Super-resolution microscopy with very large working distance by means of distributed aperture illumination

Abstract

The limits of conventional light microscopy (“Abbe-Limit“) depend critically on the numerical aperture (NA) of the objective lens. Imaging at large working distances or a large field-of-view typically requires low NA objectives, thereby reducing the optical resolution to the multi micrometer range. Based on numerical simulations of the intensity field distribution, we present an illumination concept for a super-resolution microscope which allows a three dimensional (3D) optical resolution around 150 nm for working distances up to the centimeter regime. In principle, the system allows great flexibility, because the illumination concept can be used to approximate the point-spread-function of conventional microscope optics, with the additional benefit of a customizable pupil function. Compared with the Abbe-limit using an objective lens with such a large working distance, a volume resolution enhancement potential in the order of 104 is estimated.

Nylon as an in vitro scaffold for three‐dimensional study of neural cells

“…Due to laser intensity related photobleaching and the requirement for fixation, samples do not survive well after 1 h of confocal microscopy. Live cell assays however, such as the Live/Dead assay conducted in this study, are an effective alternative . This highlights the advantageous use of nylon mesh in 3D live cell imaging assay.…”

“…Live cell assays however, such as the Live/Dead assay conducted in this study, are an effective alternative. 35 This highlights the advantageous use of nylon mesh in 3D live cell imaging assay. Furthermore, when cells were cultured in single cell type cultures or coculture, they were able to proliferate on the nylon surface and were often noted to spread along the longitudinal axis of the nylon fiber.…”

Ring Array Illumination Microscopy: Combination of Super-Resolution with Large Field of View Imaging and long Working Distances

“…Instead of holograms, distributed aperture illumination arrays can also be applied (Birk et al 2017): here, a limited number of individual collimated coherent beams are used, where intensity, direction, and phase are individually controlled compared to the very large number of diffracted beams (e.g., hundreds of thousands) generated by the hologram structures with coupled intensities, directions, and phases. This has the great advantage that the light sources can in principle be positioned at any distance from the scanning focus area ("spot") where constructive interference occurs; by changing the direction of the collimated coherent beams accordingly, the scanning area ("spot") can be moved to any position in 3D.…”

Super-Resolution Microscopy of Chromatin

“…To circumvent this, researchers have employed particular illumination schemes in order to facilitate fluorescence excitation inside the focal plane only. This technique is known from light-sheet fluorescence microscopy (LSFM) and related approaches [67,68,69], and can readily be combined with illumination grid patterns as required for SIM [70]. Apart from reducing the out-of-focus blur, the cells are much less exposed to illumination light, therefore the technique is muss less invasive.…”