The article discusses the advancements and future directions in fluorescent probe design for medical diagnostic imaging. It highlights the importance of molecular imaging, which aims to characterize and measure biological processes at the cellular and molecular levels. The text reviews the history of fluorescence imaging in medicine, noting that only a few fluorophores are currently approved for medical use. It outlines the key characteristics of successful optical molecular probes, including wavelength, brightness, stability, and pharmacokinetics. The article also classifies fluorophores into small synthetic fluorophores, genetically encoded proteins, and nanocrystals, detailing their properties and applications. Advanced applications of fluorescence probes, such as multiple-color imaging and activatable imaging probes, are discussed, emphasizing their potential to improve sensitivity and specificity in medical imaging. The text concludes by exploring the development of activatable imaging probes, which can be activated in specific environments, and their role in targeted molecular imaging.The article discusses the advancements and future directions in fluorescent probe design for medical diagnostic imaging. It highlights the importance of molecular imaging, which aims to characterize and measure biological processes at the cellular and molecular levels. The text reviews the history of fluorescence imaging in medicine, noting that only a few fluorophores are currently approved for medical use. It outlines the key characteristics of successful optical molecular probes, including wavelength, brightness, stability, and pharmacokinetics. The article also classifies fluorophores into small synthetic fluorophores, genetically encoded proteins, and nanocrystals, detailing their properties and applications. Advanced applications of fluorescence probes, such as multiple-color imaging and activatable imaging probes, are discussed, emphasizing their potential to improve sensitivity and specificity in medical imaging. The text concludes by exploring the development of activatable imaging probes, which can be activated in specific environments, and their role in targeted molecular imaging.