Cuticle Mineralization: (1) Electron-microprobe; (2) Noninvasive Ion Probe; (3) Atomic Force Microscope; (4) Reflecting Polarized LM… Looking for Latent Lesions Joe 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 Amherst Ultra-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 LL4 Cu LL4 BSI

Ca and P Images of Lobster cuticle LL4 Ca P

LL4: Na, Ca, P, K NaCa 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:

UR1 Na Ca P K

UR_1-A 2 R->pdf -> png via gimp

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

How to increase the P signal? Extract CaCO 3 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.

0.25  m diamond polished plastic embedded cuticle 0º+90º lm

B5-UL d 2 Polarlized light view 0º +90º

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

Dual Artificial H 2 SO 4 sources: Scanning Ion Electrode Technique

Nov 1, Fall06005.R R-script Plot Dual artificial H 2 SO 4 sources. 10 mM PO 3 -2 pH 6 with proton LIX.

Vector arrays viewed with kinemage Pollen tube O 2 uptake: Nitrogen bubble O 2 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+H+ HCO 3 - O2O2

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 O 2 ? 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.Ca 2+ presence but PO 4 3+ 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? Ca 2+ PO 4 3+