Quantitative Oxygen Imaging with Electron Paramagnetic/Spin Resonance Oxygen Imaging Howard Halpern

Why EPR? Bad News: native diffusible unpaired electrons: Rare Good News: rare native electrons mean no background signal Bad News: Need to infuse subjects with materials with stable unpaired electrons Good News: Can infuse materials which target pharmacologic compartments

Why EPR? Spectroscopic Imaging: Specific quantitative sensitivity to Oxygen, Temperature, Viscosity, pH, Thiol Each property of interest is measured with a specifically designed probe. Free Radicals can be detected as well

Why EPR? Spectroscopic Imaging: Specific quantitative sensitivity to Oxygen, Temperature, Viscosity, pH, Thiol No water background for signal imaging (vs MRI)

Why EPR? Spectroscopic Imaging: Specific quantitative sensitivity to Oxygen, Temperature, Viscosity, pH, Thiol No water background for signal imaging (vs MRI) Deep sensitivity at lower frequency (vs optical) 250 MHz vs 600 THz, 6 orders of magnitude lower 7 cm skin depth vs < 1 mm Vs typical EPR (0.009T vs 0.33T electromagnet) 250 MHz vs 10 GHz, factor of 40 lower 7 cm skin depth

Why Electron Paramagnetic (Spin) Resonance Imaging? More sensitive and specific reporter of characteristics of the solvent environment of a paramagnetic reporter The reporter can be designed to report (mostly) only one characteristic of the environment E.G.: oxygen concentration, pH, thiol concentrations We have chosen to image molecular oxygen.

Basic Interaction Magnetic Moment  let bold indicate vectors) Magnetic Field B Energy of interaction: – E = -µ∙B = -µB cos(θ)

Magnetic Moment ~ Classical Orbital Dipole –Orbit with diameter r, area a = πr 2 –Charge q moves with velocity v; Current is qv/2πr –Moment µ = a∙I = πr 2 ∙qv/2πr = qvr/2 = qmvr/2m mv×r ~ angular momentum: let mvr “=“ S β = q/2m; for electron, β e = -e/2m e (negative for e) µ B = β e S ; S is in units of ħ –Key 1/m relationship between µ and m µ B (the electron Bohr Magneton) = J/T

Magnitude of the Dipole Moments Key relationship: |µ| ~ 1/m Source of principle difference between –EPR experiment –NMR experiment This is why EPR can be done with cheap electromagnets and magnetic fields ~ 10mT not requiring superconducting magnets

Major consequence on image technique µ electron =658 µ proton (its not quite 1/m); Proton has anomalous magnetic moment due to “non point like” charge distribution (strong interaction effects) m proton = 1836 m electron Anomalous effect multiplies the magnetic moment of the proton by 2.79 so the moment ratio is 658

The Damping Term (Redfield) ∂ρ*/∂t= 1/iћ [H*(t’) 1, ρ*(0)]+ 0 (1/iћ) 2 ∫dt’[H* 1 (t),[H* 1 (t-t’), ρ*(t’)] Each of the H* 1 has a term in it with H= -µ∙B r and µ ~ q/m ∂ρ*/∂t~ (-) µ 2 ρ* This is the damping term: ρ*~exp(- µ 2 t with other terms) Thus, state lifetimes, e.g. T 2, are inversely proportional to the square of the coupling constant, µ 2 The state lifetimes, e.g. T 2 are proportional to the square of the mass, m 2

Consequences of the Damping Term The coupling of the electron to the magnetic field is 10 3 times larger than that of a water proton so that the states relax 10 6 times faster No time for Fourier Imaging techniques For CW we must use 1.Fixed stepped gradients 1.Vary both gradient direction & magnitude (3 angles) 2. Back projection reconstruction in 4-D

Imaging: Basic Strategy Constraint: Electrons relax 10 6 times faster than water protons Image Acquisition: Projection Reconstruction: Backprojection

Projection Acquisition in EPR Spectral Spatial ObjectSupport ~

=α=α

Projection Description With s(B sw, Ĝ) defined as the spectrum we get with gradients imposed

More Projection The integration of f sw is carried out over the hyperplane in 4-space by Defining with The hyperplane becomes with

And a Little More So we can write B = B sw + c tanα Ĝ∙x

So finally it’s a Projection with

Projection acquisition and image reconstruction Image reconstruction: backprojections in spectral-spatial space Resolution: δx=δB/G max THUS THE RESOLUTION OF THE IMAGE PROPORTIONAL TO δB each projection filtered and subsampled; Interpolation of Projections: number of projections x 4 with sinc(?) interpolation, Enabling for fitting Spectral Spatial Object Acquisition: Magnetic field sweep w stepped gradients (G) Projections: Angle  : tan(  =G*  L/  H

The Ultimate Object: Spectral spatial imaging

Preview: Response of Symmetric Trityl (deuterated) to Oxygen So measuring the spectrum/spectral width≈relaxation rate measures the oxygen. Imaging the spectrum: Oxygen Image

Why is oxygen important in cancer?

Known Since 1909 but in 1955 Thomlinson and Gray showed necrosis viable tissue

Dramatic differences in survival for patients with cervical cancer treated with radiation depending on mean tumor oxygenation: > or < 10 torr Hockel, et al, Ca Res 56 (1996), p 4509

This was thought to be the source of radiation resistance Hyperbaric oxygen trials √ (in human trials) Oxygen mimetic radiosensitizers √ (in animals only due to toxicity) Eppendorf electrode measurements (humans)

Intensity Modulated Radiation Therapy Sculpts radiation dose over distances of 5mm Able to spare normal tissues But tumor volumes are homogeneous; no accounting for different regions with different sensitivity

Biological imaging to enhance targeting of radiation therapy: oxygen imaging Intensity modulated radiation therapy allows sophisticated control over spatial distribution of radiation dose

Biological imaging to enhance targeting of radiation therapy: oxygen imaging Intensity modulated radiation therapy allows sophisticated control over spatial distribution of radiation dose But: some regions may contain hypoxic cells

Biological imaging to enhance targeting of radiation therapy: oxygen imaging Hypoxic cells are known to be radioresistant torr torr 0.25 torr 1.7 torr Air

Biological imaging to enhance targeting of radiation therapy: oxygen imaging Intensity modulated radiation therapy allows sophisticated control over spatial distribution of radiation dose Areas of hypoxia could be given extra dose if only we could identify them! So...

Why use radio-frequency for EPR? 250 MHz ~ 6 T MRI S/N    N~  1.2 IN LOSSY, CONDUCTIVE TISSUE

Aim: Image crucial physiologic information:oxygen Information: Communicated by reporter molecules which are injected into animals/humans. Communication through EPR spectrum. Simple spectrum in a complex biological system

Response of Symmetric Trityl (deuterated) to Oxygen No temp. dependence: < 0.05 mG/K Low viscosity dependence ~1mG/cP Self quenching a potential confounding variable -- Solved with Longitudinal Relaxation Rate Imaging

Information about fluid in which reporter molecule dissolved obtained from Spectral Parameters Spectral parameters extracted from spectral data using Spectral/Relaxation Rate Fitting Bonus: Fitting gives parameter uncertainties Standard penalty for deviation: χ 2 =(y i -fit(x i, a j )) 2 minimize this penalty by adjusting the Spectral Parameters a j e.g., spectral width … spectral width

Major improvement: Synthesis of selectively deuterated OX31 Improves 1. Oxygen sensitivity:  B  R/R  Spatial resolution for a given gradient: resolvable  x=  B/G 3. Sensitivity d d

Line widths pO 2 calibration Oxygen dependence of spin packet width obtained in a series of homogenous solutions of OX31: Since minimal viscosity dependence, aqueous=tissue

CW 4D IMAGE LINEWIDTH RESOLUTION A measure of the line width resolution is the distribution of linewidths from a homogeneous phantom. Here, the  of the distribution is 0.17  T, or ~ 3 torr pO 2 (8x8x8,projections in 20 minutes)

Line Width Fidelity: Agreement Between Image and Individually measured line-widths (1.6 mm) heterogeneous phantom. Side lines Approximately ±3 torr.

EPR spectral-spatial 4D imaging CW EPR imager at 250 MHz, with a loop-gap resonator

Dimensions of the resonator (1.5 cm thick) Sensitive Region of the resonator (total of just over cm)

Mouse Image (OX063) XY YZ PC3 tumor with 3D intensity image (Note no artifacts surrounding surface) XY and YZ pO2 slices corresponding to the slices in the volume

Oxylite probe Into tumor Validation with Oxylite fiberoptic probe (measuring fluorescence quenching by oxygen) Stereotactic Platform For Needle Location

Good agreement!

Adjacent tracks in areas of rapid oxygen variation agree with Oxylite

New insight into the oxygen status of tumors and tissues