Microvesicles (MV) can be defined as a heterogeneous group of materials with distinct names: microparticles, ectosomes, exosomes, exosome like vesicles, shed vesicles or oncosomes, although all of them give a rough idea of the variety of biological sources.

In recent years there has been an increasing interest in the research of submicrometer particles released to the environment by cells. Particularly, exosomes have caught the attention of scientist from different areas due to the potential source of information contained inside these particles. This information could be crucial for instance as a new tool to diagnosis purposes or biomarker studies, the investigation of their role as a signal mediators or in different biological processes, or even as a novel treatment strategies. Nevertheless, the weak scattering properties of this material along with their small size turn their analysis into a challenging task using conventional techniques. Moreover, knowing the particle concentration in the sample is as important as the size or particle size distribution (PSD) of the sample.

The isolation of exosomes is the key stage, although the sample analysis has also to be considered as an important stage. For instance, you could waste much time to isolated microvesicles, but if the selected technique to analyze the sample presents limitations, you could not take advantage of the all potential information.

One of these widespread techniques is Electron Microscopy (EM), which could achieve the necessary requirements in the analysis of exosomes. Despite being the technique with the higher resolution in the measurement of particles size, conversely the original structure of the isolated exosome sample could be modified owing to the sample preparation procedure. Some problems could crop up during the extensive sample pre-treatment, for instance particle aggregation or a reduction in the original particle size in the drying process, (some sample treatment procedures could be required fixation times to the grid up to 24 hours). Moreover, employing this technique, the particle concentration is calculated assuming that all the vesicles contained in the analyzed aliquot of sample adhered uniformly to the grid, that is, there is not any leakage during the sample immobilization. In conclusion, EM implies an extensive sample preparation procedure and high level of handling, therefore increasing the risk of sample modification or degradation, and obviously it is also time-consuming.

Another widely employed technique it is conventional flow cytometry, although the size of particles aimed to analysis could be problematic, that is, the technique could underestimate the concentration of vesicles. Another important point to be taken into account is the heterogeneous refractive index of the particles and their unknown exact value. Therefore, a reliable estimation of the particle refractive index is required to get a good conversion from the optical scatter signal to size. Furthermore, a reference material is needed to calibrate the instrument having a refractive index as close as possible to the real sample (usually polystyrene beads are employed). Normally the minimum size detectable using flow cytometry is above 200-300 nm depending on the type of instrument.

Nanoparticle tracking analysis (NTA) is a light-scattering technique designed to characterize nanoparticles and was found to be well suited for the analysis of exosomes. NTA allows real-time visualization of exosome activities, monitoring the hydrodynamic diameter of the exosomes straightaway in solution, besides obtaining at the same time an estimation of particle concentration. NTA can provide a direct idea of sample concentration and size with low costs and it has a high resolving power. Obviously the larger particles contribute more to the light scattering than the smaller one, but NTA analysis is not intensity-weighted to larger particles. NTA is a technique in which the size of the particles is obtained from the measurement of the dynamic Brownian motion, the information is not extracted from the amount of light scattered by the particles in contrast to the basis of flow cytometry.

This technique requires a minimum sample preparation procedure and also provides rapid results. It should be highlighted the importance of analyzing the fresh samples since long-term storage or conditions could contribute to sample degradation or structural changes.

Differentiation can also be performed by using of fluorophore-labeled antibodies that specifically interact with the membrane proteins of exosomes.

At Nanovex Biotechnologies facilities there is available a Nanosight LM10 instrument equipped with a laser of 405 nm which could perform Nanoparticle Tracking Analysis (NTA) of particles below 1 µm based on the analysis of Brownian motion.