Principles of EPR oxygen imagingIn Vivo Oxygen Imaging Workshop University of Chicago June 25, 2012

Boris Epel

Outline

• Principles of EPR• EPR spin probes• EPR imaging principles• Image registration and tumor localization• Image visualization and statistics

What is spin

– fundamental property of electron, like electrical charge or mass.

– the rotation of a particle around some axis– Characterized by a spin quantum number, S– electron have spin ½ – In EPR, it is unpaired spins that are of importance.

Angular and Magnetic Moments

• Electron is a moving charge –it gives rise to a magnetic moment, µ

• Electron can be described as a magnetic dipole – bar magnet

μ

SNe-

Design of EPR experiment

S

N S

N

S

N

Constant field B0

Radio frequency,

Magnet will return back in some time:longitudinal relaxation

EPR is the resonant absorption of radio frequency radiation by paramagnetic systems in the presence of an applied constant magnetic field

Electron magnetic moment isn’t free to adopt an arbitrary orientation. There is a discrete set of orientations possible.

EPR spin probes

Endogenous paramagnetic species found in mammalian bodies have very short live times, broad lines, or very low concentrations.

At present, exogenous spin probes are the only practical reporters, and appropriate spin probes are the key to successful imaging.

At present, iv injections are used for the delivery of spin probe.

The development of other means of spin probe delivery (arterial and direct injection) is under way.

EPR oxymetry probes

Soluble probes

A NitroxidesB Trityl radicals • Concentration (μM) of dissolved oxygen in the

bulk volume• Resolution 1 mmHgParticulate (Solid) probes

C Lithium phthalocyanine and its derivatives

What EPR can measure

Oxygen, pO2

Redox statusAcidosis, pHThiols (GSH)Cell viabilityViscosityTissue perfusionMolecular motion

Oxygen, pO2

Operational frequency

What is the optimum frequency? - depends on sample size

Frequency ~250 MHz ~750 MHz 1-2 GHz

Penetration > 10 cm 6-8 cm 1-1.5 cm

Object Mouse, rat, rabbit

Mouse, full body

Mouse part

Biological samples contain large proportion of water. They are aqueous andhighly dielectric. Conventional EPR spectrometers operate at X-band ~9 GHz frequencies, which result in (i) ‘non-resonant’ absorption of energy (sample heating) and (ii) poor penetration of samples. Hence the frequency of the instrumentation needs to be reduced.

EPR vs MRI

MRI EPR

Magnetic field at 250 MHz 5.9 T 9 mT

Radiofrequency pulse width μsec – msec 10 – 100 nsec

Relaxation rates msec – sec nsec - μsec

Endogenous probes Water protons -

Exogenous probes - Nitroxides, trityl

Concentration >60 M < 1 mM

Stability Stable Minutes

Line width Hz – kHz 100 kHz - MHz

In Vivo EPR Oxygen Imaging

Trityl iv line

Mousecradle

Resonator

Fiducials

Bladder flushing line

Cutaneous thermocouple

Gas anesthe-sia mask

Tumor in the cast

Spectroscopy vs Parametric Imaging

Inhomogeneous distribution

High O2

Low O2

Slow relax.

Fast relax.

One dimensional Two dimensional Three-dimensional

Image Dimensionality

Imaging Principles

• Application of the linear magnetic field gradient

B

Magnetic Field Gradient

Please do not leave metal objects close to the imager

Homogeneous field, B0 Linear gradient, 00 Linear gradient, 450

‘projection’

Imaging Principles

• Application of the linear magnetic field gradient

• Obtaining multiple projections by use of different gradients orientations

B

-0.50

0.5-0.5

0

0.5

0.2

0.4

0.6

0.8

GX

(G/cm)

GY

(G/cm)

GZ (

G/c

m)

Imaging Principles

• Application of the linear magnetic field gradient

• Obtaining multiple projections by use of different gradients orientations

• Image reconstruction (filtered backprojection)

B

-0.50

0.5-0.5

0

0.5

0.2

0.4

0.6

0.8

GX

(G/cm)

GY

(G/cm)

GZ (

G/c

m)

Imager magnet

Electron Spin Echo Oxygen Imaging

T1 or T2 [s]

pO2 [torr]

Spin-probeconcentration [mM]

Amplitude [a.u.]

Deoxygenated OXO63 spin probe

pO2 = (R – R (0 torr, 0 mM) - C)

R = 1/2T1 or 2 [mG]

Image resolution

spatial 1.2 mmtemporal 10 min (2.5 min rapid protocol)pO2 1 torr

Pulse EPR: imaging sequences

Electron Spin Echo (ESE) – T2 imaging

t t

t tT

Inversion recovery (IRESE) – T1 imaging

Concentration Dependence in Vivo

T1 shows only weak dependence on spin probe concentration

T1 – based EPR imaging is the perfect method for precise oxygen imaging

Imaging Procedures

Prepare an animal

Install animal in the resonator cradle

Install resonator into imager

Inject spin probe

Image an animal

Observe pO2 imageAcquire pO2 statistics

Determine area of interest

Acquire MRI image

Register EPRI and MRI

Acquire CT/PET image

Register CT/PET and EPR

Optional

ROI

ESE and MR Image Registration

EPRI – 3mM deuterated FINLAND fiducials (~ 0.5 mm resolution)MRI – water fiducials

Haney C. et al., Concepts in Magnetic Resonance B (2008), 33, 138-144.

Mouse leg in the resonator. Polysiloxane half-cast with inserted fiducials

3D view of MRI and amplitude ESEI image registration. Fiducials are used to establish the coordinate transformation from MRI into ESE coordinate system

24

ESE and MRI Image RegistrationFiducials

MRI

EPROI pO2

Multimodality Rat Imaging

A

B

C

D

Multi-B ESE

18F-FDG PET

T2-weighted MRI

C

pO2

EPR oxygen image visualization

Region of interestin this case area of the tumor from a registered MRI image

‘Three orthogonal slices’ view

ROI and general statistics

Colormap and view adjustment

Cursor statistics

Cursor

Summary

In vivo EPR spectroscopy and imaging methodsenable noninvasive measurement and mapping of tissue pO2.

Image resolutionspatial 1.2 mmtemporal 10 min (2.5 min rapid protocol)pO2 1 torr

Direct injection of spin probe into artery

• 51 mm diameter loop-gap resonator

• 4 cm VX2 carcinoma

• Spin probe was continuously injected directly into the artery feeding the leg. This allowed us to use only 1/4 of the calculated injection dose

B. Epel et al. Medical Physics 37 (2010) 2553-2559.

Imaging of Cycling Hypoxia

Matsumoto, S., H. Yasui, et al. (2010). "Imaging Cycling Tumor Hypoxia." Cancer Research 70(24): 10019-10023.

Yasui, H., S. Matsumoto, et al. (2010). "Low-Field Magnetic Resonance Imaging to Visualize Chronic and Cycling Hypoxia in Tumor-Bearing Mice." Cancer Research 70(16): 6427-6436.

EPR Single Point ImagingImage duration 3 minutes.

Magat, J., B. F. Jordan, et al. (2010). "Noninvasive mapping of spontaneous fluctuations in tumor oxygenation using F-19 MRI." Medical Physics 37(10): 5434-5441.

19F MRIImage duration 3 minutes.

Rapid ESE Oxygen Imaging – 1 min Resolution

0 5 10 15 20 2530

35

40

45

50

55

60

N image

0 5 10 15 20 2510

15

20

25

30

35

40

N image

0 5 10 15 20 250

5

10

15

20

25

N image

13

2

1

2

3

pO2 [torr]

T2 – based pO2 imaging

Spontaneous fluctuations of pO2 in tissues

60

0

Carbogen Challenge Experiment (2.5 min Images)

The breathing gas is switched periodically between air and carbogen (95% O2 and 5% CO2)

1 cm

10

20

30

40

50

60

0 12.5 25 37.5 50 62.5 75

minutes

21 %

95 %

0

10

20

0 12.5 25 37.5 50 62.5 75

minutes

pO2 [

torr

]pO

2 [to

rr]

O2

12

1

2

pO2

T1 +T2 pO2 imaging