This review discusses new strategies for designing fluorescent probes for medical diagnostic imaging. It highlights the importance of sensitivity and specificity in molecular imaging, which aims to monitor biological processes at the cellular and molecular level. Radionuclide and optical imaging are considered the most practical modalities for molecular imaging due to their sensitivity and target-specific detection capabilities. However, radionuclide imaging has limitations in spatial and temporal resolution and safety concerns due to radioactive compounds. Optical imaging, on the other hand, can be targeted by conjugating fluorophores with targeting ligands and offers high target-to-background ratios. However, it faces challenges in quantification due to light scattering and absorption, especially in deep tissues. The review also discusses the general requirements for fluorescence probes in medical imaging, including wavelength, brightness, stability, and pharmacokinetics. The optimal excitation wavelength for fluorophores is in the deep red or near-infrared range due to good tissue penetration and low autofluorescence. Brightness is important for depth penetration, but increased brightness often comes at the cost of increased size. Stability is crucial for in vivo imaging, as many fluorophores lose fluorescence after internalization. Pharmacokinetics are also important, as fluorophores can alter the pharmacokinetics of targeting moieties. The review classifies fluorophores into three major categories: small synthetic fluorophores, genetically encoded fluorophores, and fluorescent nanocrystals. Small molecule fluorophores are often used for in vivo imaging due to their size and brightness. Genetically encoded fluorophores are naturally occurring proteins that can be used for in vivo imaging but are limited by their size. Fluorescent nanocrystals, such as quantum dots, are highly bright but can be toxic due to heavy metals in their core. The review also discusses advanced applications of fluorescence probes in medical imaging, including multiple color imaging and activatable imaging probes. Multiple color imaging allows for the simultaneous imaging of multiple molecular targets, which is useful for in vivo imaging. Activatable imaging probes are designed to be activated in specific environments, improving the target-to-background ratio. These probes can be activated by enzymatic activity or other specific conditions. The review concludes that fluorescence probes are essential for medical imaging, and new strategies for their design are needed to improve sensitivity, specificity, and safety. The development of new fluorescent probes is crucial for the advancement of medical imaging and the diagnosis and treatment of diseases.This review discusses new strategies for designing fluorescent probes for medical diagnostic imaging. It highlights the importance of sensitivity and specificity in molecular imaging, which aims to monitor biological processes at the cellular and molecular level. Radionuclide and optical imaging are considered the most practical modalities for molecular imaging due to their sensitivity and target-specific detection capabilities. However, radionuclide imaging has limitations in spatial and temporal resolution and safety concerns due to radioactive compounds. Optical imaging, on the other hand, can be targeted by conjugating fluorophores with targeting ligands and offers high target-to-background ratios. However, it faces challenges in quantification due to light scattering and absorption, especially in deep tissues. The review also discusses the general requirements for fluorescence probes in medical imaging, including wavelength, brightness, stability, and pharmacokinetics. The optimal excitation wavelength for fluorophores is in the deep red or near-infrared range due to good tissue penetration and low autofluorescence. Brightness is important for depth penetration, but increased brightness often comes at the cost of increased size. Stability is crucial for in vivo imaging, as many fluorophores lose fluorescence after internalization. Pharmacokinetics are also important, as fluorophores can alter the pharmacokinetics of targeting moieties. The review classifies fluorophores into three major categories: small synthetic fluorophores, genetically encoded fluorophores, and fluorescent nanocrystals. Small molecule fluorophores are often used for in vivo imaging due to their size and brightness. Genetically encoded fluorophores are naturally occurring proteins that can be used for in vivo imaging but are limited by their size. Fluorescent nanocrystals, such as quantum dots, are highly bright but can be toxic due to heavy metals in their core. The review also discusses advanced applications of fluorescence probes in medical imaging, including multiple color imaging and activatable imaging probes. Multiple color imaging allows for the simultaneous imaging of multiple molecular targets, which is useful for in vivo imaging. Activatable imaging probes are designed to be activated in specific environments, improving the target-to-background ratio. These probes can be activated by enzymatic activity or other specific conditions. The review concludes that fluorescence probes are essential for medical imaging, and new strategies for their design are needed to improve sensitivity, specificity, and safety. The development of new fluorescent probes is crucial for the advancement of medical imaging and the diagnosis and treatment of diseases.