Figure 4

Tilt during sectioning and the relationship of spheroid area on histological sections vs. angle of tilt. (A) Schematic model showing the predicted cutting plane through a row of 12 spheroids at three different angles of tilt ϴ_t = 0, 0.5 & 1°. View is in the xz plane where the y-axis goes through the center of each spheroid in the row. (B) The estimated view of the cross-section of the row of spheroids (from A) as they would show up on a histological section. The color dotted lines correspond to an “ideal” cross-section through the center of each spheroid. Schematics are shown for 6 × 4, 8 × 6 & 12 × 8 arrays at angles of ϴ_t = 0, 0.5 & 1°. (C) A geometrical calculation predicts the critical angle when the outmost spheroid in an area is completely absent from the centerpoint section. (D) The coordinate system and angles of tilt in the xz (ϴ_tx)and yx (ϴ_ty) planes are show against an agarose microarray. (E-top) A schematic of five sequential sections (A–E) taken from an agarose microarray paraffin embedded block as viewed from the xz plane. (E-bottom) The corresponding row of pillars appearing on 5 consecutive histological sections used to calculate the experimental tilt for the agarose microarray system.