scholarly journals Direct Synthesis of Gold Nanoparticles on Cysteine-rich Tags in Mammalian Cells

2020 ◽  
Author(s):  
Zhaodi Jiang ◽  
Xiumei Jin ◽  
Yuhua Li ◽  
Sitong Liu ◽  
Xiao-Man Liu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Gold Nanoparticles ◽  
Single Molecule ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Direct Synthesis ◽  
Single Molecule Detection ◽  
Chemical Fixation ◽  
High Pressure Freezing ◽  
In Cells

Abstract We developed a novel auto-nucleation suppressed mechanism (ANSM) for direct synthesis of EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags (e.g., metallothionein) in cells for single-molecule detection with electron microscopy (it accompanies our Nature Method manuscript, Jiang et al. 2020 [1]). Both tagged-fusion proteins expressed in cells (e.g.bacteria, yeast and mammalian cells) and antigens stained with antibody-tag fusion proteins can be visualized by this protocol. Here we describe the typical protocols (both the chemical fixation and the high pressure freezing cases) developed for ANSM-based AuNP synthesis in HeLa cells expressing metallothionein (MTn) tags (Figure 1). This approach should be widely applicable to many systems for EM visualization of single-molecule in mammalian cells.

scholarly journals Direct Synthesis of Gold Nanoparticles on Cysteine-rich Tags in Yeast Cells

2020 ◽  
Author(s):  
Zhaodi Jiang ◽  
Xiumei Jin ◽  
Yuhua Li ◽  
Sitong Liu ◽  
Xiao-Man Liu ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Single Molecule ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Direct Synthesis ◽  
Yeast Cells ◽  
Bacterial Cells ◽  
Chemical Fixation ◽  
High Pressure Freezing ◽  
In Cells

Abstract We developed a novel auto-nucleation suppressed mechanism (ANSM) for direct synthesis of EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags (e.g., metallothionein) in cells for single-molecule detection with electron microscopy (it accompanies our Nature Method manuscript, Jiang et al. 2020 [1]). Both tagged-fusion proteins expressed in cells (e.g.bacteria, yeast and mammalian cells) and antigens stained with antibody-tag fusion proteins can be visualized by this protocol. Here we describe the typical protocols (both the chemical fixation and the high pressure freezing cases) developed for ANSM-based AuNP synthesis in yeast cells expressing metallothionein (MTn) tags (Figure 1). This approach should be useful for EM visualization of single-molecule in yeast cells, and easier adapted for bacterial cells.

scholarly journals Direct synthesis of EM-visible gold nanoparticles on genetically encoded tags for single-molecule visualization in cells

2019 ◽  
Author(s):  
Zhaodi Jiang ◽  
Xiumei Jin ◽  
Yuhua Li ◽  
Sitong Liu ◽  
Xiao-Man Liu ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Single Molecule ◽  
Direct Synthesis ◽  
Spindle Pole ◽  
Nuclear Pores ◽  
Single Molecule Level ◽  
Spindle Pole Bodies ◽  
Suppression Mechanism ◽  
Specific Synthesis ◽  
In Cells

AbstractSingle-molecule visualization in cells with genetically encoded tags for electron microscopy (EM) has been a long-awaited but unimplemented tool for cell biologists. Here, we report an approach for directly synthesizing EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags for single-molecule visualization in cells. We first uncovered an auto-nucleation suppression mechanism that allows specific synthesis of AuNPs on isolated cysteine-rich tags. We next exploited this mechanism to develop an approach for single-molecule detection of proteins in prokaryotic cells and achieved an unprecedented labeling efficiency. We then expanded it to more complicated eukaryotic cells and successfully detected the proteins targeted to various organelles, including the membranes of endoplasmic reticulum (ER) and nuclear envelope, ER lumen, nuclear pores, spindle pole bodies, and mitochondrial matrix. Thus, our implementation of genetically encoded tags for EM should allow cell biologists to address an enormous range of biological questions at single-molecule level in diverse cellular ultrastructural contexts without using antibodies.

Sizing Dna Fragments by Ultrasensitive Flow Cytometry

2001 ◽  
Vol 7 (S2) ◽  
pp. 612-613
Author(s):  
James H. Jett ◽  
Robert C. Habbersett ◽  
Xiaomei Yan ◽  
Thomas M. Yoshida ◽  
Babetta L. Marrone ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Molecule ◽  
Restriction Enzyme ◽  
Mammalian Cells ◽  
Fragment Size ◽  
Single Molecule Detection ◽  
Size Analysis ◽  
Restriction Enzyme Digestion ◽  
Flow Cytometric ◽  
Dna Fragment

As originally developed in the 1960's, flow cytometry was primarily a technique for the analysis of mammalian cells. Analysis of cellular constituents such as DNA or cell surface antigens made fluorescent by a variety of reagents has been the main stay of flow cytometric applications. Over the years, flow cytometric analysis techniques have been developed that range from multicellular spheroids containing a million or more cells down to single molecule detection. An outgrowth of single molecule detection capability is DNA fragment size analysis.DNA fragment size analysis starts with a sample of naked DNA that can be derived from a variety of sources including PCR products, double stranded viral genomes, BAC/PAC clones, and bacterial genomes. For genomic or cloned DNA, restriction enzyme digests are analyzed to produce a fingerprint pattern. The fingerprint, i. e., the distribution of fragment sizes produced by the restriction enzyme digestion, is characteristic of the source of DNA and forms the basis for identifying the source.

Genetically encoded tags for direct synthesis of EM-visible gold nanoparticles in cells

2020 ◽  
Vol 17 (9) ◽  
pp. 937-946 ◽  
Author(s):  
Zhaodi Jiang ◽  
Xiumei Jin ◽  
Yuhua Li ◽  
Sitong Liu ◽  
Xiao-Man Liu ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Direct Synthesis ◽  
In Cells

scholarly journals Author Correction: Genetically encoded tags for direct synthesis of EM-visible gold nanoparticles in cells

2020 ◽  
Vol 17 (11) ◽  
pp. 1167-1167
Author(s):  
Zhaodi Jiang ◽  
Xiumei Jin ◽  
Yuhua Li ◽  
Sitong Liu ◽  
Xiao-Man Liu ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Direct Synthesis ◽  
In Cells

scholarly journals Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Natalya Korogod ◽  
Carl CH Petersen ◽  
Graham W Knott
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Blood Vessels ◽  
Extracellular Space ◽  
Synaptic Vesicles ◽  
Adult Mouse ◽  
Chemical Fixation ◽  
High Pressure Freezing ◽  
Cellular Compartments ◽  
Brain Ultrastructure

Analysis of brain ultrastructure using electron microscopy typically relies on chemical fixation. However, this is known to cause significant tissue distortion including a reduction in the extracellular space. Cryo fixation is thought to give a truer representation of biological structures, and here we use rapid, high-pressure freezing on adult mouse neocortex to quantify the extent to which these two fixation methods differ in terms of their preservation of the different cellular compartments, and the arrangement of membranes at the synapse and around blood vessels. As well as preserving a physiological extracellular space, cryo fixation reveals larger numbers of docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses and blood vessels compared to chemical fixation. The ultrastructure of mouse neocortex therefore differs significantly comparing cryo and chemical fixation conditions.

scholarly journals A robust method for particulate detection of a genetic tag for 3D electron microscopy

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
James Rae ◽  
Charles Ferguson ◽  
Nicholas Ariotti ◽  
Richard I Webb ◽  
Han-Hao Cheng ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fusion Proteins ◽  
Ion Beam ◽  
Expression System ◽  
Internal Standard ◽  
Membrane Vesicles ◽  
Cytoskeletal Proteins ◽  
Electron Microscopic ◽  
Simple Method ◽  
In Cells

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy. Here we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal to noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins and cytoskeletal proteins. The method can be combined with different electron microscopic techniques including fast freezing and freeze substitution, focussed ion beam scanning electron microscopy, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.

High-pressure freezing of cells in suspension for low-voltage scanning electron microscopy (LVSEM)

1991 ◽  
Vol 49 ◽  
pp. 64-65
Author(s):  
Marek Malecki ◽  
James Pawley ◽  
Hans Ris
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Low Voltage ◽  
Culture Media ◽  
Cell Structure ◽  
Freeze Substitution ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Culture Media ◽  
Voltage Scanning

The ultrastructure of cells suspended in physiological fluids or cell culture media can only be studied if the living processes are stopped while the cells remain in suspension. Attachment of living cells to carrier surfaces to facilitate further processing for electron microscopy produces a rapid reorganization of cell structure eradicating most traces of the structures present when the cells were in suspension. The structure of cells in suspension can be immobilized by either chemical fixation or, much faster, by rapid freezing (cryo-immobilization). The fixation speed is particularly important in studies of cell surface reorganization over time. High pressure freezing provides conditions where specimens up to 500μm thick can be frozen in milliseconds without ice crystal damage. This volume is sufficient for cells to remain in suspension until frozen. However, special procedures are needed to assure that the unattached cells are not lost during subsequent processing for LVSEM or HVEM using freeze-substitution or freeze drying. We recently developed such a procedure.

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