scholarly journals Cryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy

2019 ◽  
Vol 116 (11) ◽  
pp. 4804-4809 ◽  
Author(s):  
Felipe Moser ◽  
Vojtěch Pražák ◽  
Valerie Mordhorst ◽  
Débora M. Andrade ◽  
Lindsay A. Baker ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Low Dose ◽  
Super Resolution ◽  
Biological Application ◽  
High Laser ◽  
Environmental Background ◽  
Special Sample Preparation ◽  
Reduce Image Quality ◽  
Resolution Imaging

Correlative light and electron cryo-microscopy (cryo-CLEM) combines information from the specific labeling of fluorescence cryo-microscopy (cryo-FM) with the high resolution in environmental context of electron cryo-microscopy (cryo-EM). Exploiting super-resolution methods for cryo-FM is advantageous, as it enables the identification of rare events within the environmental background of cryo-EM at a sensitivity and resolution beyond that of conventional methods. However, due to the need for relatively high laser intensities, current super-resolution cryo-CLEM methods require cryo-protectants or support films which can severely reduce image quality in cryo-EM and are not compatible with many samples, such as mammalian cells. Here, we introduce cryogenic super-resolution optical fluctuation imaging (cryo-SOFI), a low-dose super-resolution imaging scheme based on the SOFI principle. As cryo-SOFI does not require special sample preparation, it is fully compatible with conventional cryo-EM specimens, and importantly, it does not affect the quality of cryo-EM imaging. By applying cryo-SOFI to a variety of biological application examples, we demonstrate resolutions up to ∼135 nm, an improvement of up to three times compared with conventional cryo-FM, while maintaining the specimen in a vitrified state for subsequent cryo-EM. Cryo-SOFI presents a general solution to the problem of specimen devitrification in super-resolution cryo-CLEM. It does not require a complex optical setup and can easily be implemented in any existing cryo-FM system.

scholarly journals Super-Resolution Imaging of Higher-Order Chromatin Structures at Different Epigenomic States in Single Mammalian Cells

Cell Reports ◽  
2018 ◽  
Vol 24 (4) ◽  
pp. 873-882 ◽  
Author(s):  
Jianquan Xu ◽  
Hongqiang Ma ◽  
Jingyi Jin ◽  
Shikhar Uttam ◽  
Rao Fu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Super Resolution ◽  
Higher Order ◽  
Resolution Imaging

scholarly journals 2P265 Super-resolution imaging of chromatin domains in living mammalian cells(21B. Genome biology:Genome structure,Poster)

2014 ◽  
Vol 54 (supplement1-2) ◽  
pp. S239
Author(s):  
Tadasu Nozaki ◽  
Tomomi Tani ◽  
Sachiko Tamura ◽  
Takeharu Nagai ◽  
Kazuhiro Maeshima
Keyword(s):  
Mammalian Cells ◽  
Super Resolution ◽  
Chromatin Domains ◽  
Resolution Imaging

scholarly journals 3D Multicolor Nanoscopy at 10,000 Cells a Day

2019 ◽  
Author(s):  
Andrew E S Barentine ◽  
Yu Lin ◽  
Miao Liu ◽  
Phylicia Kidd ◽  
Leonhard Balduf ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
High Speed ◽  
Three Dimensional ◽  
Super Resolution ◽  
High Volume ◽  
Volume Data ◽  
Cell Nuclei ◽  
Fully Integrated ◽  
Resolution Imaging

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.

scholarly journals Ground State Depletion Super-resolution Imaging in Mammalian Cells

10.3791/56239 ◽  
2017 ◽  
Author(s):  
Rose E. Dixon ◽  
Oscar Vivas ◽  
Karen I. Hannigan ◽  
Eamonn J. Dickson
Keyword(s):  
Ground State ◽  
Mammalian Cells ◽  
Super Resolution ◽  
Resolution Imaging

scholarly journals Mechanistic insights into GLUT1 activation and clustering revealed by super-resolution imaging

2018 ◽  
Vol 115 (27) ◽  
pp. 7033-7038 ◽  
Author(s):  
Qiuyan Yan ◽  
Yanting Lu ◽  
Lulu Zhou ◽  
Junling Chen ◽  
Haijiao Xu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Membranes ◽  
Molecular Mechanisms ◽  
Glucose Transporter ◽  
Super Resolution ◽  
Average Diameter ◽  
Activation Mechanism ◽  
Membrane Expression ◽  
Close Relationship ◽  
Resolution Imaging

The glucose transporter GLUT1, a plasma membrane protein that mediates glucose homeostasis in mammalian cells, is responsible for constitutive uptake of glucose into many tissues and organs. Many studies have focused on its vital physiological functions and close relationship with diseases. However, the molecular mechanisms of its activation and transport are not clear, and its detailed distribution pattern on cell membranes also remains unknown. To address these, we first investigated the distribution and assembly of GLUT1 at a nanometer resolution by super-resolution imaging. On HeLa cell membranes, the transporter formed clusters with an average diameter of ∼250 nm, the majority of which were regulated by lipid rafts, as well as being restricted in size by both the cytoskeleton and glycosylation. More importantly, we found that the activation of GLUT1 by azide or MβCD did not increase its membrane expression but induced the decrease of the large clusters. The results suggested that sporadic distribution of GLUT1 may facilitate the transport of glucose, implying a potential association between the distribution and activation. Collectively, our work characterized the clustering distribution of GLUT1 and linked its spatial structural organization to the functions, which would provide insights into the activation mechanism of the transporter.

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

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.

scholarly journals Aberration correction for improving the image quality in STED microscopy using the genetic algorithm

Nanophotonics ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 1971-1980 ◽  
Author(s):  
Luwei Wang ◽  
Wei Yan ◽  
Runze Li ◽  
Xiaoyu Weng ◽  
Jia Zhang ◽  
...  
Keyword(s):  
Genetic Algorithm ◽  
Wave Front ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Aberration Correction ◽  
Sted Microscopy ◽  
Laser Wave ◽  
Resolution Imaging ◽  
Compensation Approach

AbstractWith a purely optical modulation of fluorescent behaviors, stimulated emission depletion (STED) microscopy allows for far-field imaging with a diffraction-unlimited resolution in theory. The performance of STED microscopy is affected by many factors, of which aberrations induced by the optical system and biological samples can distort the wave front of the depletion beam at the focal plane to greatly deteriorate the spatial resolution and the image contrast. Therefore, aberration correction is imperative for STED imaging, especially for imaging thick specimens. Here, we present a wave front compensation approach based on the genetic algorithm (GA) to restore the distorted laser wave front for improving the quality of STED images. After performing aberration correction on two types of zebrafish samples, the signal intensity and the imaging resolution of STED images were both improved, where the thicknesses were 24 μm and 100 μm in the zebrafish retina sample and the zebrafish embryo sample, respectively. The results showed that the GA-based wave front compensation approach has the capability of correction for both system-induced and sample-induced aberrations. The elimination of aberrations can prompt STED imaging in deep tissues; therefore, STED microscopy can be expected to play an increasingly important role in super-resolution imaging related to the scientific research in biological fields.

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