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Direct and Indirect Magnetic Force Microscopy (MFM) in

HistologyGunjan Agarwal

Mechanical and Aerospace Engineering

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Biomagnetism

Sources

– Super-paramagnetic: Iron

• Ferritin, transferritin, hemoglobin, NTBI

– Diamagnetic: Calcium

Tissues

– Liver (ferrihydrite)

– Spleen (ferrihydrite)

– Serum (?)

– Acute injury (ferrihydrite)

– Chronic plaques (maghemite)

Ferritin

Protein shell,d~12nm

Iron core,d~ 8nm

Apoferritin

IronAtoms

Ferritin

The major iron storage protein in biological systems

Globular protein complex (480 kDa): 24 subunits including heavy (H-Ferritin) and light (L-Ferritin) chains

Exists as:Apoferritin (without iron)Holoferritin (with iron)Iron core is mostly ferrihydrite, up to 4500 atomsSuperparamagnetic in nature

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Diagnostic Tests for Iron

Non-invasive:

• MRI (liver, spleen, plaques)

• Biosusceptometry

• Serum proteins (ferritin, transferrin)

• Serum iron

Problems:• Mismatch between Non-

invasive and invasive mapping

• Total iron is measured (size, density, oxidation state unknown)

• Total ferritin is measured (apo vs. holo ferritin unknown)

• Mismatch between iron vs. ferritin mapping

• Chemical environment is different in non vs. invasive imaging (eg. fixatives)

Invasive

• Histochemical stains:

– Perl’s: (Fe3+)

– Turnbull: (Fe2+),

• Immunohistochemistry (ferritin)

• Analytical TEM

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Our goal

Bridge the gap between Invasive and Non-invasive approaches for iron characterization in tissues

Non-invasive (Imaging)(MRI, Biosusceptometry)

Invasive (Histology)(Histochemical stains, TEM)

Magnetic properties Chemical properties??

Magnetic Force Microscopy

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Magnetic Force Microscopy

•High sensitivity

•High Spatial resolution

•Label free

•Available on Commercial AFMs

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Direct MFM of a Magnetic Tape

Savla et al, Israel J. of Chem., 2008

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Direct MFM of Ferritin

Bprobe

Bscanner

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Direct MFM of Ferritin and Apoferritin

Nocera et al,

Nanotechnology (2014)

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MFM in Histology

Experimental system: Rodent spleen and spinal cord

Experimental approach:Light microscopy• Tissue sections (~ 5 µm thick, in 4% PF) on glass• Histochemical stain (Perl’s)• Magnetic Force Microscopy (MFM)

- ASYMFMHM probes- Multimode AFM (Nanoscope 3a controller)

Electron microscopy• Transmission electron microscopy (TEM)• Energy Dispersive Spectroscopy (EDS)• Electron energy loss spectroscopy (EELS)

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MFM of Healthy Tissue

Objectives:

Effect of chemical fixatives

Detection of MFM signal

Verification of MFM signal

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Rodent spleen: Perl’s staining

1 mm

Area (µm2)

100 µm

100 µm

A B

CD

Size(z) of intensely stained regions ~ 20 to 40 µm2

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Rodent spleen (effect of fixative): Perl’s staining

Unfixed Fixed

*

0

0.2

0.4

0.6

0.8

4.6-23 23-46 46-69 69-92 92-460Perc

ent o

f Par

ticle

s

Particle Area (µm2)

UnfixedFixed

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MFM of fixed and unfixed tissue

200

FixedUnfixed

200 nm

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Detection of MFM signal

Bprobe

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Long range detection

z = 30 50 90 nm

z = 30 nm z = 50 nmz = 40 nm

AFM

pro

beM

FM p

robe

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Verification of MFM signal: TEM

1 µm

250 nm

BAA

5 µm

C C

0

20

40

60

80

100

120

140

160

0 0.5 1

Pixe

l Int

ensi

ty (0

-255

)

Lysosome area (µm2)

D

Size(z) of iron-rich lysosomes < 0.2 µm2

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Energy Dispersive Spectroscopy

Energy (keV)

l2

l4

l6

l8

l10

Coun

ts

200 -

400 -

800 -

1000 -

600 -

C

Fe Kα = 6.4 keVFe Kβ = 7.054 keV

• MFM signal obtained from lysosomes (regions ~ < 0.2 µm2)

• No MFM signal from mono-disperse cytoplasmic ferritin

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MFM of Healthy Tissue

Objectives: Effect of chemical fixatives

MFM signal is not affected by fixatives

Detection of MFM signalMFM signal present in iron-rich regions

AFM (non-MFM) probe cannot detect MFM signal

Verification of MFM signalSize of MFM signal corresponds to iron rich lysosomes

Blissett et al, Nanomedicine: NBM, 2017Walsh et al, J. Mag. & Mag Mater (in review)

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MFM of Diseased Tissue

Experimental system: Rodent model of acute injury (spinal cord injury)Rats: Naiive (healthy) and Injured (diseased) Tissues analyzed: spleen, spinal cord

Objective:

Is there a difference in the quality and quantityof iron in healthy vs. diseased tissue?

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Perls’ Stain (spinal cord)

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MFM analysis

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Roughness of MFM signal

Magnetic force imaging of a chain of biogenic magnetite and Monte Carlo analysis of tip–particle interactionAndré Körnig et al 2014 J. Phys. D: Appl. Phys. 47 235403 doi:10.1088/0022-3727/47/23/235403

Interparticle interactions also affect MFM signal

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Parameters affecting MFM roughness

• Density of ferritin(iron)

• Size of ferritin (iron)

• Oxidation state of ferritin iron -Magnetite (Fe2+) > Ferrihydrite (Fe3+)

TEM analysis

EELS spectroscopy

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TEM analysis: lysosome size and density

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EELS analysis: oxidative state of iron

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MFM analysis of diseased tissue

Objective: Is there a difference in the quality and quantity of iron in healthy vs. diseased

tissue?

• Size of lysosomes is reduced in injured tissues

• No major differences in oxidation state between injured and naïve animals

Blissett et al, Scientific Reports, 2018

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Indirect MFM

•Scanning at multiple lift heights not required

•Multimodal Imaging is possible (MFM, TEM, Light)

•Probe does not get contaminated•Samples can be kept in a fluids

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ID-MFM of ferritin

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Ferritin density

• Ferritin density in lysosomes (in-vivo) is much higher than that which can be achieved with purified ferritin (in-vitro)

• Direct MFM signal could only be obtained from lysosomalferritin in tissue sections

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Direct MFM of iron oxide nanoparticles

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ID MFM (10 nm thick membrane)

2.3 nm

1.05o

0.81 nm

8.05o

MFM probeAFM probe

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ID MFM (20 nm thick membrane)

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Comparison of Direct and Indirect MFM

• Minimal contribution of surface topography

• Minimal contribution from van-der Waals interactions

• No compromise in strength of MFM signal

• Multimodal imaging possible for MFM, TEM and fluorescence microscopy

Sifford et al, Nanoscale Advances, 2019

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Conclusions

• Both Direct and ID MFM can serve as high resolution, label free tools for iron-detection in histology

• MFM signal in tissues arises from clusters of ferritin(iron)

• ID MFM can serve as a artifact free, high-throughput, multimodal technique for iron-detection in histology

• ID-MFM can be adapted for samples in fluids

Height Phase

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Acknowledgements

Agarwal Lab

• Kevin Walsh

• Stavan Shah

• Brooke Ollander

• Angela Blissett

• Josh Sifford

• Rachel Novinc

Collaborators

•Dana Mctigue (OSU)

•Sam Oberdick(NIST)

•Gang Bao (Rice)

Funding

•National Science Foundation

•National Institutes of Health

•Institute of Materials Research (Ohio State University)