The Limitations of Nonlinear Fluorescence Effect in Super Resolution Saturated Structured Illumination Microscopy System

Abstract

Classically, optical systems are considered to have a fundamental resolution limit due to diffraction. Many strategies for improving both axial and lateral resolutions are based on a priori information about the input signal. These strategies lead to a numerical aperture improvement. However these are still limited by the wave nature of light. By using fluorescence technique one theoretically can reach unlimited resolution. The key point is to use the nonlinear dependence of the fluorescence emission rate on the intensity of the applied illumination. In this paper we present simulation as well as experimental results which show the advantage and the problems of using the nonlinear fluorescence effect in super resolution systems as well as discussing the nonlinear phenomena concerning the fluorescence process. The results show that the nonlinear fluorescence effect is accompanied by severe quenching, bleaching and saturation phenomena. As consequence, super resolution using saturated structured illumination method in living biological samples becomes severely restricted.

Near-field projection optical microscope (NPOM) as a new approach to nanoscale super-resolved imaging

“…In the field of super-resolution, important contributions and inventions were published in the last two decades 1 , 2 . More specifically, special methods were invented like structured illumination 3 – 5 , localized microscopy super-resolved imaging 6 – 8 , non-linear microscopy 9 , and the single pixel-based imaging 10 .…”

Compressive three-dimensional super-resolution microscopy with speckle-saturated fluorescence excitation

“…Indeed, in tightly focused beams, the vectorial nature of light cannot be neglected, especially under saturated conditions, in which case the axial field may destroy most of intensity zeros. Whether it is possible to break the diffraction barrier with saturated speckle patterns has remained an open theoretical question 30 .…”

“…Optical vortices in speckles are associated with nodal lines of intensity (in 3D) that can confine fluorescence to sub-diffraction dimensions [13], despite the contribution of the axial field. However, whether it is possible to break the diffraction barrier with saturated speckle patterns has remained an open theoretical question [30].…”

Sub-100 nm resolution microscopy based on proximity projection grating scheme

“…In order to improve the resolution of SIM further, nonlinear SIM (NSIM) 15 or saturated patterned excitation microscopy (SPEM) 16 have been proposed. However, these techniques employ nonlinear saturation effects in fluorophores, which can lead to severe photobleaching and has therefore restricted application in biological imaging 17 .…”