WORKSHOP, JANUARY 16 – 18, 2011, Ithaca, NY “ESR MICROSCOPY & ITS APPLICATION IN BIOMEDICAL ESR IMAGING”

Electron Spin Resonance Electron Spin Resonance Mi t ACERTMi t ACERTMicroscopy at ACERTMicroscopy at ACERT

Jack FreedJack FreedJack FreedJack FreedNational Biomedical Center for Advanced ESR Technology (ACERT)National Biomedical Center for Advanced ESR Technology (ACERT)

Department of Chemistry and Chemical BiologyDepartment of Chemistry and Chemical BiologyCornell University,Cornell University,Co e U e s ty,Co e U e s ty,Ithaca, NY, 14853Ithaca, NY, 14853

www.acert.cornell.eduwww.acert.cornell.edu

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AcertAcert ImageImage

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OutlineOutlineI.I. Introduction to ESRMIntroduction to ESRMIIII ESRM vs NMRM & Optical MicroscopyESRM vs NMRM & Optical MicroscopyII.II. ESRM vs. NMRM & Optical MicroscopyESRM vs. NMRM & Optical MicroscopyIII.III. ESRM: ESRM: Small is Better; Higher Frequencies are BetterSmall is Better; Higher Frequencies are Better

IV.IV. Some ESRM ExamplesSome ESRM ExamplesV.V. Motivating Biomedical ESR Imaging Motivating Biomedical ESR Imaging g g gg g g

StudiesStudiesVI.VI. A Medical Goal of ESRMA Medical Goal of ESRMVI.VI. A Medical Goal of ESRMA Medical Goal of ESRMVII.VII. NanoscaleNanoscale ESRMESRMVIIIVIII S & C l iS & C l iVIII.VIII. Summary & ConclusionsSummary & Conclusions

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What is ESR microscopy (ESRM)?What is ESR microscopy (ESRM)?It isIt is MRIMRI but with micron resolution !!but with micron resolution !!

ESR Microscopy (ESRM) is an

It is It is MRIMRI, but with micron resolution !!, but with micron resolution !!ESR Microscopy (ESRM) is an imaging method aiming at obtaining spatially resolved spectroscopic magneticspectroscopic magnetic resonance information from small samples with micron-scale resolution at ambient conditions Scale down for smallerresolution, at ambient conditions.The ESR signal originates from paramagnetic molecules/centers i th l th t

Scale down for smaller samples

in the sample that may occur naturally, or can be added to the sample (similar to dyes in optics or contrast agents in NMR).

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BackgroundBackgroundggWe can distinguish between 3 regimes We can distinguish between 3 regimes

with respect to the resolution inwith respect to the resolution inwith respect to the resolution in with respect to the resolution in magnetic resonance imaging:magnetic resonance imaging:

Conventional in-vivo MRI, ESRI well

MRFM, ESR-STM,

extensive

NMR microscopy well established only for >50 μm, ESRI, well

established activity, initial results

y μ ,not practical below

10 μm

Resolutionmm scale nm scaleμm scale

Ambient Conditions Extreme ConditionsAmbient Conditions(desirable for biological systems)

Extreme Conditions

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ESR MicroscopyESR MicroscopypypyBy applying the same principles of clinical MRI/ESI, p p ,but with small samples, one can approach and even exceed micron resolution atexceed micron resolution, at ambient conditions.

In ESR microscopy, unlike in clinical applications, issues such as paramagnetic “dyes”such as paramagnetic dyes and elevated RF frequencies (sample penetration depth),

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are not substantial.

Motivation for ESR MicroscopyMotivation for ESR MicroscopyBiophysicalBiophysical, , inin--vitro/exvitro/ex--vivovivo–– Intracellular Intracellular pOpO22

MiMi–– Micro Micro oror macroviscosity in living viscosity in living cells cells

–– EnzmeEnzme--mediatedmediated–– EnzmeEnzme--mediated mediated hydrolysishydrolysis

–– RedoxRedox propertiesproperties–– SelfSelf ddiffusioniffusionSelfSelf ddiffusioniffusion

measurementsmeasurementsBiomedicalBiomedical, , inin--vitrovitro–– Molecular imagingMolecular imaging

10-100 μm

g gg g–– Tissue Tissue

characterizationcharacterization–– Drug release Drug release μ

Taken from http://genetic-identity.com/Basic_Genetics/basic_genetics.html

processesprocesses

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Motivation:Motivation:Motivation:Motivation:ESR Microscopy is a ESR Microscopy is a

complementary tool for complementary tool for interpreting the results ofinterpreting the results ofinterpreting the results of interpreting the results of larger scale ESR imaging:larger scale ESR imaging:Distribution of Contrast AgentsDistribution of Contrast AgentsNonNon--Uniformity in ConcentrationUniformity in ConcentrationyyNonNon--Uniformity in Oxygen ContentUniformity in Oxygen Content

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ESR vs. NMR MicroscopyESR vs. NMR MicroscopyESRESR moremore sensitivesensitive perper spinspin (higher(higher magneticmagnetic moment)moment)..ESRESR resonatorsresonators havehave higherhigher QQ thanthan NMRNMR micromicro--coilscoils (Q(Qgg ((~~10001000 vsvs.. ~~1010))..ESRESR resolutionresolution isis notnot limitedlimited byby diffusiondiffusion (e(e..gg.. diffusiondiffusionlimitlimit inin ESRESR ~~200200nmnm vsvs ~~ 1010μμmm inin NMR)NMR)limitlimit inin ESRESR 200200nmnm vsvs.. 1010μμmm inin NMR)NMR)

K. Golman et. al., JMR 133, pp. 1-12, 1998

ResolutionResolution betterbetter thanthan [[11μμm]m]33 inin severalseveral minutesminutes..MoreMore sensitivesensitive toto dynamicdynamic effectseffects..yyUniqueUnique probesprobes withoutwithout “background”“background” protonproton signalsignal(radicals(radicals areare addedadded toto thethe sample)sample)..MuchMuch lessless expensiveexpensive magnetmagnet technologytechnologyMuchMuch lessless expensiveexpensive magnetmagnet technologytechnology..

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ESR vs. Optical MicroscopyESR vs. Optical Microscopy3D imaging without the effects of absorption/reflection/scattering.Accurate voxel assignment.Non transparent and thick samplesNon-transparent and thick samples.Flow, self diffusion tensor.Oxygen concentration-sensitive probes; Reactive oxygen and nitrogen species with high chemical specificityand nitrogen species with high chemical specificity.Dynamic effects on lineshape.Magnetic resonance parameters (T1/T2) correlated with cell/tissue state.

Richer sample information, improved clinical conclusiveness, can be correlated with animals in-vivo imaging.Typical acquisition time of min, compared to sec.St t l/ h l i l i f ti h ld b bt i d

1010

Structural/morphological information should be obtained from optical image.

Sample/Resonator SizeSample/Resonator Size

Frequency: from ~1 GH t 35 GH

9 9 GHz GHz cavitycavityGHz to ~35 GHz →

large increase in SNR due to Boltzmann and

cavity cavity resonatorresonator

Faraday factors.Resonator active volume: from ~1000 mm3 to ~ 0.06 mm3 → ~130 times increase

9 9 GHz high GHz high permittivity permittivity dielectricdielectric~130 times increase

in the “small voxel SNR”.

dielectric dielectric resonatorresonator

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Pulse electronics & Spin ProbesPulse electronics & Spin Probes

1. Strong CW and pulsed gradients due to uniquegradients due to unique miniature resonator geometry and drivers → gradients of more than 50gradients of more than 50 T/m.

2. Unique spin probes, Trityl, LiPc derivativesLiPc derivatives.

3. Pulse operation with state-of-the-art averager.

4. New system architectural design and efficient image acquisition algorithms.

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The experimental setupThe experimental setup

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The Imaging ProbeThe Imaging Probe

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The The IImaging Probemaging Probe at 16GHzat 16GHz

3 Axes Gradient Coils

High Q Resonator

Flat sampleFlat sample holders

1 cmThermocouple1515

Pulse experimental results, Pulse experimental results, 1616 GHzGHzLiPcLiPc crystalcrystal (( 101088 Spins inSpins in m3))LiPcLiPc crystalcrystal ( ~( ~10108 8 Spins in Spins in μm3).).50 50 avg. per gradient set ( ~avg. per gradient set ( ~1919minmin.. of of data collection)data collection)Resolution of ~Resolution of ~00..77××00..7575××77..5 5 μm3 ~ca

MW

Gy > 45 T/m → 1.1 μm

900 1800 echo signal

1.0 μs

4μm3..Image size of Image size of 250250xx250250xx6464 voxelsvoxels..SNR ~SNR ~1717//voxelvoxel..

Gx > 50 T/m → 1.3 μm

Gy 45 T/m 1.1 μm

0.7 μs

Gz > 1.5 T/m → 8 μm

Typical Pulse Imaging Scheme

Optical image of LiPc (55x67x15μm3)

F.T.

Time domain signal Reconstructed image ESR Microscopy image of LiPc

Applications for Biomedical ResearchApplications for Biomedical Research1 mM trityl solution in a micro-fluidic device for cell imaging applications.~26 min of acquisition time.Resolution of 7×8×10 μm3.Image size of 128×128×128 voxels.SNR ~30 (~3×108 spins/voxel).

Optical Image of a phantom sample filled with Trityl solution

Cells, suspended in the solution can be introduced to the fluidic channel for imagingintroduced to the fluidic channel for imaging

ESRM image of phantom sampleMouse leg tissue bearing a PC3 Tumor in the sample holder

0.5mm

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Initial Work on Applications Initial Work on Applications 16 16 GHz Pulsed ProbeGHz Pulsed Probe Air Bubbles

DrugDrug releaserelease:: inin--vitrovitro observationobservation ofof slowslowreleaserelease ofof trityltrityl fromfrom polymerpolymer micromicro--spheresspheres andand relatedrelated phenomenaphenomena

Sphere 1Air Bubbles

spheres,spheres, andand relatedrelated phenomenaphenomena..HereHere wewe observedobserved thethe TT22 weightedweighted imageimage..

Sphere 2Sphere 2Resonator

Shorter T2, corresponds tocorresponds to “effective” viscosity of ~10 cP inside thecP inside the sphere.

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Mouse leg cancerMouse leg cancerMouse leg cancerMouse leg cancer

1919With H. Halpern

EPR Imaging at 1.2GHz by J. Zweier

EPR image of a live mouse fed with paramagnetic activated carbon A Surface rendering of the 3D EPR imageEPR image of a live mouse fed with paramagnetic activated carbon. A. Surface rendering of the 3D EPR image superimposed with 3D rendering of the NMR image. B. Slices from the same 3D image. EPR images rendered in color superimposed with corresponding slices of NMR image rendered in gray scale. The parameters used for the EPRI acquisition were as follows: frequency 1.19 GHz, scan width 1.2 mT, microwave power ~300 mW, modulation amplitude 0.1 mT, maximum field gradient 1.5 mT/cm along X and Z directions, and 0.1 mT/cm along Y, scan time 2.6 s, 32×32 projections. The MRI data was acquired at 0.38 T magnetic field, using gradient echo pulse sequence with the following parameters: 12.5 kHz bandwidth, matrix 128 × 128 × 128, repetition time TR = 70 ms, echo delay time TE = 10 ms, number of excitations (NEX) = 1, field of view (FOV) in plane 50 × 50 mm, FOV in slice direction 130 mm (transverse slice thickness of 1.01 mm), flip angle 65 degree. J Magn Reson. Author manuscript; available in PMC 2009 July 22.

Published in final edited form as: J Magn Reson. 2007 September; 188(1): 68–73.

CW EPRI of a Tumor bearing mouse at 300MHz by Krishna (7.5min measuring time)

“Dancing with the Electrons: Time-Domain and CW IN Vivo EPR Imaging,” Krishna, Subramanian, et. al., Magnetic Resonance Insights, 2008.

EPR Imaging at 250MHz by Halpern

(a) Co-registration of electron paramagnetic resonance fiducial images (red wire mesh) and magnetic resonance imaging (MRI) fiducial images (green shaded surface). Also shown in green is whole leg surface from MRI. (b) Comparison of tumor contours from MRI (red contour) and stereotactic needle (“tumor touch”, yellow contour) in electron paramagnetic resonance ( , y ) p gimage slice. Pixel volume (0.66 m). (spatial resolution of ~1mm), “Electron Paramagnetic Resonance Oxygen Image Hypoxic Fraction Plus Radiation Dose Strongly Correlates With Tumor Cure in FSa Fibrosarcomas,” International Journal of Radiation Oncology Biology Physics, 2008

EPR Imaging at 250MHz by Halpern

Surface rendering of the intensity from an EPR image is shown in tan. Tissue pO2 values are represented as colors in the orthogonal planes. A quantitative color bar relates color to pO2.p g p q p

EPR Dosimetry by Swartz & Grinberg

EPR dosimetry in fingernails Oximetry for Peripheral Vascular Disease

Tooth Dosimetry

PEDRI Images at 567MHz by Kuppusamy

PEDRI images of a living mouse infused with 3-CP. (a) The posterior slice of 3-CP: (a-f) EPR off, (g-l) 12 W EPR irradiation power. (b) The anterior slice of TEMPONE: (a-f) EPR off, (g-l) 12 W EPR irradiation power. The acquisition parameters were described under Materials and Methods. “In vivo proton electron double resonance imaging of the distribution and clearance of nitroxide radicals in mice,” by Haihong Li, Guanglong He Yuanmu Deng Periannan Kuppusamy Jay L Zweier * Magnetic Resonance inHe, Yuanmu Deng, Periannan Kuppusamy, Jay L. Zweier , Magnetic Resonance in Medicine, 2006

Prostate Tissue: Motivation & ObjectiveProstate cancer is one of the most common cancers in Prostate cancer is one of the most common cancers in men.men.Information on the tissue architecture can used to Information on the tissue architecture can used to di ti i h li t ( ) ti f b idi ti i h li t ( ) ti f b idistinguish malignant (or cancerous) tissue from benign distinguish malignant (or cancerous) tissue from benign (or normal) tissue.(or normal) tissue.Tissue structure including the size of the prostate Tissue structure including the size of the prostate capsule nerve distribution along the prostatic capsulecapsule nerve distribution along the prostatic capsulecapsule, nerve distribution along the prostatic capsule capsule, nerve distribution along the prostatic capsule etc. is also important to determine the prostate cancer etc. is also important to determine the prostate cancer staging, staging, & & the possibility of local treatment.the possibility of local treatment.** Prostate cancer staging is determined based on the** Prostate cancer staging is determined based on the Prostate cancer staging is determined based on the Prostate cancer staging is determined based on the

size of tumor, the extent of invaded lymph nodes, size of tumor, the extent of invaded lymph nodes, & & metastasis (or distant spread).metastasis (or distant spread).

ESRM can provide images of tissue structure with aESRM can provide images of tissue structure with aESRM can provide images of tissue structure with a ESRM can provide images of tissue structure with a resolution of a few resolution of a few micrometers.micrometers.Oxygen distribution image of the fresh prostate tissue is Oxygen distribution image of the fresh prostate tissue is also a possibility that can provide further information for also a possibility that can provide further information for p y pp y pthe determination of cancer, etcthe determination of cancer, etc..

Prostate Tissue Sample Preparationp pFor initial experiments, normal prostate tissues are prepared as follows:

1. Biopsied tissue was frozen either w/o using cryoprotectant medium (named: dry sample) or in OCT medium (2 to 1 of OCT and sucrose). **OCT (optimal cutting temperature) compound is water-soluble glycols & resins.water soluble glycols & resins.

2. Tissue was sliced in 60 micron thickness by cryo-sectioning & immersed in 10% of Formaldehyde for fixation & transporting from campus-to-campus at room temperature

3. Tissue samples in Formaldehyde were brought into the anaerobic chamber (oxygen is < 1000ppm).

4. Tissue was rinsed 3-4 times with Trityl solution (1.3mM of OX63 d24 in 5mM NaOH T2 ~3 7 microseconds) & any excessOX63_d24 in 5mM NaOH, T2 3.7 microseconds) , & any excess amount of Trityl was removed by using Kimberly tissue & loaded in the flat capillary. The sample was immediately sealed with UV curing epoxy for imaging experiment.

5 Experiments were performed at 20°C5. Experiments were performed at 20 C.

ESRM Image

Optical image of the tissue sample ESRM image of the tissue sampleExperimental condition:

Rep rate: 8KHz with 8 step phase cycling, Total average:200, Image size: 200x200x64, p p p y g, g , g ,

Total experimental time ~1.1hour (Net data acq. Time ~10min), Temp: 20 deg. C, Overall T2 ~1.4 microseconds. Estimated resolution ~6.25x6.25x20μm Dr. Tewari, Weill/Cornell

LGR at LGR at 1515GHzGHz

r = 750umzw = 650umz = 1000umt = 100umd

d = 300um (distance from LGR to coupling loop)L~1.326nH, C~0.0730455 pF

wrt

- expected resonant frequency ~16.17GHzR~0.17Ohm

Critically coupled loaded Q~400

Discrete Port 1 with port pimpedance of 50Ohm

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ScannedScanned--Probe Detection of Electron Spin Probe Detection of Electron Spin Resonance from a Resonance from a NitroxideNitroxide Spin ProbeSpin Probepp

Scanned-probe ESR experiment schematic.

A microstrip line half-wave resonator delivers a transverse magneticdelivers a transverse magnetic field, B 1, oscillating at 17.7 GHz.

In the center of the resonator, the microwave field oscillates along the x direction.

A longitudinal Zeeman field of magnitude B 0 ≈ 0.6 T is applied along the z axis.

The high-compliance cantilever has its long axis along y & oscillates in the x direction. The cantilever's 4 μm-diameter nickel tip was affixed by hand.

The sample is a 230 nm thick film of 40 From: Scanned-probe Detection of ElectronThe sample is a 230 nm-thick film of 40 mM TEMPAMINE in perdeuterated polystyrene, coated with 20 nm of gold. The sample film was spin-coated onto a 250 μm-thick quartz

From: Scanned-probe, Detection of Electron Spin Resonance from a Nitroxide Spin Probe, E. Moore, S.-G. Lee, S. Hickman, S. Wright, L.E.Harrell, P.P. Borbat, J.H. Freed, J.A. Marohn PNAS 106, 22251-22256 (2009)

wafer. (For clarity, sample and substrate are

not drawn to scale. )3030

Summary & ASummary & Applicationspplications1) ESR microscopy has several virtues over

NMR and complements optical and fluorescence microscopyfluorescence microscopy.

2) We demonstrated a pulsed 3D ESR microscope operating at 16 GHz withmicroscope operating at 16 GHz with resolution of ~1 μm.

3) There are a variety of ESRI applications3) There are a variety of ESRI applications for ESRM.

4) Further improvements are expected to ) p pincrease resolution to sub-micron.

5) True nano-scale awaits developments in M ti R F MiMagnetic Resonance Force Microscopy.

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ESRM GROUPESRM GROUPESRM GROUPESRM GROUPPeter BorbatPeter BorbatPeter BorbatPeter BorbatCurt Curt DunnamDunnamBoris DzikovskiBoris Dzikovski

Past Contributors:Past Contributors:Past Contributors:Past Contributors:Aharon BlankAharon BlankChang Seok ShinChang Seok Shin

3232FUNDED BY NIH/NCRR

The The The The EndEnd