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 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 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 Ground-State Depletion Nanoscopy of Nitrogen-Vacancy Centres in Nanodiamonds

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jelle Storterboom ◽  
Martina Barbiero ◽  
Stefania Castelletto ◽  
Min Gu
Keyword(s):  
Single Molecule ◽  
Ground State ◽  
Nitrogen Concentration ◽  
Super Resolution ◽  
Optical Power ◽  
Stimulated Emission ◽  
Nitrogen Vacancy ◽  
Resolution Imaging ◽  
Localisation Microscopy ◽  
Bulk Diamond

AbstractThe negatively charged nitrogen-vacancy ($${\text{NV}}^{ - }$$ NV - ) centre in nanodiamonds (NDs) has been recently studied for applications in cellular imaging due to its better photo-stability and biocompatibility if compared to other fluorophores. Super-resolution imaging achieving 20-nm resolution of $${\text{NV}}^{ - }$$ NV - in NDs has been proved over the years using sub-diffraction limited imaging approaches such as single molecule stochastic localisation microscopy and stimulated emission depletion microscopy. Here we show the first demonstration of ground-state depletion (GSD) nanoscopy of these centres in NDs using three beams, a probe beam, a depletion beam and a reset beam. The depletion beam at 638 nm forces the $${\text{NV}}^{ - }$$ NV - centres to the metastable dark state everywhere but in the local minimum, while a Gaussian beam at 594 nm probes the $${\text{NV}}^{ - }$$ NV - centres and a 488-nm reset beam is used to repopulate the excited state. Super-resolution imaging of a single $${\text{NV}}^{ - }$$ NV - centre with a full width at half maximum of 36 nm is demonstrated, and two adjacent $${\text{NV}}^{ - }$$ NV - centres separated by 72 nm are resolved. GSD microscopy is here applied to $${\text{NV}}^{ - }$$ NV - in NDs with a much lower optical power compared to bulk diamond. This work demonstrates the need to control the NDs nitrogen concentration to tailor their application in super-resolution imaging methods and paves the way for studies of $${\text{NV}}^{ - }$$ NV - in NDs’ nanoscale interactions.

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