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

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.

UMass AmherstUltra-Chron

Electron Microprobe

• Electron-backscatter• X-ray backscatter, elemental• Maps of elements• 100 millimicron resolution *

* Ultra-chron Project

LL3 {latent lesion?}

Na Ca

P K

microbes

LL3 LL4Cu

Cu

LL4BSI

BSI

Ca and P Images of Lobster cuticle

LL4Ca P

LL4: Na, Ca, P, K

Na Ca

P K

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

LL 4:

1 2 3

3 2 1

UR1

Na Ca

P K

UR_1-A2R->pdf -> png via gimp

Lobster Dermal Gland Canal Ca/Mg/Sr/Ba P/Fe/Cl

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…

• Micro-scale core of cuticle.

• Reduce the cuticle/canal ratio.

Coring Drill Press

Leitz Ortholux Polarizing microscope, 3.5, 8, 16, 45x objectives.

0.25m diamond polished plastic embedded cuticle

0º +90ºlm

B5-UL d2

Polarlized light view

0º +90º

Non-invasive Ion Probe: Measure Size and Strength of an Ion Source

Dual Artificial H2SO4 sources:

Scanning Ion Electrode Technique

Nov 1, Fall06005.R R-script PlotDual artificial H2SO4 sources. 10 mM PO3

-2 pH 6 with proton LIX.

Vector arrays viewed with kinemage• Pollen tube O2 uptake:

• Nitrogen bubble O2 sink:

• Proton secreting cell in zebrafish yolk sac

• (not shown) toad skin

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

AFM Atomic Force Microscope

AFM Display Screens

Backscatter Electron Image

• Recognize homologous layers between methods

In

Out

Post-ecdysial endocuticle

Pre-ecdysial endocuticle

Exocuticle

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

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

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

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+