RESUMEN
We study the conditions under which fluorescent beads can be used to emulate single fluorescent molecules in the calibration of optical microscopes. Although beads are widely used due to their brightness and easy manipulation, there can be notable differences between the point spread functions (PSFs) they produce and those for single-molecule fluorophores, caused by their different emission patterns and sizes. We study theoretically these differences for various scenarios, e.g., with or without polarization channel splitting, to determine the conditions under which the use of beads as a model for single molecules is valid. We also propose methods to model the blurring due to the size difference and compensate for it to produce PSFs that are more similar to those for single molecules.
Asunto(s)
Colorantes Fluorescentes , CalibraciónRESUMEN
Complete bases that are useful for beam propagation problems and present the distinct property of being spatially confined at the initial plane are proposed. These bases are constructed in terms of polynomials of Gaussians, in contrast with standard alternatives, such as the Hermite-Gaussian basis, that are given by a Gaussian times a polynomial. The property of spatial confinement implies that, for all basis elements, the spatial extent at the initial plane is roughly the same. This property leads to an optimal scaling parameter that is independent of truncation order for the fitting of a confined initial field. Given their form as combinations of Gaussians, the paraxial propagation of these basis elements can be modeled analytically.
RESUMEN
We derive bases constructed from simple vortices and complex focus fields and show that they are useful in the description of strongly focused fields. Both scalar and electromagnetic fields are considered, and in each case two types of basis are discussed: bases that use standard polynomials but whose orthogonality condition requires a non-uniform directional weight factor, and bases that are orthogonal with uniform weight but that require new polynomials. Their performance is studied by fitting prescribed fields, where it is seen that the accuracy provided by both types of bases is comparable.
RESUMEN
A novel type of discrete basis for paraxial beams is proposed, consisting of monomial vortices times polynomials of Gaussians in the radial variable. These bases have the distinctive property that the effective size of their elements is roughly independent of element order, meaning that the optimal scaling for expanding a localized field does not depend significantly on truncation order. This behavior contrasts with that of bases composed of polynomials times Gaussians, such as Hermite-Gauss and Laguerre-Gauss modes, where the scaling changes roughly as the inverse square root of the truncation order.
