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UCSF Hyperpolarized MR #2: DNP Physics and Hardware (2019
- 1. Hyperpolarized 13C MRI: Polarization Physics & Hardware BioE 297: Summer Seminar 07/12/2019
- 2. Overview 1. DNP Physics 2. Polarizer Hardware 3. Imaging Approaches
- 3. § In the absence of a magnetic field, no preferred orientation The Magnetic in MRI
- 4. § What happens when spins are placed into a magnetic field? • Zeeman splitting • Precess about B0 ‒ ω = γB0 Bulk Magnetization
- 5. § So why the need for a strong magnet in MRI? • Earth’s magnetic field? § Boltzmann equilibrium! Bulk Magnetization
- 6. § Boltzmann equilibrium: Boltzmann Distribution € n+ n− = exp γ!B0 kBT ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ ÷÷ ø ö çç è æ = + - = -+ -+ Tk B nn nn P B o th 2 tanh !g Net Magnetization (parallel - antiparallel) Total # of spins
- 7. SNR in MRI ( ) ( ) ( )a l xrw µ g w sin 2 24 )1 23 * 2 23 21 0 23 2 ÷ ÷ ø ö ç ç è æ ÷ ÷ ÷ ø ö ç ç ç è æ ÷ ÷ ø ö ç ç è æ + = F QV T TN k II R c!
- 8. SNR in MRI §Signal in an MR experiment is proportional to n, γ, and Pth. §Polarization is a function of γ, Bo and T: At 3T and 37°C: §Pth(1H) ≈ 9 x 10-4 % §Pth(13C) ≈ 2.3 x 10-4 % ÷÷ ø ö çç è æ = + - = -+ -+ Tk B nn nn P B o th 2 tanh !g thPnSNR gµ
- 9. Sensitivity in MRI § Water, fat have strong signal § 13C, 15N, 31P inform about biochemistry • Inherently poor SNR § Carbon is the backbone of life • 12C has no NMR signal • 13C abundance = 1.1% Bottomley, Radiology 170(1): 1989
- 10. Thermal Polarization § Polarization is a function of γ, Bo and T: § How can we increase polarization? • Increase B0 • Decrease temperature ÷÷ ø ö çç è æ = + - = -+ -+ Tk B nn nn P B o th 2 tanh !g (Upper limit of ~30-50T)
- 11. Thermal Polarization § Polarization is a function of γ, Bo and T: § How can we increase polarization? • Increase B0 • Decrease temperature ÷÷ ø ö çç è æ = + - = -+ -+ Tk B nn nn P B o th 2 tanh !g (Upper limit of ~30-50T)
- 12. Brute-Force Polarization § Force spins into high polarization at low temperature/high B0 § Pros: • Straightforward § Cons: • Engineering challenge (3He/4He refrigerator) • LONG buildup times
- 13. Dynamic Nuclear Polarization § Exogenously increase 13C polarization via Dynamic Nuclear Polarization (DNP1) § Yields 20+% 13C polarization in 1-2 hours • 105 signal enhancement 1Ardenkjær-Larsen et al ., PNAS 2003; 100(18).
- 14. DNP Hyperpolarization t = 1s
- 15. DNP Hyperpolarization t = 1s t = 65h
- 16. DNP Requirements • Transfer polarization from e- to n • Requirements: • Low temperature • High field • Free radical (unpaired electron) • 1:1000 ratio • Forms a neat glass (no crystallization) • MW source to transfer polarization from e- to nuclei • Three main mechanisms: • Solid Effect • Cross Effect • Thermal Mixing Forbidden passage (quenching): e- to local nuclei (grey). First passage (SE, CE, TM): e- to near nuclei (blue). Second passage (spin diffusion): nuclei to nuclei (green). In every passage: tuned relaxation! e-e- nuclei nuclei 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C e- 13C 13C 13C 13C 13C 13C 13C 13C 13C T1(e-)<T1(n)T1(e-)~T1(n) 13C T1(e-) short T1(n) long e- nuclei T1(e-)>T1(n)
- 17. Solid Effect • Transfer polarization from e- to n by driving a forbidden ‘flip-flop’ two- quantum transition • Electrons relax to full polarization but nuclei remain polarized; T1,e << T1,n • Apply microwaves at ѡ = ѡe ± ѡn for optimal enhancement • Dominant at low field and low EPA concentrations w 0(n) w0(e-) + w0(n) w0(e-) - w0(n) w0(e-) E 1 2 4 3 |"#$%#$& |"'()*%#$& |"#$%'()*& |"'()* %'()* &
- 18. Cross Effect w0(n) 1 2 4 3 5 6 8 7 w0(e- 1) w0(e- 2) • Three spin process: 2 electrons and 1 nucleus • Requirements: • ѡn = ѡe1 – ѡe2 • EPR linewidth that is broader than the nuclear Larmor frequency • Coupling between the two electrons • MW irradiation at one of the e- resonances leads to triple spin flips, transferring polarization from e- to n • Dominant at high field and low EPA concentrations E
- 19. Thermal Mixing • Extension of the Cross Effect • Interaction between MANY electrons and one nucleus • Apply microwaves near the electron resonance • Puts the nuclear spin system in contact with the electron spin system • Leads to dynamic cooling of the nuclear spins à increases nuclear polarization • Requires lower MW power than the solid effect • Dominant effect at intermediate field and high EPA concentration
- 20. Spin Diffusion 230 W. T. Wenckebach only once. But there are many nuclear spins which we wish to polarize. To en- able this, the electron spin has to be repolarized. This is achieved by the elec- tron spin–lattice relaxation process. Thus, DNP occurs in three steps. First, the electron spins are polarized via electron spin–lattice relaxation. Next, this polarization is transferred to the nearby nuclear spins by means of the microwave field. Finally, nuclear spin diffusion transports the polarization to the far nuclear spins. Inevitably, all these processes have to counter nuclear spin–lattice relaxation, which is not shown in Fig. 1. 3.2 The Hamiltonian Electron Spins Lattice Local Nuclear Spins Near Nuclear Spins Far Nuclear SpinsSpin Lattice Relaxation Microwave Pumping Nuclear Spin Diffusion Fig. 1. Overview of the processes involved in DNP. e- 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C e- 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13 13C 13C 13C 13C 13C13C • DNP process is efficient from electrons to near nuclei (blue) • Spin diffusion transfers polarization to remote nuclei not coupled to e- • Optimum concentration of nuclear spins • Minimum to allow spin diffusion • Maximum to prevent nuclear T1 shortening
- 21. § Solid-state polarization builds up over time § Analogous to T1 recovery • 𝑃𝑜𝑙 𝑡 = 𝑃𝑜𝑙&'( × 1 − 𝑒 -./ 01 • Want >3 𝜏b for >95% Polmax § Buildup time is ~17m for pyruvate (1.4K / 3.35T) • Typically longer for other substrates DNP: Buildup
- 22. § How do we determine which microwave frequency to use? • Run a microwave sweep (DNP spectrum) • Short polarization time + loop through μ-wave frequencies § Useful when: • Developing new probes • Using a new system (even if same field strength!) • Using a different radical • Routine check DNP: Optimum Microwave Frequency
- 23. DNP: Effect of Temperature and Field Strength • Polarizers operate at different field strengths and temperatures • Hypersense: 3.35T/1.4K • SPINlab: 3.35T/0.8K 5.00T/0.8K • Others operate at 7T-10.1T, 0.8K • In general, high field/lower temperature lead to higher polarization. • Why does this make a difference when P(e-) already 100%?
- 24. DNP: Effect of Temperature and Field Strength Forbidden passage (quenching): e- to local nuclei (grey). First passage (SE, CE, TM): e- to near nuclei (blue). Second passage (spin diffusion): nuclei to nuclei (green). In every passage: tuned relaxation! e-e- nuclei nuclei 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C 13C e- 13C 13C 13C 13C 13C 13C 13C 13C 13C T1(e-)<T1(n)T1(e-)~T1(n) 13C T1(e-) short T1(n) long e- nuclei T1(e-)>T1(n) § Comes down to interplay between T1(e-) and T1(n) § High field/low temperature predominantly leads to an increase in T1(n), increasing polarization § Doping with paramagnetic lanthanides (Gd, Ho) also increase polarization by preferentially reducing T1(e-) T1(e)/T1(n) 'Leakage Factor’ Effects of nuclear relaxation
- 25. DNP: Effect of Temperature and Field Strength § Comes down to interplay between T1(e-) and T1(n) § High field/low temperature predominantly leads to an increase in T1(n), increasing polarization § Doping with paramagnetic lanthanides (Gd, Ho) also increase polarization by preferentially reducing T1(e-) T1(e)/T1(n) 'Leakage Factor’ Effects of nuclear relaxation
- 26. DNP: Effect of Temperature and Field Strength § Sample temperature is critical to maximize polarization • Roughly 10% change / 0.1K ‒ e.g. 1.35K à 1.55K lowers SNR by 20% § Always check polarizer temperature when running an experiment!
- 27. DNP: Effect of Radical § Many different stable radicals can be used § Choice of solvent may dictate which radical can be used (i.e. some radicals are hydrophobic, etc) § Narrow linewidth radicals (like trityl) are most effective for direct polarization of low-ɣ nuclei, like 13C § Radical concentration can also influence polarization • Tradeoff between faster buildup times/increased T1(n) relaxation ‒ Data shown are at 7T
- 28. DNP: Effect of Radical § Many different stable radicals can be used § Choice of solvent may dictate which radical can be used (i.e. some radicals are hydrophobic, etc) § Narrow linewidth radicals (like trityl) are most effective for direct polarization of low-ɣ nuclei, like 13C § Radical concentration can also influence polarization • Tradeoff between faster buildup times/increased T1(n) relaxation ‒ Data shown are at 7T
- 29. DNP: Effect of Radical § Many different stable radicals can be used § Choice of solvent may dictate which radical can be used (i.e. some radicals are hydrophobic, etc) § Narrow linewidth radicals (like trityl) are most effective for direct polarization of low-ɣ nuclei, like 13C § Radical concentration can also influence polarization • Tradeoff between faster buildup times/increased T1(n) relaxation ‒ Data shown are at 7T
- 30. § Operates at 3.35T / 1.4K § Designed for small samples § 5-300μL sample volume § 4-10mL output volume § Key components: – Magnet (1) – Vacuum pump (2) – Microwave source (4) – Dissolution stick (10) § Crucial step is rapid dissolution – Frozen sample is rapidly thawed with superheated solution Hypersense Polarizer
- 31. 3.35T magnet Hypersense Polarizer 1. Take 13C enriched compound 2. Add small amount of stable unpaired electron – Typically 15mM trityl radical 3. Pressurize sample space, insert sample – Minimize this time! 4. Cool to 1.4K at 3.35T 5. Irradiate with microwaves
- 33. • The buffer is heated and pressurized • The sample space is pressurized • The sample is raised out of the liquid helium • The dissolution stick is lowered, docking with the sample holder • The solvent is injected, dissolving the sample with He chase gas, while preserving the enhanced polarization ~ 3.35T ~ 1.4K Dissolution Procedure Resulting sample: • High polarization • Physiologic temperature • Physiologic pH
- 34. Hypersense: Key Considerations 1. Always check helium gas tank • Used to pressurize sample space • Used as chase gas for dissolution
- 35. Hypersense: Key Considerations 1. Always check helium gas tank 2. Always check solid-state buildup • No solid-state signal? ‒ Sample not present ‒ Microwaves not on ‒ Polarimeter not working • Lower than expected solid-state signal? ‒ Sample spilled/incorrect volume ‒ Incorrect microwave frequency 3. Always check temperature (1.35K) • Elevated temperature à reduced polarization • Typically fixed with a bakeout (system process to remove contaminants from the insert)
- 36. Spinlab Polarizer §Two Spinlab polarizers (Surbeck Lab) • 3.35T / 0.8K à ~20% [1-13C]pyruvate polarization • 5T / 0.8K à ~45% [1-13C]pyruvate polarization § Designed for larger samples § 50 - 1500μL sample volume § 10 - 45mL output volume §Differences between Hypersense & Spinlab?
- 37. • Key components: • Magnet • Microwave source • Vacuum pump • Sorption pump Hypersense Polarizer • Main differences from Hypersense: • Simultaneous polarization (up to 4 samples) • Sample space NOT pressurized • No helium boiloff
- 38. § Sample space is not pressurized for dissolution • Minimizes cryogen consumption • With no dissolution stick, how do we retrieve and dissolve the sample? • Requires integrated fluid path for dissolution § Fluid Path: • Sample vial ßà Sample cup • Dynamic seal ßà Sample port • Coaxial tubing ßà Dissolution Line • Receiving vessel (for clinical use) ßà Flask Spinlab Polarizer: Fluid Path
- 39. § Sample space is not pressurized for dissolution • Minimizes cryogen consumption • With no dissolution stick, how do we retrieve and dissolve the sample? • Requires integrated fluid path for dissolution § Fluid Path: • Sample vial ßà Sample cup • Dynamic seal ßà Sample port • Coaxial tubing ßà Dissolution Line • Receiving vessel (for clinical use) ßà Flask Spinlab Polarizer: Fluid Path
- 40. § Sample preparation • More time consuming than the Hypersense 1. Load sample vial 2. Glue to fluid path outer lumen 3. Fill syringe with dissolution media 4. Pressure test fluid path 5. Dry fluid path 6. Load the sample § For dissolution • Open valve, hot water via inner lumen • Outer lumen: dissolved sample • No He chase gas (key difference w/ Hypersense) Spinlab Polarizer: Fluid Path
- 41. Spinlab: Key Considerations § Many of the Hypersense points are pertinent here 1. Always check solid-state buildup 2. Always check temperature (0.8K) § Need to inspect fluid path before loading 1. Liquid/moisture in inner lumen à ice blockage 2. Sample vial not glued properly à vial ruptures during dissolution 3. No output tube à can’t collect polarized solution
- 42. Hypersense SPINlab Small volumes (5-300 µL) Large volumes (50 µL – 1500 µL) 5 min sample prep time 15 min sample prep time ~1L LHe / dissolution No LHe consumption Shorter buildup time, lower polarization Longer buildup time, higher polarization 1 dissolution / hour (compound dependent) Up to 4 dissolutions / hour 24 hour duty cycle 12 hour duty cycle (+12 hour regen) High Field Lab Surbeck Lab Polarizer Comparison
- 43. SPINlab Polarizer: Clinical Applications Sterile fluid pathSpinLab + QC QC Unit: radical, oC, % polarization, pH
- 44. GMP Chemicas Low Bio- Burden Fluid Path (FP) Spinlab: Polarize FP/Dissolve /terminal sterilization Spin Pharmacy Lab Acquire mp-1H MRI and 13C MRI Data inject < 1min Recon & relate to other MR data Filled FP dispense < 24hrs
- 45. [113C]pyruvate 40,000 increase in MR signal at 3T1 1Ardenkjaer-Larsen et al. PNAS. 2003 2 Kurhanewicz et al. Neoplasia, 2011 3Nelson et al., STM, 2013 Clinical MR Scanner Volume 13C MRI in 10’s of secs3 Inject i.v. – Fast 13C MRI SPINlab Polarizer: Clinical Applications
- 46. Hyperpolarized 13C has been able to… Measure pH in tumors3 Measure cellular transport rates6 Give indicator to treatment response4 6Day et al. 20074Harris et al. 2009 Measure redox potential7 Measure blood flow and perfusion1,2 3Gallagher et al. 20082Ishii et al. 2007 Measure cardiac ischemia5 5Golman et al. 20081Mansson et al. 2006 7Keshari et al. 2011
- 47. kPL sec-1 0.016 0.004 0.008 Phase 2 Trial - 3D Dynamic HP 13C MRI in Patients Prior to and Following 6 months of Androgen Deprivation Therapy (ADT) T2 wt. Image ADC Image Overlaid kpl Image HP 13C spectral Array Pre-treatment 3 months post ADT (Lurpon + Casodex) + Doxcetaxel Patient with Gleason 4+5 cancer and lymph node metasatses
- 49. Imaging Probes
- 50. Now that we have all this signal… and all these probes… how do we image them?
- 51. Take Home Messages §DNP enables all of our pre-clinical and clinical metabolic studies • Three main mechanisms: Solid Effect, Cross Effect, and Thermal Mixing • Efficiency depends on lots of variables! EPR linewidth, B0, temperature, electron and nuclear concentration, T1e and T1n, etc §Polarizers are robust but care must be taken when operating them • Double check sample prep and the polarizer before dissolution §When in doubt, don’t be afraid to ask for help
- 52. Further Reading: A Brief List § Design and Performance of a DNP Prepolarizer Coupled to a Rodent MRI Scanner: https://onlinelibrary.wiley.com/doi/abs/10.1002/cmr.b.20099 § Increase in signal-to-noise ratio of > 10,000 times in liquid-state NMR: https://www.pnas.org/content/100/18/10158 § Dynamic Nuclear Polarization Polarizer for Sterile Use Intent: https://onlinelibrary.wiley.com/doi/full/10.1002/nbm.1682 § NMR Spectroscopy Unchained - Attaining the Highest Signal Enhancements in Dissolution Dynamic Nuclear Polarization: https://pubs.acs.org/doi/10.1021/acs.jpclett.8b01687