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Figure 5. PalmGRET enables multimodal, multiresolution imaging and analysis of extracellular particles (EPs)’ distribution in organs. (A) Schematic of in vivo time-lapsed imaging of EPs. Immunocompetent C3H mice were administered with EP-293T-PalmGRET (100 µg) into the tail vein, followed by Fz injection for in vivo imaging at 5, 10, 20, and 30 min post-EP administration. (B) Bolus IV-injected EP-293T-PalmGRET (100 µg) was visualized under BLI and BRET-FL (GFP using BRET) channels from 5 to 20 min post-administration. The majority of the EP signals were detected in the lungs and spleen. By contrast, imaging under epi-illumination resulted in mostly scattered, non-EP-specific signals. (C) Ex vivo imaging of organs harvested at 30 min post-injection of EP-293T-PalmGRET (100 µg) (Mice No. 1 and No. 2) and WT subjects. EP-293T-PalmGRET signals were readily detected in the lungs, liver, and spleen. (D) Super-resolution radial fluctuations (SRRF) nanoscopy of lung sections at 30 min post-injection of EP-293T-PalmGRET (100 µg) or PBS (control). Enlarged images (dashed boxes) of boxed regions I.ii) reveal injected EPs in lung tissues. The nuclei were stained by DAPI, and EP-293T-PalmGRET was immunoprobed by anti-GFP antibody followed by AlexaFluor 568-secondary antibody to minimize the background signal. Bar, 10 µm; in enlarged images, 500 nm. (E) Quantification of EP signals by SRRF imaging demonstrated a significant increase in EP counts in the lungs followed by the spleen and liver at 30 min post-EP injection. The kidneys showed no significant increase in EP counts. 293T-PalmGRET signals were quantified by ImageJ from seventy images of tissue sections of mice injected with EP-293T-PalmGRET or PBS. The EP counts were normalized against organ weight. N.S., P > 0.05; *P < 0.05; **P < 0.01 with two-tailed Studen’s t-test. Reprinted with permission from^[80].