Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter.

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The optical diffraction limit has been the dominant barrier to achieving higher optical resolution in the fields of microscopy, photolithography, and optical data storage. We present here an approach toward imaging below the diffraction barrier. Through the exposure of photosensitive films placed a finite and known distance away from nanoscale, zero-mode apertures in thin metallic films, we show convincing, physical evidence that the propagating component of light emerging from these apertures shows a very strong degree of collimation well past the maximum extent of the near-field (0/4n-0/2n). Up to at least 2.5 wavelengths away from the apertures, the transmitted light exhibits subdiffraction limit irradiance patterns. These unexpected results are not explained by standard diffraction theory or nanohole-based ''beaming'' rationalizations. This method overcomes the diffraction barrier and makes super-resolution fluorescence imaging practical.fluorescence microscopy ͉ nanoholes ͉ subdiffraction limit ͉ subwavelength imaging ͉ super-resolution T he spatial resolution of an optical system operating outside of the optical near-field can given by the Rayleigh criterion (1),where is the wavelength of the emitted photons and NA is the numerical aperture of the system. This criterion says that two point sources can be ''just resolved'' when their separation is W. The 0.61 term arises from the superposition of two Fraunhofer diffraction patterns for circular apertures (Airy disks) such that the principle maximum of one of the patterns coincides with the first minimum of the other. This superposition of two sources at distance W results in an intensity distribution of two peaks with a valley between with a contrast ratio (ratio of peak to valley intensities) of 0.81.In an effort to circumvent this limit, a method was first proposed in which light is leaked through an aperture much smaller than the wavelength in an opaque screen (2). Because the light through such an aperture is known to diffract heavily and the power flux through such an aperture is evanescent, the aperture must be placed in the optical near-field of the probe. This method, practically realized, is called near-field scanning optical microscopy (NSOM) (3-5). Super-resolution (resolution values smaller than W, above) with NSOM has been demonstrated often. NSOM, however, suffers from the low photon flux through a zero-mode waveguide (6, 7) and the requirement of strict maintenance of surface-to-aperture distance within a few nanometers (8). Parallel probe NSOM approaches have been proposed and developed in which a plate with periodic perforations of zero-mode waveguides is placed on top of a sample in the optical near-field (9, 10).The behavior of light emitted from periodically perforated arrays of subwavelength apertures in metal films on mesoscopic length scales, between the length regime where light classically propagates (d Ͼ Ͼ ; the far-field) and the range where it displays evanescent properties (shorter than half of a wavelength; the near-field), is ...