Combination of structured illumination and single molecule localization microscopy in one setup

Abstract

Understanding the positional and structural aspects of biological nanostructures simultaneously is as much a challenge as a desideratum. In recent years, highly accurate (20 nm) positional information of optically isolated targets down to the nanometer range has been obtained using single molecule localization microscopy (SMLM), while highly resolved (100 nm) spatial information has been achieved using structured illumination microscopy (SIM). In this paper, we present a high-resolution fluorescence microscope setup which combines the advantages of SMLM with SIM in order to provide high-precision localization and structural information in a single setup. Furthermore, the combination of the wide-field SIM image with the SMLM data allows us to identify artifacts produced during the visualization process of SMLM data, and potentially also during the reconstruction process of SIM images. We describe the SMLM-SIM combo and software, and apply the instrument in a first proof-of-principle to the same region of H3K293 cells to achieve SIM images with high structural resolution (in the 100 nm range) in overlay with the highly accurate position information of localized single fluorophores. Thus, with its robust control software, efficient switching between the SMLM and SIM mode, fully automated and user-friendly acquisition and evaluation software, the SMLM-SIM combo is superior over existing solutions.

Superresolution light microscopy shows nanostructure of carbon ion radiation‐induced DNA double‐strand break repair foci

“…The technical specifications of the SIM/SPDM combination microscope used here are described in detail elsewhere (34). Briefly, the microscopy mode can be switched by selecting between 2 separate excitation light beam paths using electronically controlled flip mirrors.…”

“…Our microscope setup combines structured illumination microscopy (SIM) and spectral precision distance microscopy (SPDM), allowing subsequent acquisitions of the same cell in both modes (34). SIM uses a special illumination pattern to improve the resolution up to 2-fold (27,28,35).…”

Correlative multicolor 3D SIM and STORM microscopy

“…Since both methods are based on wide field microscopy, they are particularly amenable to a combined approach. That this can be useful has been exemplified by a proof-of-principle study using a custom built microscope [5]. However, this setup did not implement the use of multicolor labeling or of 3D information, despite these modalities being critical for solving most biological questions.…”

Perspectives in Super-Resolved Fluorescence Microscopy: What Comes Next?

“…Development of novel probes with low molecular weight and increased photostability in switching-based super-resolution microscopy is currently a major field of research. A combination of structured illumination excitation and other SRM techniques [38] such as "photostability" based localization microscopy at low intensities may be extremely useful also to further enhance substantially the optical resolution [39]: Computer simulations as well as proof-of-principle experiments have shown that the localization of a fluorescent point source by structured illumination excitation may enhance the localization accuracy and hence the optical resolution up to an order of magnitude, depending on the photon yield [40]. The underlying principle is similar to determining the position of the central maximum of the Airy disk from stochastic sampling of the diffraction pattern in 2D localization microscopy [41,42].…”