Optimized Properties and Synthesis of Photoactivatable Diazoketorhodamines Facilitate and Enhance High-Throughput Single-Molecule Tracking.

Photoactivatable (PA) rhodamine dyes are widely used in single-molecule tracking (SMT) and a variety of other fluorescence-based imaging modalities. One of the most commonly employed scaffolds uses a diazoketone to lock the rhodamine in the nonfluorescent closed form, which can be activated with 405 nm light. However, poor properties of previously reported dyes require significant washing, which can be resource- and cost-intensive, especially when performing microscopy in a large scale and high-throughput fashion. Here, we report improved diazoketorhodamines that perform exceptionally well in single-molecule tracking microscopy. We also report on the optimization of an improved synthetic method for further iteration and tailoring of diazoketorhodamines to the requirements of a specific user.

A Photoactivatable Formaldehyde Donor with Fluorescence Monitoring Reveals Threshold To Arrest Cell Migration.

Controlled light-mediated delivery of biological analytes can enable the investigation of highly reactivity molecules within living systems. As many biological effects are concentration dependent, it is critical to determine the location, time, and quantity of analyte donation. In this work, we have developed the first photoactivatable donor for formaldehyde (FA). Our optimized photoactivatable donor, photoFAD-3, is equipped with a fluorescence readout that enables monitoring of FA release with a concomitant 139-fold fluorescence enhancement. Tuning of photostability and cellular retention enabled quantification of intracellular FA release through cell lysate calibration. Application of photoFAD-3 uncovered the concentration range necessary for arresting wound healing in live cells. This marks the first report where a photoactivatable donor for any analyte has been used to quantify intracellular release.

NitroxylFluor: A Thiol-Based Fluorescent Probe for Live-Cell Imaging of Nitroxyl.

Detection of nitroxyl (HNO), the transient one-electron reduced form of nitric oxide, is a significant challenge owing to its high reactivity with biological thiols (with rate constants as high as 109 M-1 s-1). To address this, we report a new thiol-based HNO-responsive trigger that can compete against reactive thiols for HNO. This process forms a common N-hydroxysulfenamide intermediate that cyclizes to release a masked fluorophore leading to fluorescence enhancement. To ensure that the cyclization step is rapid, our design capitalizes on two established physical organic phenomena; the alpha-effect and the Thorpe-Ingold effect. Using this new trigger, we developed NitroxylFluor, a selective HNO-responsive fluorescent probe. Treatment of NitroxylFluor with an HNO donor results in a 16-fold turn-on. This probe also exhibits excellent selectivity over various reactive nitrogen, oxygen, and sulfur species and efficacy in the presence of thiols (e.g., glutathione in mM concentrations). Lastly, we successfully performed live cell imaging of HNO using NitroxylFluor.

Synthetic Methods for the Preparation of a Functional Analogue of Ru360, a Potent Inhibitor of Mitochondrial Calcium Uptake.

The mixed-valent oxo-bridged ruthenium complex [(HCO2)(NH3)4Ru(µ-O)Ru(NH3)4(O2CH)]3+, known as Ru360, is a selective inhibitor of mitochondrial calcium uptake. Although this compound is useful for studying the role of mitochondrial calcium in biological processes, its widespread availability is limited because of challenges in purification and characterization. Here, we describe our investigations of three different synthetic methods for the preparation of a functional analogue of this valuable compound. We demonstrate that this analogue, isolated from our procedures, exhibits potent mitochondrial calcium uptake inhibitory properties in permeabilized HeLa cells and in isolated mitochondria.

Thienylpiperidine Donor NIR Xanthene-Based Dye for Photoacoustic Imaging.

Few xanthene-based near-infrared (NIR) photoacoustic (PA) dyes with absorbance >800 nm exist. As accessibility to these dyes requires long and tedious synthetic steps, we designed a NIR dye (XanthCR-880) with thienylpiperidine donors and a xanthene acceptor that is accessible in 3-4 synthetic steps. The dye boasts a strong PA signal at 880 nm with good biological compatibility and photostability, yields multiplexed imaging with an aza-BODIPY reference dye, and is detected at a depth of 4 cm.

Surveillance of Cancer Stem Cell Plasticity Using an Isoform-Selective Fluorescent Probe for Aldehyde Dehydrogenase 1A1.

Cancer stem cells (CSCs) are progenitor cells that contribute to treatment-resistant phenotypes during relapse. CSCs exist in specific tissue microenvironments that cell cultures and more complex models cannot mimic. Therefore, the development of new approaches that can detect CSCs and report on specific properties (e.g., stem cell plasticity) in their native environment have profound implications for studying CSC biology. Herein, we present AlDeSense, a turn-on fluorescent probe for aldehyde dehydrogenase 1A1 (ALDH1A1) and Ctrl-AlDeSense, a matching nonresponsive reagent. Although ALDH1A1 contributes to the detoxification of reactive aldehydes, it is also associated with stemness and is highly elevated in CSCs. AlDeSense exhibits a 20-fold fluorescent enhancement when treated with ALDH1A1. Moreover, we established that AlDeSense is selective against a panel of common ALDH isoforms and exhibits exquisite chemostability against a collection of biologically relevant species. Through the application of surface marker antibody staining, tumorsphere assays, and assessment of tumorigenicity, we demonstrate that cells exhibiting high AlDeSense signal intensity have properties of CSCs. Using these probes in tandem, we have identified CSCs at the cellular level via flow cytometry and confocal imaging, as well as monitored their states in animal models.