cuticle mineralization: (1) electron-microprobe; (2) noninvasive ion probe; (3) atomic force...
TRANSCRIPT
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Cuticle Mineralization:(1) Electron-microprobe;
(2) Noninvasive Ion Probe; (3) Atomic Force Microscope;(4) Reflecting Polarized LM…
Looking for Latent LesionsJoe Kunkel, Director, UMass Ion Probe Facility
Mike Jercinovic, Director, UMass Electron Microprobe
Lynne A. McLandsborough, Director, UMass Food Science AFM
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Analysis of Cuticle Minerals• Treat cuticle as a
mineral specimen.• Premise: An intact
mineral layer is partially dissolved in latent shell disease.
• The attack may occur through surface irregularities which break the intact mineral layer.
• Ergo: Mineral irregularities latent lesions.
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UMass AmherstUltra-Chron
Electron Microprobe
• Electron-backscatter• X-ray backscatter, elemental• Maps of elements• 100 millimicron resolution *
* Ultra-chron Project
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LL3 {latent lesion?}
Na Ca
P K
microbes
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LL3 LL4Cu
Cu
LL4BSI
BSI
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Ca and P Images of Lobster cuticle
LL4Ca P
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LL4: Na, Ca, P, K
Na Ca
P K
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LL 4: Ca/P plots
• Regions with different ratios of Ca to P.
• Is there a mineral corespondence?
• Outer dense layer of close to highly aligned mineral which shows birefringence.
• High-P layers?• Pure vs Composites?
Ca
P
Ca-scan
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LL 4:
1 2 3
3 2 1
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UR1
Na Ca
P K
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UR_1-A2R->pdf -> png via gimp
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Lobster Dermal Gland Canal Ca/Mg/Sr/Ba P/Fe/Cl
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How to increase the P signal?• Extract CaCO3 with
formic acid.• Micro-X-ray powder
analysis: no apatite!• Image formic acid
tubes in dissecting microscope.
• Drill core the dermal gland canals.
• Collect cores for micro-X-ray powder analysis…
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• Micro-scale core of cuticle.
• Reduce the cuticle/canal ratio.
Coring Drill Press
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Leitz Ortholux Polarizing microscope, 3.5, 8, 16, 45x objectives.
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0.25m diamond polished plastic embedded cuticle
0º +90ºlm
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B5-UL d2
Polarlized light view
0º +90º
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Non-invasive Ion Probe: Measure Size and Strength of an Ion Source
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Dual Artificial H2SO4 sources:
Scanning Ion Electrode Technique
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Nov 1, Fall06005.R R-script PlotDual artificial H2SO4 sources. 10 mM PO3
-2 pH 6 with proton LIX.
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Vector arrays viewed with kinemage• Pollen tube O2 uptake:
• Nitrogen bubble O2 sink:
• Proton secreting cell in zebrafish yolk sac
• (not shown) toad skin
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Latent lesion hypothetical properties:
• Site of microbial attack, a proton source and carbonate source; probe them.
• Oxidative burst in response to attack results in an oxygen sink; probe it.
• Etching of minerals results in a soft texture spot viewed by AFM, demineralization by electron microprobe and deformed birefringence by polarized light.
• Use localizing methods to target microbe collection and develop in situ staining.
H+
HCO3-
O2
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AFM Atomic Force Microscope
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AFM Display Screens
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Backscatter Electron Image
• Recognize homologous layers between methods
In
Out
Post-ecdysial endocuticle
Pre-ecdysial endocuticle
Exocuticle
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Montage of AFM images
• Pore canals in endo-, exo-cuticle to epicuticle surface. A living connection to surface?
• Oxidative burst sensable at the surface?• Reactive oxygen species at surface? … Quinones?
• Phenoloxidases at surface consuming O2?
• Scanning Oxygen electrode application?
ExocuticlePost-exuvial
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AFM Magnification series
• Zooming in to submicron structure and texture.• What are the limits of resolution of this approach?
100 m
50 m
25 m
5 m
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Highest current AFM image resolution:
• What are the limits of resolution and approach?• Currently at sub-micron resolution with low-res cantilever.• Use higher resolution cantilever. Currently using crayon!
1 m
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End
1. Ca2+ presence but PO43+ absence in the cuticle
outer layer which is birefringent! Calcite/Aragonite?2. Is there apatite in the lobster cuticle canals?3. Where do microbes attack the mineral layers?4. Use mineral sensitive technology to explore.5. How do mineral trace elements affect their
properties?
Ca2+ PO43+