Intracellular Dynamics of Extracellular Vesicles by Segmented Trajectory Analysis.

The analysis of nanoparticle (NP) dynamics in live cell studies by video tracking provides detailed information on their interactions and trafficking in the cells. Although the video analysis is not yet routinely used in NP studies, the equipment suitable for the experiments is already available in most laboratories. Here, we compare trajectory patterns, diffusion coefficients, and particle velocities of NPs in A549 cells with a rather simple experimental setup consisting of a fluorescence microscope and openly available trajectory analysis software. The studied NPs include commercial fluorescent polymeric particles and two subpopulations of PC-3 cell-derived extracellular vesicles (EVs). As bioderived natural nanoparticles, the fluorescence intensities of the EVs limited the recording speed. Therefore, we studied the effect of the recording frame rate and analysis parameters to the trajectory results with bright fluorescent commercial NPs. We show that the trajectory classification and the apparent particle velocities are affected by the recording frame rate, while the diffusion constants stay comparable. The NP trajectory patterns were similar for all NP types and resembled intracellular vesicular transport. Interestingly, the EV movements were faster than the commercial NPs, which contrasts with their physical sizes and may indicate a greater role of the motor proteins in their intracellular transports.

Crystallization Kinetics of an Amorphous Pharmaceutical Compound Using Fluorescence-Lifetime-Imaging Microscopy.

Pharmaceutical scientists are increasingly interested in amorphous drug formulations especially because of their higher dissolution rates. Consequently, the thorough characterization and analysis of these formulations are becoming more and more important for the pharmaceutical industry. Here, fluorescence-lifetime-imaging microscopy (FLIM) was used to monitor the crystallization of an amorphous pharmaceutical compound, indomethacin. Initially, we identified different solid indomethacin forms, amorphous and γ- and α-crystalline, on the basis of their time-resolved fluorescence. All of the studied indomethacin forms showed biexponential decays with characteristic fluorescence lifetimes and amplitudes. Using this information, the crystallization of amorphous indomethacin upon storage in 60 °C was monitored for 10 days with FLIM. The progress of crystallization was detected as lifetime changes both in the FLIM images and in the fluorescence-decay curves extracted from the images. The fluorescence-lifetime amplitudes were used for quantitative analysis of the crystallization process. We also demonstrated that the fluorescence-lifetime distribution of the sample changed during crystallization, and when the sample was not moved between measuring times, the lifetime distribution could also be used for the analysis of the reaction kinetics. Our results clearly show that FLIM is a sensitive and nondestructive method for monitoring solid-state transformations on the surfaces of fluorescent samples.

Correction: Addressing challenges in the removal of unbound dye from passively labelled extracellular vesicles.

[This corrects the article DOI: 10.1039/D1NA00755F.].

Addressing challenges in the removal of unbound dye from passively labelled extracellular vesicles.

Studies of extracellular vesicles (EVs), their trafficking and characterization often employ fluorescent labelling. Unfortunately, little attention has been paid thus far to a thorough evaluation of the purification of EVs after labelling, although the presence of an unbound dye may severely compromise the results or even lead to wrong conclusions on EV functionality. Here, we systematically studied five dyes for passive EV labelling and meticulously compared five typical purification methods: ultracentrifugation (UC), ultracentrifugation with discontinuous density gradient (UCG), ultrafiltration (UF), size exclusion chromatography (SEC), and anion exchange chromatography (AEC). A general methodology for evaluation of EV purification efficiency after the labelling was developed and tested to select the purification methods for the chosen dyes. Firstly, we found that some methods initially lead to high EV losses even in the absence of the dye. Secondly, the suitable purification method needs to be found for each particular dye and depends on the physical and chemical properties of the dye. Thirdly, we demonstrated that the developed parameter E rp (relative purification efficiency) is a useful tool for the pre-screening of the suitable dye-purification method combinations. Additionally, it was also shown that the labelled EVs properly purified from the unbound dye may show significantly reduced contrast and visibility in the target application, e.g. in the live cell fluorescence lifetime imaging.