SPDM: light microscopy with single-molecule resolution at the nanoscale (original) (raw)

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

Journal of Optics, 2013

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.

SPDM: single molecule superresolution of cellular nanostructures

2009

Novel methods of visible light microscopy have overcome the limits of resolution hitherto thought to be insurmountable. The localization microscopy technique presented here based on the principles of Spectral Precision Distance Microscopy (SPDM) with conventional fluorophores under special physical conditions allows to measure the spatial distribution of single fluorescence labeled molecules in entire cells with macromolecular precision which is comparable to a macromolecular effective optical resolution. Based on detection of single molecules, in a novel combination of SPDM and Spatially Modulated Illumination (SMI) microscopy, a lateral (2D) effective optical resolution of cellular nanostructures around 10 - 20 nm (about 1/50th of the exciting wavelength) and a three dimensional (3D) effective optical resolution in the range of 40 - 50 nm are achieved.

A common framework for single-molecule localization using sequential structured illumination

Biophysical Reports, 2022

Localization of single fluorescent molecules is key for physicochemical and biophysical measurements such as single-molecule tracking and super-resolution imaging by singlemolecule localization microscopy (SMLM). Recently a series of methods have been developed in which the localization precision is enhanced by interrogating the molecular position with a sequence of spatially modulated patterns of light. Among them, the MINFLUX technique outstands for achieving a ~10-fold improvement compared to wide-field camera-based singlemolecule localization, reaching ~1 − 2 nm localization precision at moderate photon counts. Here, we present a common mathematical framework for this type of measurement that allows a fair comparison between reported methods and facilitates the design and evaluation of new methods. With it, we benchmark all reported methods for single-molecule localization using sequential structured illumination, including long-established methods such as orbital tracking, along with two new proposed methods: orbital tracking and raster scanning with a minimum of intensity.

Doubling the resolution of single-molecule localization microscopy with image scanning microscopy

Single-molecule localization microscopy (SMLM) is a widely used super-resolution microscopy technique, renowned for its simplicity and impressive achievable resolution. It is typically based on a wide-field fluorescence microscope and relies on emitter photoswitching to capture individual snapshots of a sample with sparse distributions of fluorescent labels. These labels can then be precisely identified and localized. Recently, we demonstrated that SMLM can also be realized with a fast confocal laser-scanning microscope (CLSM), opening the door to fluorescence lifetime SMLM. This technique has found applications in lifetime-based image multiplexing and metal-induced energy transfer SMLM.In this work, we present an extension of CLSM-based SMLM by incorporating a single-photon detector array into the CLSM. This enables the combination of CLSM-based SMLM with Image Scanning Microscopy (ISM), a powerful technique for doubling the lateral resolution of a laser-scanning confocal microscop...

Single Molecule Light Field Microscopy

bioRxiv, 2020

We introduce single molecule light field microscopy (SMLFM), a novel 3D single molecule localization technique that is capable of up to 20 nm isotropic precision across a 6 μm depth of field. SMLFM can be readily implemented by installing a refractive microlens array into the conjugate back focal plane of any widefield single molecule localization system. We demonstrate that 3D localization can be performed by post-processing 2D localization data generated by common, widely-used, algorithms. In this work we benchmark the performance of SMLFM and finally showcase its capabilities by imaging fluorescently labeled membranes of fixed eukaryotic cells below the diffraction limit.

Simultaneous near-field and far-field fluorescence microscopy of single molecules

Optics Express, 2011

A new microscope objective is presented for the parallel fluorescence detection below and above the critical angle of total internal reflection with single molecule sensitivity. The collection of supercritical angle fluorescence (SAF) leads to a strongly surface confined detection volume whereas the collection of undercritical angle fluorescence (UAF) allows for the observation of deeper axial sections of the specimen. By simultaneous detection of the near-field-mediated SAF and the far-field UAF emission modes the z-position of emitters can be obtained on the nanometer scale. We investigate the point spread function of the optics and demonstrate nanoscopic z-localization of single molecules. The oil immersion objective, developed for use on common microscope bodies, opens up new possibilities for the study of topographies and dynamics at surfaces and on membranes.