The study of a new family of phase masks for three-dimensional fluorescence nanoscopy

Aleksei Gorshelev, Ivan Eremchev, Sergei Kulik, Andrei Naumov, Evgeniy Vorontsov, Vladimir Volostnikov, Svetlana Kotova, K.R. Karimullin, A.V. Naumov, M.G. Gladush
2018 EPJ Web of Conferences  
The question of the axial coordinate determination accuracy is quite sophisticated in far field 3D fluorescence nanoscopy. The accuracy of a point emitter axial coordinate reconstruction depends among other things on the conversion efficiency of the phase mask and the microscope objective instrumental function. It was found that the instrumental functions of different microscope objectives differ significantly from each other, most have a strongly non-uniform spatial distribution of the
more » ... tion of the radiation intensity in a parallel beam created by an objective focused on a point emitter. It was shown that taking into account the actual microscope objective instrumental function when calculating the phase masks allows to increase significantly the axial coordinate reconstruction accuracy. Fluorescence microscopy with ultra-high spatial resolution (far field fluorescence nanoscopy, FFN) is currently one of the most effective and in-demand tools for research in the fields of biological and medical physics, biology, and chemical physics. It is also of interest for applications in solid state physics. Many of these far field fluorescence nanoscopy techniques (STORM, PALM, etc.) are based on the possibility of fluorescence imaging of single point-like emitting labels with nanoscale reconstruction of their lateral spatial coordinates, and subsequent computer rendering of object nanostructure by mapping plenty single labels positions (see [1, 2] and references therein). Application of the technique based on the theory of spiral beam optics allows to reconstruct all three spatial coordinates of a single point emitter with nanometer accuracy. In this technique the spatial phase modulation of the light beam is used for the transformation of the single emitter point spread function into double helix point spread function [3] . The accuracy of a point emitter axial coordinate reconstruction depends on a variety of experiment parameters: the number of recorded photons, the conversion efficiency of a phase mask, the microscope objective instrumental function, and the characteristics of the optical scheme used in the registration channel. FFN methods can be especially informative if fluorescent single molecules (SM) of organic dyes are used as emitting point light sources at cryogenic temperatures, when zero-* Corresponding author: gorshelev@isan.troitsk.ru
doi:10.1051/epjconf/201819004007 fatcat:625hlvptpzcmjkpykjwpjpdkc4