Super-resolution observation of lysosomal dynamics with fluorescent gold nanoparticles

Abstract

Because lysosomes play critical roles in multiple cellular functions and are associated with many diseases, studying them at the subcellular level could elucidate their functionality and support the discovery of therapeutic drugs for treating those diseases. The commonly used dyes for super-resolution imaging of lysosomes are the commercial molecular LysoTrackers. But the tolerance to changes in the lysosomal microenvironment and to lysosomal membrane permeabilization (LMP) and the photostability of the LysoTrackers are worrisome. The purpose of our study was to evaluate the feasibility of performing a fluorescent gold nanoprobe for super-resolution observation of lysosomal dynamics in living cells and compare it to the commercial LysoTrackers. Methods: The nanoprobe Cy5@Au NP contained three parts: a bio-inert gold core, a biocompatible polyethylene glycol spacer, and a fluorophore cyanine 5. Structured illumination microscopy (SIM) was employed to capture the fluorescence of Cy5@Au NPs in cells. The tolerance assays to changes in the lysosomal microenvironment and to LMP, the photobleaching assay, and the long-term lysosomes labelling assay of Cy5@Au NPs were compared with commercial LysoTrackers. The super-resolution observation of lysosomal dynamics with Cy5@Au NPs was performed. Results: Cy5@Au NPs can light up lysosomes specifically under SIM. Compared with commercial lysosomal molecular probes, Cy5@Au NPs exhibited stronger tolerance in lysosomes during various treatments, and changes in the lysosomal microenvironment and LMP did not cause Cy5@Au NPs to lose track of their targets. Cy5@Au NPs demonstrated an excellent anti-photobleaching ability, and a long-term labelling assay revealed that they could label lysosomes more than 3 d. Biological events of lysosomes such as the kiss-and-run process, fusion, fission, and mitophagy were recorded with the fluorescent Cy5@Au NPs under SIM. Conclusions: The nanoprobe Cy5@Au NP was successfully used as a lysosomal probe for the super-resolution observation in living cells and found to overcome the limitations of commercial LysoTrackers. Our results thus confirm that nanoparticles can be useful tools for subcellular super-resolution imaging and highlight new avenues for using nanoparticles in biology.

Keywords: long-term labelling; lysosomes; mitophagy; structured illumination microscopy; super-resolution imaging.

Figure 1

(A) Schematic illustration of the compositions of a Cy5@Au NP. (B) SIM images of HeLa cells stained by Cy5@Au NPs (1.36 × 10^-10 M) for different treatment periods. (C) SIM images of HeLa cells stained by Cy5@Au NPs in different concentrations for 24 h. The dilution ratios between original nanoparticles solution and DMEM (with FBS) were 1:2000, 1:1000, and 3:2000 and the corresponding molar concentrations of nanoparticles were 6.8 × 10^-11 M, 1.36 × 10^-10 M, and 2.04 × 10^-10 M, respectively.

Figure 2

(A) Images of COM and SIM. (B) SIM images of a colocalization experiment with HeLa cells costained with Cy5@Au NPs and LTR. (C) SIM images of a colocalization experiment with HeLa cells costained with Cy5@Au NPs and MTG.

Figure 3

(A) SIM images of untreated and chloroquine-treated HeLa cells stained with Cy5@Au NPs and LTR, respectively. (B) SIM images of untreated and 4% paraformaldehyde-treated HeLa cells (i.e., for cell fixation) stained with Cy5@Au NPs and LTR, respectively.

Figure 4

(A) SIM image of HeLa cells stained with Cy5@Au NPs before photobleaching. (B) Magnified images of the dashed box in (A) during photobleaching. (C) SIM image of HeLa cells stained with LTR before photobleaching. (D) Magnified images of the dashed box in (C) during photobleaching. (E) Normalized intensity during photobleaching of Cy5@Au NPs and LTR. (F) SIM images of HeLa cells stained with Cy5@Au NPs and LTR for 3 d.

Figure 5

The dynamics of lysosomes in living cells. (A) SIM image of HeLa cells stained with Cy5@Au NPs before kiss-and-run process. (B) Magnified images of the dashed box in (A) during kiss-and-run process. (C) SIM image of HeLa cells stained with Cy5@Au NPs before fusion. (D) Magnified images of the dashed box in (C) during fusion. (E) SIM image of HeLa cells stained with Cy5@Au NPs before fission. (F) Magnified images of the dashed box in (E) during fission.

Figure 6

(A) SIM images of untreated and CCCP-treated HeLa cells stained with Cy5@Au NPs and MTG. (B) SIM images of CCCP-treated HeLa cells stained with Cy5@Au NPs and DAPGreen (DAPG).