Fig. 2: Simultaneous measurement of phagosome-lysosome fusion and phagosome transport dynamics.
From: Kinetics of phagosome maturation is coupled to their intracellular motility
a Schematic illustration of the Förster resonance energy transfer (FRET)-based phagosome-lysosome fusion assay. The FRET-RotSensor was coated with SAv-Alexa568 (FRET donor). BSA-Alexa647-biotin (FRET acceptor) was loaded into lysosomes. Phagosome fusion with lysosomes leads to intermixing between donor fluorophore (Alexa568) and fluid phase acceptor fluorophore (Alexa647) generating FRET emission (680 nm) under the donor excitation of 561 nm. b and c Fluorescence images and intensity plots showing the change of FRET emission (ex/em: 561/680 nm) and donor emission (magenta, ex/em: 561/586 nm) from a FRET-RotSensor (100 nm yellow-green nanoparticle in green) in a RAW264.7 macrophage. Scale bar, 2 μm. d FRET ratio vs. time plot is fitted with sigmoidal function (shown as the black solid line) to determine the initial time point of phagosome-lysosome fusion (tinitial), the time point where FRET-signal reaches a plateau (tfinal), and the FRET rate, as indicated by the red dotted line. Scatter plots showing normalized FRET rate against translational (e) and rotational velocities (f) of single phagosomes during the period of its fusion with lysosomes. Each data point represents data from a single phagosome. Data points from multiple phagosomes within the same cells are shown in the same solid color. Data points from cells containing only one phagosome are shown as black circles. For translational tracking, N = 40 phagosomes from 22 cells. For rotational tracking, N = 11 phagosomes from 10 cells. The black lines indicate linear regression with a Pearson’s coefficient of 0.75 in e and of 0.86 in f.
