Fig. 68. Multistep FRET network. (A) Assembly consisting of a central QD and biotin–streptavidin assembled DNA tetrahedrons with three different dyes. Excitation of the QD can excite all three dyes via direct QD-dye FRET or QD-dye-dye-dye FRET cascades. (B) The tetrahedron DNAs contain biotin and dye functionalities as well as specific cleavage sites for three endonucleases, which will specifically release the different dyes from the tetrahedron. (C) Fluorescence images of the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA in the (1) absence of any endonucleases and presence of (2) HaeIII and (3) HaeIII + EcoRV + PvuII. (a)–(c) Fuorescence images of the same field in the 525QD channel (500–550 nm) and the Cy3/Texas Red channel I (573–617 nm) are acquired simultaneously by using dual-view optics at an excitation wavelength of 405 nm. Then (d–f) fluorescence images in the Cy3/Texas Red channel II (565–605 nm) and Cy5 channel (672–712 nm) are acquired simultaneously. Fluorescence spots shown in the Cy3/Texas Red channel I/II (b, d, green color) result from FRET from the 525QD to Cy3/Texas Red (yellow arrows); fluorescence spots shown in the Cy5 channel (e, red color) result from FRET from the 525QD to Cy5 (white arrows). Fluorescence images with blended colors in (c) cyan indicate the colocalization of 525QD and Cy3/Texas Red; fluorescence images with blended colors in (f) yellow indicate the colocalization of Cy3/Texas Red and Cy5. The scale bar is 2 μm. Reproduced with permission from ref. 431 Copyright 2019 American Chemical Society.