Fig. 1. FRET reaction of BSA-DOX nanoparticles (acceptor) with sfGFP fluorescent protein (donor) labeled on cell membranes during endocytosis

Fig. 2. Characteristics of BSA-DOX nanoparticles. (a) Standard concentration curves of DOX solutions; (b) absorption and emission spectra of DOX solutions (lines 1 and 3) and BSA-DOX nanoparticles (lines 2 and 4); (c) hydrodynamic particle size and TEM image of BSA-DOX nanoparticles; (d) CCK-8 cytotoxicity assay result

Fig. 3. Fluorescence images and flow cytometry results of cellular uptake of BSA-DOX nanoparticles in presence of four endocytosis inhibitors

Fig. 4. Analysis of fluorescence lifetime data. (a)(d) Two-photon fluorescence maps, (b)(e) fluorescence lifetime pseudo-color maps and (c)(f) two-dimensional lifetime histograms for control and BSA-DOX 8 h; (g)(i) fluorescence lifetime pseudo-color images and (h)(j) two-dimensional lifetime histograms for FRET region and non-FRET region

Fig. 5. Relationship between cellular endocytosis process and FRET effect. (a) Two-photon fluorescence maps and fluorescence lifetime pseudo-color images and (b) lifetime decay curves of cells incubated with BSA-DOX for 0,8,20 h and with DOX solution for 1 h; (c) average lifetime of FRET regions; (d) FRET efficiency

Fig. 6. Relationship between starvation treatment and cellular endocytosis. (a) Two-photon fluorescence map and fluorescence lifetime pseudo-color images; (b) average lifetime of FRET regions; (c) FRET efficiency; (d) line graph of FRET efficiency; (e) time serieTP-FLIM images of BSA-DOX nanoparticles uptake by normal and starved cells