Multiphoton microscopy in retinal research: Advances, applications, and future directions

The retina, a critical component of the eye, converts light into neural signals through an intricate network of cells, including photoreceptors, bipolar cells, and retinal ganglion cells. The complex layering of the retina presents significant challenges for imaging, particularly due to light scattering and phototoxicity, which limit the resolution and depth of conventional optical techniques. Multiphoton microscopy (MPM), introduced in the 1990s, has emerged as a transformative solution to these challenges. This technique, utilizing two-photon and three-photon excitation, offers high-resolution, deep tissue imaging with minimal photodamage, making it ideal for retinal research. MPM allows for detailed visualization of retinal neurons, circuits, and cellular structures, even in live models, without the need for contrast agents. Recent advancements, such as the integration of adaptive optics and fluorescence lifetime imaging, have further enhanced its capabilities, enabling the study of retinal development, function, and disease. MPM has provided valuable insights into retinal disorders like diabetic retinopathy and retinal degeneration, supporting the development of therapeutic interventions. Despite its potential, MPM faces challenges in clinical adaptation, including cost and complexity. Nonetheless, ongoing advancements in imaging depth, speed, and precision suggest that MPM will play a pivotal role in both fundamental retinal research and clinical diagnostics.