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Polarity-sensitive probes (PAS) were synthesized through the attachment of azetidine and sulfonyl substituents to the pyrene fluorescent core. The emission peaks and fluorescence lifetimes of these PAS probes exhibit high sensitivity to polarity, enabling the visualization of microenvironmental characteristics and dynamics across multiple organelles. [ABSTRACT FROM AUTHOR]

Resolving complex three-dimensional (3D) subcellular dynamics noninvasively in live tissues demands imaging tools that balance spatiotemporal resolution, field-of-view and phototoxicity. Image scanning microscopy (ISM), as an advancement of confocal laser scanning microscopy, provides a 2-fold 3D resolution enhancement. Nevertheless, the relatively low imaging speed has been the major obstacle for ISM to be further employed in in vivo imaging of biological tissues. Our proposed solution, multi-confocal image scanning microscopy (MC-ISM), aims to overcome the limitations of existing techniques in terms of spatiotemporal resolution balancing by optimizing pinhole diameter and pitch, eliminating out-of-focus signals, and introducing a frame reduction reconstruction algorithm. The imaging speed is increased by 16 times compared with multifocal structured illumination microscopy. We further propose a single-galvo scan, akin to the Archimedes spiral in spinning disk confocal systems, to ensure a high-speed and high-accuracy scan without the galvanometer's inertial motion. Benefitting from its high photon efficiency, MC-ISM allows continuous imaging of mitochondria dynamics in live cells for 1000 frames without apparent phototoxicity, reaching an imaging depth of 175 μm. Noteworthy, MC-ISM enables the observation of the inner membrane structure of living mitochondria in Arabidopsis hypocotyl for the first time, demonstrating its outstanding performance. [ABSTRACT FROM AUTHOR]

Mitochondria, as essential organelles in cells, play a crucial role in cellular growth and apoptosis. Monitoring mitochondria is of great importance, as mitochondrial dysfunction is often considered a hallmark event of cell apoptosis. Traditional fluorescence probes used for mitochondrial imaging and sensing are mostly intensity-based and are susceptible to factors such as concentration, the probe environment, and fluorescence intensity. Probes based on fluorescence resonance energy transfer (FRET) can effectively overcome external interference and achieve high-contrast imaging of mitochondria as well as quantitative monitoring of mitochondrial microenvironments. This review focuses on recent advances in the application of FRET-based probes for mitochondrial structure imaging and microenvironment sensing. [ABSTRACT FROM AUTHOR]

Due to their strong hydrophobicity and the aggregation-caused quenching effect, the application of perylene diimide (PDI) dyes in biological and medicinal fields lags far behind that of other dyes. Based on a multifunctional encapsulation strategy, we prepared isopropylphenyl sulfone encapsulated PDI dyes (SFPDIs). The four hydrophilic sulfone groups on the bay position of the PDIs not only effectively inhibit the fluorescence quenching caused by π-aggregation but also endow the SFPDIs with good live-cell permeability. The six lipophilic isopropylphenyl groups encapsulate PDI emitters and confer SFPDIs with excellent lipid droplet-targeting ability. Furthermore, the strong electron-withdrawing sulfone groups give the PDIs excellent anti-oxidation ability. [ABSTRACT FROM AUTHOR]

Mitochondrial membrane environmental dynamics are crucial for understanding function, yet high-resolution observation remains challenging. Here, HBimmCue is introduced as a fluorescent probe localized to inner mitochondrial membrane (IMM) that reports lipid polarity and membrane order changes, which correlate with cellular respiration levels. Using HBimmCue and fluorescence lifetime imaging microscopy (FLIM), IMM lipid heterogeneity is uncovered across scales, from nanoscale structures within individual mitochondria to mouse pre-implantation embryos. At the sub-organelle level, stimulated emission depletion (STED)-FLIM imaging highlights nanoscale polarity variations within the IMM. At the sub-cellular and cellular level, reduced IMM lipid polarity is observed in damaged mitochondria marked for lysosomal degradation and distinct IMM lipid distributions are identified in neurons and disease models. Additionally, metabolic dysfunction associated with oocytes aging and metabolic reprogramming from zygote to blastocyst is detected. Together, the work demonstrates the broad applicability of HBimmCue, offering a new paradigm for investigating lipid polarity and respiration level at multiple scales., (© 2025 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Mitochondrial cristae, formed by folding the mitochondrial inner membrane (IM), are essential for cellular energy supply. However, the observation of the IM is challenging due to the limitations in spatiotemporal resolution offered by conventional microscopy and the absence of suitable in vitro probes specifically targeting the IM. Here, we describe a detailed imaging protocol for the mitochondrial inner membrane using the Si-rhodamine dye HBmito Crimson, which has excellent photophysical properties, to label live cells for imaging via stimulated emission depletion (STED) microscopy. This allows for STED imaging over more than 500 frames (approximately one hour), with a spatial resolution of 40 nm, enabling the observation of cristae dynamics during various mitochondrial processes. The protocol includes detailed steps for cell staining, image acquisition, image processing, and resolution analysis. Utilizing the superior resolution of STED microscopy, the structure and complex dynamic changes of cristae can be visualized. Key features • The protocol is designed to visualize mitochondrial cristae in living cells using STED microscopy. • The protocol enables nanoscale observation of dynamic mitochondrial cristae. • Real-time observation of mitochondrial morphological changes, fusion, and fission events., Competing Interests: Competing interestsB.G. is an inventor of the awarded patent (ZL202011401937.1) of HBmito Crimson. The other authors declare no competing interests., (©Copyright : © 2025 The Authors; This is an open access article under the CC BY-NC license.)