /2

6. Fund BioImag 2013 10-6 10-2. When is SNR (sensitivity) optimal ? Situation: RF pulses  applied every TR seconds n timesQuestion: M xy (=signal) maximal ?  Calculate the optimum flip angle  =f(TR) Immediately after n th TR After RF Flip  After T 1 recovery Assume M xy =M  ~0 after TR seconds In equilibrium (steady-state condition): M z (n+1)=M z (n)=M z Equilibrium transverse Magnetization: M xy (0)=M z sin  TR  M xy (0)  M z (0) M z (n) M z (n+1)

7. Fund BioImag 2013 10-7 How does the signal depend on TR, T 1 and flip angle ? Ernst Angle  E M xy (signal) → maximum at  E Where  E = Ernst Angle Richard Ernst Physical Chemist 1991 dM xy /d  =0: Ernst Angle vs TR/T 1 EE

8. Fund BioImag 2013 10-8 10-3. What role does the chemical environment play? Chemical shift: Effect of B 0 on e-cloud H Proton: nucleus of 1 H H Proton + e cloud: 1 H atom H Reorientation of e cloud in magnetic field: e produce a small magnetic field  B at the proton: B0B0 Resonance frequency of the pure proton: L=B0L=B0  =  L (1+  ) (Larmor frequency) Chemical shift  H O Hydrogen e - H Little shielding HOHCH H C More shielding Nearby electronegative atoms (e.g. O, Cl): attract electrons → lower electron density → deshielding of nearby H → Resonance frequency is higher in OH than CH  B=  B 0

9. Fund BioImag 2013 10-9 How is chemical shift  linked to electronegativity ? Example: Protons Compound, CH 3 X CH 3 F CH 3 O H CH 3 Cl CH 3 Br CH 3 ICH 4 (CH 3 ) 4 Si XFOClBrIHSi Electronegativity of X 4.03.53.12.82.52.11.8 Chemical shift, δ / ppm 4.263.43.052.682.160.230  =(  -  L ref )10 6 /  L ref 0 ppm is defined by resonance frequency of referencecompound  L ref (e.g. tetramethylsilane (TMS) for 1 H)

10. Fund BioImag 2013 10-10 10-4. How can we measure chemical shift ? MR spectroscopy Free induction decay (FID) signal: distinguish resonance frequency → Fourier transformation (real part only): Area of resonance  M(0)  number of nuclei  concentration (if relaxation can be neglected) == Integral (area) Ratio: 1:2:3 (relative number of spins in molecule) 1 2 3 Example: Ethanol 1/  T 2

11. Fund BioImag 2013 10-11 Ex. illustration of chemical proximity (triplet & quartet) Hyperfine splitting nucleus  tiny magnetic field linked to its dipole: changes polarity if spin is “up” or “down”  affects the e cloud in the molecule → alters the magnetic field at a nearby nucleus: ↑ H ↓ H CH 2 group → four combinations (with equal probability) : up-up, down-down, up-down, down-up (The latter two produce the same magnetic field) → methyl triplet (relative intensity ratio 1:2:1) Nearby spin-1/2: 1 H resonance will split into two of equal magnitude (doublet) Example: Ethanol 1 1 2 B0B0 e - cloud nuclear spin-induced field 1 1 3 3

12. Fund BioImag 2013 10-12 Ex. 31 P NMR spectroscopy Phosphate metabolism is at the heart of cellular energetics 1: phosphocreatine (PCr) 13: phospho-ethanolamine (PE) 14: phosphocholine (PC) 15: glycerophosphocholine (GPC) 22: glycerophosphoethanolamie(GPE) 23: ATP 24: inorganic phosphate (Pi) 25:dinucleotides (NAD(P)[H]) 1 23 24 13,14 15,22 25 Also measured: Intracellular pH Creatine kinase activity ATPase activity

13. Fund BioImag 2013 10-13 What can MR spectroscopy measure ? Concentration of biochemical compounds –signal is proportional to the number of spins present, i.e. concentration After FT, integrate (measure the area of the peak). Rules for a compound to be detectable: 1.Concentration > 1mM 2.Water-soluble compounds (mobile) 3. 1 H is most sensitive nucleus (gyromagnetic ratio) Concentration of biochemical compounds –signal is proportional to the number of spins present, i.e. concentration After FT, integrate (measure the area of the peak). Rules for a compound to be detectable: 1.Concentration > 1mM 2.Water-soluble compounds (mobile) 3. 1 H is most sensitive nucleus (gyromagnetic ratio) Spatial Resolution Voxel volume ~ 1/Signal Water (80M 1 H concentration) ~1mm (human) ~50µm (rodent) Biochemical compounds (~mM concentration) ~ cm (human) ~ mm (rodent) Why is spatial resolution better for rodent studies ? Induced emf  depends on RF coil size (Lesson 9)

14. Fund BioImag 2013 10-14 How can the huge water signal be suppressed in 1 H NMR ? NB. Resonance suppression: 1.Minimize M z : “selective” 90 0 pulse applied on- resonance on the signal to be suppressed. 2.Selectivity achieved by using weak B 1 (lecture 9), i.e. long RF pulse. 3.90 0 (selective) followed by  0 for excitation and detection [assume the suppressed signal is dephased (see Lecture 11)] Full signal (no suppression) Scaled signal (no suppression) Scaled signal (with suppression) Water + Phe suppres sion fat water  90 0 M z (water)=0

15. Fund BioImag 2013 10-15 54321 (ppm) Ex. Proton spectroscopy of the brain Biochemical compounds detectable in vivo 1 9 8 5 4 3 12,13 17 6,7,18 2 10 1,2 16 6 6,7 7 20 14,15 21 11,20 19 Energy metabolism: 1: phosphocreatine (PCr) 2: creatine (Cr) 3: glucose (Glc) 4: lactate (Lac) 5: alanine (Ala) Neurotransmission: 6: glutamate (Glu) 7: glutamine (Gln) 8: GABA 9: N-acetyl-aspartyl- glutamate (NAAG) 10: aspartate (Asp) 11: glycine (Gly) 12: serine (Ser) Membrane metabolism: 13: phospho-ethanolamine (PE) 14: phosphocholine (PC) 15: glycerophosphocholine (GPC) 16: N-acetyl-aspartate (NAA) Antioxidants/osmolytes: 17: glutathione (GSH) 18: vitamin C (Asc) 19: taurine (Tau) 20: myo-inositol (Ins) 21: scyllo-inositol (s-Ins) Incompletely suppressed H 2 O signal 14,1516 3 8

16. Fund BioImag 2013 10-16 How can biochemical compounds be measured in vivo ? Analysis of 1 H NMR spectroscopy of the brain in vivo NAA 4.5ppm4.03.53.02.52.01.51.0 fit residual Lac Glu Gln

17. Fund BioImag 2013 10-17 Ex. Metabolic phenotyping of transgenic animals R6/2 mouse (human Huntingtin gene knockin) at wk 8 0 2 4 6 8 10 12 14 16 Ala Asp Cr PCr GABA Glc Gln Glu GSH Ins Lac NAA NAAG PE Tau concentrations (µmol/g) Wild-typeHD transgenic Standard deviation N=7

18. Fund BioImag 2013 10-18 Brain tumor 1H1H 31 P Muscle Example: Human brain and muscle

19. Fund BioImag 2013 10-19 Ex. 1 H NMR spectroscopy in vivo Mouse brain Mouse model of human glioma Mouse model of stroke