bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/049403

Ex-STORM: Expansion Single Molecule Nanoscopy

Hu Cang·Zhisong Tong·Paolo Beuzer·Qing Ye·Josh Axelrod·Zhenmin Hong
View original source ↗Export metadata ↓

Abstract

We present critical improvements to recently invented Expansion Microscopy (ExM), which resolve the incompatibility between ExM and single molecule super-resolution microscopy STORM. Specifically, the improved ExM circumvents the massive, 50-100%, bleaching of fluorophores during sample preparations, and preserve the efficiency of enzymatic oxygen scavenging systems in expanded samples. These improvements open up new avenues for Ex-STORM - expanding a sample mechanically, and then visualizing the sample with STORM.

Explore related subjects

BioengineeringPreprint

Keep this discovery

BibTeXRIS

Hu Cang, Zhisong Tong, Paolo Beuzer, Qing Ye, Josh Axelrod, Zhenmin Hong. 2016-04-20. Ex-STORM: Expansion Single Molecule Nanoscopy. https://doi.org/10.1101/049403

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2016

QTL-guided metabolic engineering of a complex trait

Engineering complex phenotypes for industrial and synthetic biology applications is difficult and often confounds rational design. Bioethanol production from lignocellulosic feedstocks is a complex trait that requires multiple host systems to utilize, detoxify, and metabolize a mixture of sugars and inhibitors present in plant hydrolysates. Here, we demonstrate an integrated approach to discovering and optimizing host factors that impact fitness of Saccharomyces cerevisiae during fermentation of a Miscanthus x giganteus plant hydrolysate. We first used high-resolution Quantitative Trait Loci (QTL) mapping and systematic Bulk Reciprocal Hemizygosity analysis (bRHA) to discover 17 loci that differentiate hydrolysate tolerance between an industrially related (JAY291) and a laboratory (S288C) strain. We then used this data to identify a subset of favorable allelic loci that were most amenable for strain engineering. Guided by this \"genetic blueprint\", and using a dual-guide Cas9-based method to efficiently perform multi-kilobase locus replacements, we engineered an S288C strain with superior hydrolysate tolerance than JAY291. Our methods should be generalizable to engineering any complex trait in S. cerevisiae, as well as other organisms.

Bioengineering↗
bioRxiv2016

Development of a modular automated system for maintenance and differentiation of adherent human pluripotent stem cells.

Patient-specific induced pluripotent stem cells (iPSCs) have tremendous potential for development of regenerative medicine, disease modelling and drug discovery. However, the processes of reprogramming, maintenance and differentiation are labour intensive and subject to inter-technician variability. To address these issues, we established and optimised protocols to allow for the automated maintenance of reprogrammed somatic cells into iPSCs to enable the large-scale culture and passaging of human pluripotent stem cells (PSCs) using a customized TECAN Freedom EVO. Generation of iPSCs was performed offline by nucleofection followed by selection of TRA-1-60 positive cells using a Miltenyi MultiMACS24 Separator. Pluripotency markers were assessed to confirm pluripotency of the generated iPSCs. Passaging was performed using an enzyme-free dissociation method. Proof of concept of differentiation was obtained by differentiating human PSCs into cells of the retinal lineage. Key advantages of this automated approach are the ability to increase sample size, reduce variability during reprogramming or differentiation, and enable medium to high-throughput analysis of human PSCs and derivatives. These techniques will become increasingly important with the emergence of clinical trials using stem cells.

Bioengineering↗
bioRxiv2016

An Electrochemistry Study of Cryoelectrolysis in Frozen Physiological Saline

This is the first quantitative study on the fundamental physical and electrochemical processes that occur during cryoelectrolysis. Cryoelectrolysis is a new minimally invasive tissue ablation surgical technique that combines the processes of electrolysis and solid/liquid phase transformation (freezing). We measured the pH front propagation and the changes in resistance in a tissue simulant made of physiological saline gel with a pH dye as a function of the sample temperature in the high subzero range above the eutectic. Results demonstrate that effective electrolysis can occur in a high subzero freezing milieu and that the propagation of the pH front is only weakly dependent on temperature. These observations are consistent with a mechanism involving ionic movement through the concentrated saline solution channels between ice crystals at subfreezing temperatures above the eutectic. The Joule heating in these microchannel may cause local microscopic melting, the observed weak dependence of pH front propagation on temperature, and the large changes in resistance with time. In addition, we observed that the pH front propagation from the anode is more rapid than from the cathode. The explanation is the electro-osmotic flow from the cathode to the anode. The findings in this paper may be of fundamental value for designing future cryoelectrolytic ablation surgery protocols.

Bioengineering↗