Atomic Force Microscopy Measurement of Extracellular Vesicles Derived from Plasma

The remarkable feature of atomic force microscopy (AFM) is its abil-ity to “view” details at the atomic and molecular levels. This makes AFM a suitable method to detect and quantify extracellular vesicles (EVs). AFM operated in fluid tapping mode allows the detection of EVs, while applying a minimal force, thereby preserving their natural state. Monoclonal antibodies immobilized on a modified mica surface enable capturing subsets of EVs. AFM allows nanoscale meas-urements of individual EVs and simultaneously measures the 3D size of the EVs. The numbers of EVs attached to the antibody-coated surface can be quantified by using image processing software. For the first time we have shown that AFM detects 1,000-fold more CD41-positive EVs than flow cytometry (FCM) does. These EVs have sizes ranging between 10 nm and 475 nm with a peak at 67.5 nm, which is clearly below the detection limit of conventional FCM. This AFM method was also used to detect EVs bearing tissue factor (TF) antigen. We also demonstrated the feasibility of the AFM method combined with a microfluidic method to detect and quantify CD41positive EVs directly in plasma, reducing time between venepuncture and EV measurement and also preventing EV loss during the isolation procedure. Future research will focus on further ways to improve and standardize the AFM method for EV measurement, its use to optimize preanalytical variables of EV preparation, and quantification of EV subsets in clinical samples. Ultimately, the accurate measurement of EVs may contribute to the development of EVs as a diagnostic tool and possible prognostic and/or predictive (bio)markers in various diseases. 9.1 Introduction: Atomic Force Microscopy Invented in 1986 by Binnig, Quate, and Gerber,1 the atomic force microscope is one of a family of scanning probe microscopes that produces three-dimensional (3D) images. The atomic force microscope, with its ability to produce images of exquisitely high resolution (down to the Ångstrom range), was originally developed for its use in the physical sciences. An atomic force microscope measures the forces between a nanostylus (fine tip) and a sample immobilized on an atomically flat substrate, usually mica or gold.2,3 The tip is attached to the free end of a microcantilever and is brought very close to the surface of the sample. Attractive or repulsive forces resulting from interactions between the tip and the surface will cause positive or negative bending of the cantilever. A laser beam is reflected on the back of the cantilever, and the upward and downward deviations of the cantilever are read by a sensitive photosensor monitor.4 Figure 9.1 shows the basic concept of an atomic force microscope. The piezoelectric ceramic transducer, probe, and photodiode are the three major components in atomic force microscopy (AFM).4The piezoelectric ceramic transducer expands or contracts in the presence of a voltage gradient, and conversely, it develops an electrical potential in response to mechanical pressure, enabling movements in x-, y-, and z-directions. The probe represents a microcantilever bearing a sharp tip at one end. Cantilevers are usually made from silicon (Si) or silicon nitride (Si3N4). Different cantilever lengths, materials, and shapes allow for varied spring constants and resonant frequencies. A photodiode collects the signal of the laser beam deflection from the back of the cantilever.