Super-resolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a classical widefield microscope. Previously, three-dimensional (3D) SOFI has been demonstrated by sequential imaging of multiple depth positions. Here we introduce a multiplexed imaging scheme for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. The simultaneous acquisition of multiple focal planes significantly reduces the acquisition time and thus the photobleaching. We demonstrate multiplane 3D SOFI by imaging fluorescently labelled cells over an imaged volume of up to 65 × 65 × 3.5 μm3 without depth scanning. In particular, we image the 3D network of mitochondria in fixed C2C12 cells immunostained with Alexa 647 fluorophores and the 3D vimentin structure in living Hela cells expressing the fluorescent protein Dreiklang.
Ubiquitin carboxy-terminal hydrolase
L1 (UCHL1) is a deubiquitylating
enzyme that is proposed as a potential therapeutic target in neurodegeneration,
cancer, and liver and lung fibrosis. Herein we report the discovery
of the most potent and selective UCHL1 probe (IMP-1710) to date based
on a covalent inhibitor scaffold and apply this probe to identify
and quantify target proteins in intact human cells. IMP-1710 stereoselectively
labels the catalytic cysteine of UCHL1 at low nanomolar concentration
in cells. We further demonstrate that potent and selective UCHL1 inhibitors
block pro-fibrotic responses in a cellular model of idiopathic pulmonary
fibrosis, supporting the potential of UCHL1 as a potential therapeutic
target in fibrotic diseases.
The discovery of biocompatible reactions has had a tremendous impact on chemical biology, allowing the study of numerous biological processes directly in complex systems. However, despite the fact that multiple biocompatible reactions have been developed in the past decade, very few work well in living mice. Here we report that D-cysteine and 2-cyanobenzothiazoles can selectively react with each other in vivo to generate a luciferin substrate for firefly luciferase. The success of this “split luciferin” ligation reaction has important implications for both in vivo imaging and biocompatible labeling strategies. First, the production of a luciferin substrate can be visualized in a live mouse by bioluminescence imaging (BLI), and furthermore allows interrogation of targeted tissues using a “caged” luciferin approach. We therefore applied this reaction to the real-time non-invasive imaging of apoptosis associated with caspase 3/7. Caspase-dependent release of free D-cysteine from the caspase 3/7 peptide substrate Asp-Glu-Val-Asp-D-Cys (DEVD-(D-Cys)) allowed selective reaction with 6-amino-2-cyanobenzothiazole (NH2-CBT) in vivo to form 6-amino-D-luciferin with subsequent light emission from luciferase. Importantly, this strategy was found to be superior to the commercially-available DEVD-aminoluciferin substrate for imaging of caspase 3/7 activity. Moreover, the split luciferin approach enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. Lastly, the luciferin ligation reaction is three orders of magnitude faster than Staudinger ligation suggesting further applications for both bioluminescence and specific molecular targeting in vivo.
In a pinch: The nickel pincer complex 1 catalyzes the cross‐coupling of the title compounds with remarkable substrate scope and functional group tolerance. A nickel/alkynyl species was isolated and shown to be catalytically competent. THF=tetrahydrofuran, O‐TMEDA=bis[2‐(N,N‐dimethylaminoethyl)] ether.
Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.