NanoSight

Direct Visualization, Sizing and Counting of Exosomes

Contents

1. Introduction
2. Detecting and Counting Exosomes
3. Alternative Techniques
4. Selectivity of Measurement
5. In Conclusion
6. Recent Publications on Exosome Detection and Analysis Citing NanoSight Data 

Introduction

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 100nm to 1μm, whilst exosomes are in the range 30-100nm. 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 30—1000nm 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-labelled particles using a range of excitation wavelengths.

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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).

Figure 1*. Sample of diluted platelet-free plasma.  A) A typical image produced by the NanoSight technique. The image allows the users to instantly recognise certain features about their sample including concentration and level of polydispersity. B)  Particle size distribution and calculated original concentration from the sample.

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 Alternative Techniques

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)

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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 analysing such populations through, for instance, the use of antibodymediated fluorescent labelling. This approach allows the user to detect, analyse and count only the specific nanoparticles to which the fluorescently-labelled 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 highefficiency, high stability quantum dot labels for best results.</p> <p>This is demonstrated in Figure 2A 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 (405nm). These quantum dots were used to label an antibody (NDOG II) specific to the target biomarker present on a syncytiotrophoblast microvesicle (STBM).</p> <p>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-labelled NDOG II antibody had bound specifically, as measured under fluorescence mode (red line); and iii) a control (green line) comprising a similar QDotlabelled 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 labelled by the STBM-specific Q-Dot-NDOG II antibody and that the control successfully showed a very low signal.

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In Conclusion

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 analyse specific types of particle with a complex sample.

Figure 2*:

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.

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Recent Publications on Exosome Detection and Analysis Citing NanoSight Data

1. Chris Gardiner, Rebecca Dragovic, Alexandra Brooks, Lydia Alvarez, Paul Harrison, and Ian Sargent (2009) Visualisation, sizing and counting of cellular microparticles and exosomes using Nanoparticle Tracking Analysis, Oxford Biomedical Imaging Festival, St John’s College, University of Oxford, Oxford UK 16th September 2009
2. Harrison, P. (2009) - " Circulating Microparticles : measurement and clinical significance". UKNEQAS for Blood Coagulation Meeting, June 2009, Sheffield.
3. Y. Yuana, T. H. Oosterkamp, S. Bahatyrova, B. Ashcroft, P. Garcia Rodriguez, R. M. Bertina and S. Osanto (2010) Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles, Journal of Thrombosis and Haemostasis, Volume 8 Issue 2, Pages 315 - 323
4. Harrison P, Dragovic R, Albanyan A, Lawrie AS, Murphy M, Sargent I. (2009) Application of dynamic light scattering to the measurement of microparticles. Journal of Thrombosis and Haemostasis, Volume 7, Supplement 2: Abstract OC-TU-056
5. Chris Gardiner, Rebecca Dragovic, Alexandra Brooks, Dionne Tannetta, Chris Redman, Paul Harrison, and Ian Sargent. (2010) Nanoparticle Tracking Analysis For The Measurement And Characterisation Of Cellular Microvesicles And Nanovesicles, BSHT and NVTH Joint Symposium, June 23-25th, 2010, NH Hotel Leeuwenhorst, Noordwijkerhout, The Netherlands, submitted
6. E. van der Pol, A.G. Hoekstra, A. Sturk, C. Otto, T.G. van Leeuwen and R. Nieuwland (2010) Optical and non-optical methods for detection and characterisation of microparticles and exosomes Journal of Thrombosis and Haemostasis, Accepted for publication, doi: 10.1111/j.1538-7836.2010.04074.x
7. Don A. Gabriel and Karen Giordano (2010) Microparticle Sizing and Counting Using Light Scattering Methods, Semin Thromb Hemost 36(8): 824-832
8. Helen Sheldon, Emily Heikamp, Helen Turley, Rebecca Dragovic, Peter Thomas, Chern Ein Oon, Russell Leek, Mariola Edelmann, Benedikt Kessler, Richard C. A. Sainson, Ian Sargent, Ji-Liang Li and Adrian L. Harris (2010) New mechanism for notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes, Blood, pre-published online June 17, 2010; DOI 10.1182/blood-2009-08-239228
9. Helen Vallhov, Cindy Gutzeit, Sara M. Johansson, Noémi Nagy, Mandira Paul, Qin Li, Sherree Friend, Thaddeus C. George, Eva Klein, Annika Scheynius and Susanne Gabrielsson (2010) Exosomes Containing Glycoprotein 350 Released by EBV-Transformed B Cells Selectively Target B Cells through CD21 and Block EBV Infection In Vitro The Journal of Immunology November 24, 2010 doi: 10.4049/jimmunol.1001145
10. Pia R.M. Siljander (2011) Platelet-derived microparticles – an updated perspective Proceedings of the 43rd Nordic Coagulation Meeting - Interaction between clinic and laboratory, Thrombosis Research, Volume 127, Supplement 2, January 2011, Pages S30-S33,
11. Rebecca Dragovic, Chris Gardiner, Alexandra Brooks, Dionne Tannetta, David Ferguson, Patrick Hole, Bob Carr, Chris Redman, Adrian Harris, Pete Dobson, Paul Harrison and Ian Sargent (2011) Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis, NanoMedicine, submitted

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