Despite their advantages, the use of fluorescent proteins also comes with some limitations and challenges: 1. Phototoxicity: Prolonged exposure to excitation light can damage cells. 2. Photobleaching: Fluorescent proteins can lose their fluorescence over time when exposed to light. 3. Spectrum Overlap: Overlapping emission spectra of different fluorescent proteins can complicate multiplexing. 4. Expression Levels: Achieving optimal expression levels without affecting cellular function can be challenging.
