Nutrient flow and localization of ECM within colonies. Vertical transverse colony cross sections. (A) Areas of galactose or Cu²⁺ induction in colonies (the vertical arrow indicates diffusion into the colony). (B) Timeline of ECM formation. (A and B) Green, GFP fluorescence marks areas in which the inducer reached the cells; red, autofluorescence of all colony cells visible in areas where ECM prevented the inducer from accessing the cells. Intact colonies were induced from the bottom by placing them for 5 h on agar soaked with 2% galactose or 5 mM CuSO₄. GFP fluorescence was detectable by 2P-CM as early as 45 min after induction. (C) The exposed area of the vertical transverse colony section was placed flat on galactose-soaked agar (a 2-h induction), after which internal cells were induced as well. (D) A model scheme featuring impermeable ECM (yellow). Two (B [55 h] and C) or three (A) individual images spanning the width of the colony were acquired and assembled after acquisition to generate the composite image shown.