While the principles underpinning the Nanoparticle Tracking Analysis (NTA) technique have been described in an earlier white paper, it must be reiterated that the use of high intensity laser beams combined with a low-background optical configuration allows particles of deeply sub-micron dimensions to be visualized, the lower range of particle sizes measureable depending on particle refractive index. While for very high refractive index particles, such as colloidal gold, accurate determination of size can be achieved down to 15 nm diameter, for lower refractive index particles, such as those of biological origin such as exosomes, the smallest detectable size might only be 30-40 nm. This minimum size limit allows, however, the analysis of microvesicles and exosomes of a size which would normally be far below the detection threshold of 300 nm for most commercially available flow cytometers. The upper size limits are approached when the Brownian motion of a particle becomes too limited to track accurately, typically 1-2 μm diameter.

The laser with which the nanoparticles are illuminated can be exchanged for one with which fluorescence could be excited, allowing nanoparticles labelled with fluorescent molecules to be visualized, tracked and thus sized and concentration measured specifically through the use of appropriate optical filters. Accordingly, instead of the usual 638 nm red laser, a 532 nm green laser diode can be used to excite a range of organic fluorophores, while a deep blue/violet 405 nm laser diode allows semiconductor CdSe nanocrystals (also known as quantum dots) to be detected on an individual basis. A 488 nm laser diode can similarly be used to excite more conventional dyes as used historically in flow cytometry.

Through the use of antibody-mediated fluorophore labelling of specific sub-populations of exosomes, phenotyping within complex mixtures can therefore be achieved. Of specific importance in this regard is the ability to speciate a particular exosome type by means of Antibody(Ab)-labelling, while simultaneously measuring the size of the exosome by analyzing its Brownian motion, the two measurements being independent of each other. Note also that the concentrations of such labelled exosomes can still be recovered and compared to the total number of similar sized structures whether labelled or not.