direct and indirect magnetic force microscopy (mfm) in ... · ferritin. protein shell, d~12nm. iron...
TRANSCRIPT
![Page 1: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/1.jpg)
1
Direct and Indirect Magnetic Force Microscopy (MFM) in
HistologyGunjan Agarwal
Mechanical and Aerospace Engineering
![Page 2: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/2.jpg)
2
Biomagnetism
Sources
– Super-paramagnetic: Iron
• Ferritin, transferritin, hemoglobin, NTBI
– Diamagnetic: Calcium
Tissues
– Liver (ferrihydrite)
– Spleen (ferrihydrite)
– Serum (?)
– Acute injury (ferrihydrite)
– Chronic plaques (maghemite)
![Page 3: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/3.jpg)
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
![Page 4: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/4.jpg)
4
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
![Page 5: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/5.jpg)
5
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
![Page 6: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/6.jpg)
6
Magnetic Force Microscopy
•High sensitivity
•High Spatial resolution
•Label free
•Available on Commercial AFMs
![Page 7: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/7.jpg)
7
Direct MFM of a Magnetic Tape
Savla et al, Israel J. of Chem., 2008
![Page 8: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/8.jpg)
8
Direct MFM of Ferritin
Bprobe
Bscanner
![Page 9: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/9.jpg)
9
Direct MFM of Ferritin and Apoferritin
Nocera et al,
Nanotechnology (2014)
![Page 10: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/10.jpg)
10
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)
![Page 11: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/11.jpg)
11
MFM of Healthy Tissue
Objectives:
Effect of chemical fixatives
Detection of MFM signal
Verification of MFM signal
![Page 12: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/12.jpg)
12
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
![Page 13: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/13.jpg)
13
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
![Page 14: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/14.jpg)
14
MFM of fixed and unfixed tissue
200
FixedUnfixed
200 nm
![Page 15: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/15.jpg)
15
Detection of MFM signal
Bprobe
![Page 16: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/16.jpg)
16
Long range detection
z = 30 50 90 nm
z = 30 nm z = 50 nmz = 40 nm
AFM
pro
beM
FM p
robe
![Page 17: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/17.jpg)
17
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
![Page 18: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/18.jpg)
18
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
![Page 19: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/19.jpg)
19
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)
![Page 20: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/20.jpg)
20
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?
![Page 21: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/21.jpg)
21
Perls’ Stain (spinal cord)
![Page 22: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/22.jpg)
22
MFM analysis
![Page 23: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/23.jpg)
23
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
![Page 24: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/24.jpg)
24
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
![Page 25: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/25.jpg)
25
TEM analysis: lysosome size and density
![Page 26: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/26.jpg)
26
EELS analysis: oxidative state of iron
![Page 27: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/27.jpg)
27
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
![Page 28: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/28.jpg)
28
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
![Page 29: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/29.jpg)
29
ID-MFM of ferritin
![Page 30: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/30.jpg)
30
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
![Page 31: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/31.jpg)
31
Direct MFM of iron oxide nanoparticles
![Page 32: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/32.jpg)
32
ID MFM (10 nm thick membrane)
2.3 nm
1.05o
0.81 nm
8.05o
MFM probeAFM probe
![Page 33: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/33.jpg)
33
ID MFM (20 nm thick membrane)
![Page 34: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/34.jpg)
34
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
![Page 35: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/35.jpg)
35
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
![Page 36: Direct and Indirect Magnetic Force Microscopy (MFM) in ... · Ferritin. Protein shell, d~12nm. Iron core, d~ 8nm. Apoferritin. Iron. Atoms. Ferritin The major iron storage protein](https://reader033.vdocument.in/reader033/viewer/2022052615/6081ac3dc4c97133c54698a3/html5/thumbnails/36.jpg)
36
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)