RESUMEN

Accurate characterization of extracellular vesicles (EVs) is critical to explore their diagnostic and therapeutic applications. As the EV research field has developed, so too have the techniques used to characterize them. The development of reference materials are required for the standardization of these techniques. This work, initiated from the ISEV 2017 Biomarker Workshop in Birmingham, UK, and with further discussion during the ISEV 2019 Standardization Workshop in Ghent, Belgium, sets out to elucidate which reference materials are required and which are currently available to standardize commonly used analysis platforms for characterizing EV refractive index, epitope abundance, size and concentration. Due to their predominant use among EV researchers, a particular focus is placed on the optical methods nanoparticle tracking analysis and flow cytometry.

RESUMEN

The judgment of the imaging quality of an optical system can be carried out by examining its through-focus intensity distribution. It has been shown in a previous paper that a scalar-wave analysis of the imaging process according to the extended Nijboer-Zernike theory allows the retrieval of the complex pupil function of the imaging system, including aberrations as well as transmission variations. However, the applicability of the scalar analysis is limited to systems with a numerical aperture (NA) value of the order of 0.60 or less; beyond these values polarization effects become significant. In this scalar retrieval method, the complex pupil function is represented by means of the coefficients of its expansion in a series involving the Zernike polynomials. This representation is highly efficient, in terms of number and magnitude of the required coefficients, and lends itself quite well to matching procedures in the focal region. This distinguishes the method from the retrieval schemes in the literature, which are normally not based on Zernike-type expansions, and rather rely on point-by-point matching procedures. In a previous paper [J. Opt. Soc. Am. A 20, 2281 (2003)] we have incorporated the extended Nijboer-Zernike approach into the Ignatowsky-Richards/Wolf formalism for the vectorial treatment of optical systems with high NA. In the present paper we further develop this approach by defining an appropriate set of functions that describe the energy density distribution in the focal region. Using this more refined analysis, we establish the set of equations that allow the retrieval of aberrations and birefringence from the intensity point-spread function in the focal volume for high-NA systems. It is shown that one needs four analyses of the intensity distribution in the image volume with different states of polarization in the entrance pupil. Only in this way will it be possible to retrieve the "vectorial" pupil function that includes the effects of birefringence induced by the imaging system. A first numerical test example is presented that illustrates the importance of using the vectorial approach and the correct NA value in the aberration retrieval scheme.

Asunto(s)

RESUMEN

Taking the classical Ignatowsky/Richards and Wolf formulas as our starting point, we present expressions for the electric field components in the focal region in the case of a high-numerical-aperture optical system. The transmission function, the aberrations, and the spatially varying state of polarization of the wave exiting the optical system are represented in terms of a Zernike polynomial expansion over the exit pupil of the system; a set of generally complex coefficients is needed for a full description of the field in the exit pupil. The field components in the focal region are obtained by means of the evaluation of a set of basic integrals that all allow an analytic treatment; the expressions for the field components show an explicit dependence on the complex coefficients that characterize the optical system. The electric energy density and the power flow in the aberrated three-dimensional distribution in the focal region are obtained with the expressions for the electric and magnetic field components. Some examples of aberrated focal distributions are presented, and some basic characteristics are discussed.