PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking

ABSTRACTDiffraction-unlimited single-molecule switching (SMS) nanoscopy techniques like STORM /(F)PALM enable three-dimensional (3D) fluorescence imaging at 20-80 nm resolution and are invaluable to investigate sub-cellular organization. They suffer, however, from low throughput, limiting the output of a days worth of imaging to typically a few tens of mammalian cells. Here we develop an SMS imaging platform that combines high-speed 3D single-molecule data acquisition with an automated, fully integrated, high-volume data processing pipeline. We demonstrate 2-color 3D super-resolution imaging of over 10,000 mammalian cell nuclei in about 26 hours, connecting the traditionally low-throughput super-resolution community to the world of omics approaches.

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Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling

10.1101/374058 ◽

2018 ◽

Cited By ~ 2

Author(s):

Guy Nir ◽

Irene Farabella ◽

Cynthia Pérez Estrada ◽

Carl G. Ebeling ◽

Brian J. Beliveau ◽

...

Keyword(s):

Single Molecule ◽

Three Dimensional ◽

Super Resolution ◽

Imaging Data ◽

Chromosomal Dna ◽

Contact Maps ◽

Integrative Modeling ◽

Resolution Imaging ◽

Three Dimensional Models ◽

Single Molecule Localization Microscopy

AbstractChromosome structure is thought to be crucial for proper functioning of the nucleus. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. We begin by applying Oligopaint probes and the single-molecule localization microscopy methods of OligoSTORM and OligoDNA-PAINT to image 8 megabases of human chromosome 19, discovering that chromosomal regions contributing to compartments can form distinct structures. Intriguingly, our data also suggest that homologous maternal and paternal regions may be differentially organized. Finally, we integrate imaging data with Hi-C and restraint-based modeling using a method called integrative modeling of genomic regions (IMGR) to increase the genomic resolution of our traces to 10 kb.One Sentence SummarySuper-resolution genome tracing, contact maps, and integrative modeling enable 10 kb resolution glimpses of chromosome folding.

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Restoring single-molecule localizations with wavefront sensing adaptive optics for deep-tissue super-resolution imaging

10.1101/2021.11.18.469175 ◽

2021 ◽

Author(s):

Sanghyeon Park ◽

Yonghyeon Jo ◽

Minsu Kang ◽

Jin Hee Hong ◽

Sangyoon Ko ◽

...

Keyword(s):

Adaptive Optics ◽

Single Molecule ◽

Fluorescence Emission ◽

Super Resolution ◽

Light Modulator ◽

Application Range ◽

Wavefront Distortion ◽

Resolution Imaging ◽

Localization Precision ◽

Major Factors

Specimen-induced aberration has been one of the major factors limiting the imaging depth in single-molecule localization microscopy (SMLM). In this study, we measured the wavefront of intrinsic reflectance signal at the fluorescence emission wavelength to construct a time-gated reflection matrix and find complex tissue aberration without resorting to fluorescence detection. Physically correcting the identified aberration via wavefront shaping with a liquid-crystal spatial light modulator (SLM) enables super-resolution imaging even when the aberration is too severe for initiating localization processes. We demonstrate the correction of complex tissue aberration, the root-mean-square (RMS) wavefront distortion of which is more than twice the 1 rad limit presented in previous studies; this leads to the recovery of single molecules by 77 times increased localization number. We visualised dendritic spines in mouse brain tissues and early myelination processes in a whole zebrafish at up to 102 μm depth with 28-39 nm localization precision. The proposed approach can expand the application range of SMLM to thick samples that cause the loss of localization points owing to severe aberration.

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Light Sheet Illumination for 3D Single-Molecule Super-Resolution Imaging of Neuronal Synapses

Frontiers in Synaptic Neuroscience ◽

10.3389/fnsyn.2021.761530 ◽

2021 ◽

Vol 13 ◽

Author(s):

Gabriella Gagliano ◽

Tyler Nelson ◽

Nahima Saliba ◽

Sofía Vargas-Hernández ◽

Anna-Karin Gustavsson

Keyword(s):

Single Molecule ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Three Dimensional ◽

Super Resolution ◽

Light Sheet ◽

Three Dimensions ◽

Single Molecule Imaging ◽

Single Molecule Tracking ◽

Resolution Imaging

The function of the neuronal synapse depends on the dynamics and interactions of individual molecules at the nanoscale. With the development of single-molecule super-resolution microscopy over the last decades, researchers now have a powerful and versatile imaging tool for mapping the molecular mechanisms behind the biological function. However, imaging of thicker samples, such as mammalian cells and tissue, in all three dimensions is still challenging due to increased fluorescence background and imaging volumes. The combination of single-molecule imaging with light sheet illumination is an emerging approach that allows for imaging of biological samples with reduced fluorescence background, photobleaching, and photodamage. In this review, we first present a brief overview of light sheet illumination and previous super-resolution techniques used for imaging of neurons and synapses. We then provide an in-depth technical review of the fundamental concepts and the current state of the art in the fields of three-dimensional single-molecule tracking and super-resolution imaging with light sheet illumination. We review how light sheet illumination can improve single-molecule tracking and super-resolution imaging in individual neurons and synapses, and we discuss emerging perspectives and new innovations that have the potential to enable and improve single-molecule imaging in brain tissue.

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