We observed three different pathways for ILV formation, summarised in Fig. . In Fig. (Movie S3), internal lipid nanotubes that include the external CBlue probe remodel into discrete ILVs that also encapsulate CBlue from the bulk medium. A second mechanism saw two separate GUVs undergo a fusion mechanism, where two independent fusion pores form in the adhesion contact area, such that the membrane in the adhesion plaque becomes scissioned inside the new GUV and encapsulates the AF488 probe (Fig. , Movie S4). A final ILV formation mechanism was observed when a non-spherical GUV with larger excess membrane area undergoes membrane fluctuations that lead to a discocyte GUV shape folding around such that the spheroidal end-caps of this structure contact and fuse. This topological transition leaves a CBlue-encapsulating ILV trapped inside an outer membrane (Fig. , Movie S5).