Recently, the teams led by Professor Heping Cheng and Professor Peng Xi at Peking University, in collaboration with Professor Baoxiang Gao's team at Hebei University, published a research article in PhotoniX entitled "Imaging mitochondrial electric flickers in intact cells with a membrane-anchored indicator." The study reports HBmito Crimson (HBmito), a novel ultrasensitive fluorescent voltage probe anchored to the inner mitochondrial membrane. This probe enables ultrasensitive optical monitoring of near-field potential signals at the inner mitochondrial membrane interface and, for the first time, reveals a class of extremely small, millisecond-scale transient electrical activities in mitochondria. These findings provide a powerful imaging tool for understanding mitochondrial electrophysiology.
Mitochondria are central hubs of cellular energy metabolism and signal transduction, and their membrane potential dynamics represent important physiological signals. Conventional detection of mitochondrial membrane potential mainly relies on Nernstian probes that report bulk transmembrane voltage changes. However, the response kinetics of these probes are limited by diffusion and equilibration processes. As a result, suitable probes have been lacking for detecting faster and more refined electrical activities at the mitochondrial membrane interface.
HBmito is a membrane-anchored small-molecule voltage indicator with mitochondrial enrichment, near-infrared emission, and high photostability. By optimizing the fatty chain length, fluorophore scaffold, and spectral properties, the research team ultimately obtained this high-performance probe. Experimental results showed that HBmito anchors near the inner mitochondrial membrane interface and reports near-field potential-related signals at this interface, rather than merely reflecting bulk transmembrane voltage changes as conventional probes do.
HBmito exhibits outstanding signal amplitude and dynamic range. During mitochondrial action potentials, its average fluorescence change is approximately 0.55 ΔF/F0; among the brightest top 10% of events, its peak fluorescence response reaches up to a 1.93-fold increase. Compared with previously reported small-molecule membrane voltage indicators, HBmito improves the dynamic range by approximately one order of magnitude. In terms of detection principle, the signal recorded by HBmito does not solely represent bulk transmembrane voltage changes; it also contains faster surface-potential components near the inner membrane interface. Therefore, HBmito shows unique advantages in detecting fast and localized electrical activities.
Leveraging the high sensitivity and large dynamic range of HBmito, the research team captured a previously unreported class of transient miniature mitochondrial electrical activities—"mitochondrial electric flickers"—under high-speed imaging at 1000 frames per second. Compared with mitochondrial action potentials, which last for approximately 10 seconds, these events typically last only tens of milliseconds and have a peak amplitude of about 0.03 ΔF/F0. The unitary signal integral of these electric flickers is more than 1000-fold smaller than that of mitochondrial action potentials. Further experimental results suggest that a single electric flicker may reflect the gating activity of one or a few ion channels, serving as a fundamental unit of mitochondrial electrophysiological activity.
The significance of this study lies not only in the development of a novel ultrasensitive mitochondrial membrane voltage probe, but also in advancing mitochondrial electrical activity research from observing "bulk membrane potential changes" to resolving "fast, localized electrical signals at the membrane interface." Obtaining membrane voltage probes that combine high sensitivity, fast response, and large dynamic range has long been a key challenge in cellular electrophysiology. The membrane-anchored near-field potential sensing strategy proposed in this study provides important inspiration for addressing this challenge.
Dr. Wei Ren, a postdoctoral researcher at the College of Future Technology, Peking University, and Dr. Lin Zhang, a 2025 PhD graduate, are co-first authors of the article. Professor Heping Cheng of Peking University, Professor Baoxiang Gao of Hebei University, and Professor Peng Xi of Peking University are co-corresponding authors. Researcher Xianhua Wang and Associate Professor Zhixing Chen from the College of Future Technology, Peking University, also contributed to the study. This work was supported by the National Natural Science Foundation of China, the National Key Research and Development Program of China, and imaging support from the National Center for Protein Sciences at Peking University.
