Insufficient brightness of fluorophores poses a major bottleneck for the advancement of super-resolution microscopes. Despite being widely used, many rhodamine dyes exhibit sub-optimal brightness due to the formation of twisted intra­molecular charge transfer (TICT) upon photoexcitation. Herein, we have developed a new class of quaternary piperazine-substituted rhodamines with outstanding quantum yields (Φ = 0.93) and superior brightness (ε × Φ = 8.1 × 104 L·mol–1·cm–1), by utilizing the electronic inductive effect to prevent TICT. We have also successfully deployed these rhodamines in the super-resolution imaging of the microtubules of fixed cells and of the cell membrane and lysosomes of live cells. Finally, we demonstrated that this strategy was generalizable to other families of fluorophores, resulting in substantially increased quantum yields.

A fundamental, highly fluorescent, and easily accessible scaffold named BOPPY is reported. The use of hydrazine as a bridging linkage between pyrrole and N-heteroarenes enables the binding of two BF 2 units to provide sufficient rigidity of the unsymmetric core skeleton. These resultant unsymmetrical BOPPYs are thus highly fluorescent in their solutions and solid powder states and exhibit high molar absorption coefficients (42200−47000 M −1 cm −1 ), large Stokes shifts, excellent photostability, and insensitivity to pH. More importantly, these BOPPYs showed efficient two-photon absorption in the wide spectral range of 700−900 nm, making them well suited for two-photon fluorescence microscopy imaging in living cells.Letter pubs.acs.org/OrgLett

A change of mitochondrial temperature can be an important indicator of mitochondrial metabolism that generates considerable heat. For this reason, development of fluorescent probes to detect mitochondrial temperature has become an attractive topic. Previous efforts have successfully addressed the major issues, such as temperature sensitivity and mitochondrial targetability. However, there remains a key obstacle to practical applications. Considering the highly dynamic features of mitochondria, especially the variation of the inner-membrane potential, it is quite necessary to permanently immobilize a temperature probe in mitochondria in order to avoid unstable intracellular localization along with the changes of mitochondrial status. Herein, we report Mito-TEM, the first fixable, fluorescent molecular thermometer. Mito-TEM is based on a positively charged rhodamine B fluorophore that has the tendency of being attracted to mitochondria, which have negative potential. This fluorophore containing rotatable substituents also contributes to the temperature-responsive fluorescence property. Most importantly, a benzaldehyde is introduced in Mito-TEM as an anchoring unit that condenses with aminos of the protein and thus immobilizes the probe in mitochondria. The specific immobilization of Mito-TEM in mitochondria is unambiguously demonstrated in colocalization imaging. By using Mito-TEM, a method of visualizing and quantifying a temperature distribution through grayscale imaging of mitochondria is established and further applied to monitor the temperature changes of live cells under light heating and PMA stimulation.

No abstract

Immobilizable probes represent a valuable trend.

Temperature in mitochondria can be a critical indicator of cell metabolism. Given the highly dynamic and inhomogeneous nature of mitochondria, it remains a big challenge to quantitatively monitor the local temperature changes during different cellular processes. To implement this task, we extend our strategy on mitochondria-anchored thermometers from “on–off” probe Mito-TEM to a ratiometric probe Mito-TEM 2.0 based on the Förster resonance energy transfer mechanism. Mito-TEM 2.0 exhibits not only a sensitive response to temperature through the ratiometric changes of dual emissions but also the specific immobilization in mitochondria via covalent bonds. Both characters support accurate and reliable detection of local temperature for a long time, even in malfunctioning mitochondria. By applying Mito-TEM 2.0 in fluorescence ratiometric imaging of cells and zebrafishes, we make a breakthrough in the quantitative visualization of mitochondrial temperature rises in different inflammation states.

AIE-active and bright solid-state red-emissive meso-2-ketopyrrolyl BODIPYs have been developed as viscosimeters in live cells for real-time quantification of intracellular viscosities.

As a new form of regulated cell death, ferroptosis is closely related to various diseases. To interpret this biological behavior and monitor related pathological processes, it is necessary to develop appropriate detection strategies and tools. Considering that ferroptosis is featured with remarkable lipid peroxidation of various cell membranes, it is logical to detect membranes’ structural and environmental changes for the direct assessment of ferroptosis. For this sake, we designed novel polarity-sensitive fluorescent probes Mem-C 1 C 18 and Mem-C 18 C 18 , which have superior plasma membrane anchorage, high brightness, and sensitive responses to environmental polarity by changing their fluorescence lifetimes. Mem-C 1 C 18 with much less tendency to aggregate than Mem-C 18 C 18 outperformed the latter in high resolution fluorescence labeling of artificial vesicle membranes and plasma membranes of live cells. Thus, Mem-C 1 C 18 was selected to monitor plasma membranes damaged along ferroptosis process for the first time, in combination with the technique of fluorescence lifetime imaging (FLIM). After treating HeLa cells with Erastin, a typical ferroptosis inducer, the mean fluorescence lifetime of Mem-C 1 C 18 displayed a considerable increase from 3.00 to 4.93 ns, with a 64% increase (corresponding to the polarity parameter Δf increased from 0.213 to 0.232). Therefore, our idea to utilize a probe to quantitate the changes in polarity of plasma membranes proves to be an effective method in the evaluation of the ferroptosis process.