Much interest centers around microvesicles and nanovesicles (exosomes) as they are increasingly cited as potential biomarkers. Whilst definitions in this emerging field currently lack formality, these two classes of bio-nanoparticle are differentiated both by their size ranges and their biogenesis. Typically microvesicles are described as being 100 nm to 1 μm, whilst exosomes are in the range 30-100 nm. Microvesicles are typically formed by blebbing of the plasma membrane, whereas exosomes are released by exocytosis from multivesicular bodies of the endosome. Both appear involved in cell signaling, carrying as they do a range of signaling proteins as well as messenger and microRNAs. Circulating levels are found to be elevated in various disorders, including atherosclerosis and coronary artery disease, haematological and inflammatory diseases, diabetes and cancer.
To date, exosomes research has been constrained by a lack of suitable methods for characterization. NanoSight addresses this need with unique and proven technology. Nanoparticle Tracking Analysis (NTA) allows specific exosomes and microvesicles in the range of 50-1000 nm in liquid suspension to be directly and individually visualized and counted in real-time. Simultaneously, NTA provides high-resolution particle size distribution profiles and concentration measurements. The technique is easy to use, fast, robust, accurate and cost effective, representing an attractive alternative or complement to existing methods. Operation in fluorescence mode enables characterization and speciation of suitably-labeled particles using a range of excitation wavelengths.
Detecting and Counting Exosomes
Both Dynamic Light Scattering (DLS) and NTA measure the Brownian motion of nanoparticles whose speed of motion, or diffusion coefficient (Dt), is related to particle size through the Stokes-Einstein equation.
In NTA laser light illuminates particles in suspension and a video camera captures the scattered light produced. The particle diffusion is determined by tracking individual particle positional changes in two dimensions. Knowing Dt, the particle hydrodynamic diameter can then be determined.
The images show particles moving under Brownian motion. Initial visual inspection reveals the presence of larger particles or aggregated material (Figure 1A). The NTA software then rapidly generates a high resolution size distribution on a particle-by-particle basis and a count (in terms of absolute number concentration) of the vesicles seen (Figure 1B).
Figures 1A & 1B. Sample of diluted platelet-free plasma. A) A typical image produced by the NanoSight technique. The image allows the users to instantly recognize certain features about their sample including concentration and level of polydispersity. B) Particle size distribution and calculated original concentration from the sample.
Traditionally a number of techniques have been used to characterize micro– and nano- vesicles with more techniques being available to the micro sized particles. These include:
- Flow Cytometry
- Dynamic Light Scattering (DLS)
- Electron Microscopy (EM)
- Enzyme Linked Immune-Sorbent Assays (ELISA)
Most widespread is Flow Cytometry. Commercial flow cytometry typically has a lower practical size limit (for polystyrene beads) of around 300 nm at which point the signal is indistinguishable from the baseline noise level. Whilst this detection limit can be extended with the use of fluorescent labels, at lower sizes the ability to accurately size such particles is severely limited. Dynamic Light Scattering (DLS) has also been used in this application but being an ensemble measurement, the results comprise either a simple z-average (intensity weighted) particle size and polydispersity or a very limited-resolution particle size distribution profile. Crucially, no particle concentration information is available and DLS cannot measure fluorescently-labeled particles. Electron microscopy is a useful research tool for studying micro– and nanovesicles but at the expense of capital running costs, extensive sample preparation, slow throughput time and sample integrity following sample preparation.
Selectivity of Measurement
Whilst it is frequently adequate to determine merely whether particles of a certain size or size range are present in a sample, it is often much more important to identify and discriminate specific sub-populations of particles within the sample. The NanoSight technique is capable of selectively analyzing such populations through, for instance, the use of antibody-mediated fluorescent labeling. This approach allows the user to detect, analyze and count only the specific nanoparticles to which the fluorescently-labeled antibody binds, with background non-specific particulates being excluded through the use of appropriate optical filters. Whilst a range of fluorophores can be used, it is advantageous to employ high-efficiency, high stability quantum dot labels for best results.
This is demonstrated in Figure 2A (below) which shows a single video frame of fluorescent light emitted from quantum dots excited by a NanoSight instrument fitted with an appropriate blue laser diode (405 nm). These quantum dots were used to label an antibody (NDOG II) specific to the target biomarker present on a syncytiotrophoblast microvesicle (STBM).
Figure 2B shows three size distributions showing: i) all particles present in the STBM sample (blue line) as detected by (non-fluorescent) light scatter; ii) the particles to which the fluorescent QDot-labeled NDOG II antibody had bound specifically, as measured under fluorescence mode (red line); and iii) a control (green line) comprising a similar QDot-labeled antibody but where the antibody has no affinity to the target biomarker on the STBM (also measured in fluorescence mode). This shows that the majority of the particles present in the sample were successfully and specifically labeled by the STBM-specific Q-Dot-NDOG II antibody and that the control successfully showed a very low signal.
A) Fluorescence image from quantum dots attached via antibody to STBM particles.
B) Particle size distributions from scattered light (blue), correct antibody (red) and incorrect (control) antibody (green). Note the number concentration vertical axis.
NanoSight NTA can size and count both microvesicles and exosomes at a low concentration and, when used in conjunction with fluorescent labels, can selectively determine and analyze specific types of particle with a complex sample.