NTA goes co-localization: Characterization of double-labelled extracellular vesicles
Characterization of bio-nanoparticles (BNPs) such as extracellular vesicles (EVs), exosomes or virions has usually been a demanding task requiring expertise, significant time and sample amount. While nanoparticle tracking analysis (NTA) is a comparatively fast technique, it has been predominantly used for measurement of particle size distribution and concentration determination in unspecific scatter mode. With the possibility of fluorescence detection, NTA reached a milestone where fluorescently labelled particles can be detected offering enhanced resolution and bio-specific results compared to scatter-based NTA. This communication describes the most current progress of NTA: quantification of colocalization ratios of two complementary fluorescently labelled compartments present on BNPs. Technical principles of the advanced setup of a PMX-230 TWIN ZetaView® instrument with colocalization software feature are presented and experimental guidelines such as staining procedures and software settings for the characterization of fluorescently double-labelled BNPs are discussed. For the first time we present quantitative colocalization results of selected EVs (cell line and donor) and phages realized with NTA.
Nanoparticle Tracking Analysis, Fluorescence, Labelling, Immunolabelling, Tetraspanin, Membrane dye, Colocalization, Biomarker, Exosomes, Phenotyping
Introduction
Techniques incorporating fluorescence as detection principle can be regarded as the analytical backbone in almost every bioanalytical laboratory. For the characterization of exosomes, micro vesicles (MV) and virions, resumed as bio-nanoparticles (BNPs), fluorescent staining techniques allow fast and specific staining of membranes, surface proteins or nucleic acids by applying lipid dyes, antibodies, and intercalating RNA/DNA dyes [1, 2]. The presence or absence of characteristic targets is associated with the biological function of such BNPs, therefore labelling procedures and bioanalytical techniques following labelling need to be well-matched to achieve optimum results.
In general, the term colocalization refers to the simultaneous detection of two or more specific targets in the same space. Each target is labelled with a specific fluorescent probe, having individual fluorescent excitation wavelengths and emission characteristics [3]. Quantitative colocalization measurements on BNPs are challenged by their size and the low number of available fluorochromes. Assuming an EV of 100 nm and an antibody with 3 nm in size, the number of antibodies conjugated to an EV is ~4000 for a monolayer. Presumably an average of 2.5 fluorochromes per antibody results in ~10000 fluorochromes attached to an EV. In real samples such numbers are considerably lower due to epitope density, steric hinderance, inactivation and multi-labelled EVs. To resolve such challenging characteristics with fluorescence microscopy (FM) significant enhancement of resolution and limit of detection is necessary. Stochastic optical reconstruction microscopy (STORM) provides approx. 10-fold higher resolution to conventional fluorescence microscopy and even allows resolution of the distribution of tetraspanins on a single EV in 3D [4, 5]. While characterization of blood cells or HIV is routinely performed with flow cytometry (FCM), detection of EVs in the size range of 30 – 200 nm remains challenging. Experiments need careful consideration with respect to correct EV count, neither not to miss the EV population nor to pick up noise instead of real signal. EV analysis performed by FCM started more than a decade ago and requires both high performance instrumentation and a certain level of expertise [6–9].
