Figure 2: Spatial arrangement of lipidaceous and proteinaceous components of the expressed adhesive.

(a) CLSM volume projection of a single antennular adhesive plaque stained for proteins showing a clear void between the proteinaceous phase (green) and bacterial growth on the true outer phase of the adhesive plaque, which is not stained for proteins (b., bacteria; l.p., lipid phase; p.p., protein phase; a.d., adhesive disc). (b–d) Isosurface rendering of a viewed from various angles. It is clear that proteinaceous phase (blue) and the bacterial growth (green) are separated by a material that does not stain for proteins. (e,f) Imaged during the adhesive secretion; the cyprid adhesive discs (blue) are embedded in a phase of the cyprid adhesive that did not stain for proteins but which accrued bacterial growth (green). (g) XY view from a TPEFM volume projection of the adhesive plaque embedding the paired antennules stained with lysochrome ORO. Relative fluorescence intensity units (RFU) plot (inset) shows lower intensity values when compared with the labelling of the proteinaceous phase j. (h,i) Isosurface renderings of the lipidaceous phase viewed from above (h) and beneath the plaque (i). The lipidaceous phase appears to entirely cover the bulk phase of the adhesive plaque, which is proteinaceous. (j) XY view from a TPEFM volume projection of an adhesive plaque stained for phosphoproteins shows that the bulk of cement plaque is phosphoproteinaceous and with much higher RFU as compared with g. (k,l) Isosurface renderings of phosphoprotein-stained plaque from j, viewed from above (k) and beneath the plaque (l), shows that the bulk of the plaque is proteinaceous (blue) with bacterial growth (green) separated from the proteinaceous phase. (m–o) Pseudocoloured BCARS chemical image (m) with 500 nm lateral resolution taken at 30 ms per pixel acquisition times at 5 μm from coverslip surface. RGB image chemical contrast comes from total C-H stretch at 2,920 cm⁻¹ (red channel), the ratio of C-H to Amide III of 2,920:1,262 cm⁻¹ (green channel), and the ratio of C-H to nucleotide bases of 2,920:1,486 cm⁻¹ (blue channel). Bright-field image (n) from identical location as chemical image as well as spectra (o) obtained from individual pixels within BCARS chemical image to clarify distinct spectral differences between outer lipidaceous and inner regions of the adhesive plaque that are pointed to by arrows in chemical image. Scale bars (μm), 5 (a–c), ~2 (d), ~5 (e,f), ~20 (g,j), ~10 (h,i,k,l,) and 20 (m,n).