Abstract
Carbon dots (CDs) have emerged as an exceptional alternative to traditional fluorescent probes for cellular imaging due to their high brightness, photostability, tunable fluorescence emission, and low toxicity. These properties make CDs ideal for applications requiring high sensitivity and minimal phototoxicity. However, their optimal potential is realized when combined with advanced fluorescence imaging techniques like fluorescence lifetime imaging microscopy (FLIM). FLIM provides deep insights into dynamic cellular behaviors by measuring fluorescence lifetimes, offering information that traditional intensity-based methods cannot measure. Thus, this review explores the optical properties and fluorescence mechanisms of CDs. The use of UV-vis absorption and photoluminescence (PL) spectroscopy is also discussed to illustrate the unique behavior of CDs. Their applications in cellular imaging, including organelle visualization and real-time tracking of intracellular processes, are examined. The combination of CDs with FLIM enhances the sensitivity of cellular imaging, enabling label-free, time-resolved measurements of cellular dynamics. This integration allows for precise monitoring of metabolic shifts, molecular interactions, and bacterial detection via multicolor imaging. Finally, we addressed the challenges and future directions in optimizing CDs and FLIM, with a focus on improving temporal and spatial resolution. The combination of CDs and FLIM holds transformative potential for advancing biomedical diagnostics and therapeutic monitoring.