Towards single-photon deep-tissue microscopy

Fluorescence light-sheet microscopy is gaining rapid adoption in developmental biology. With irradiation levels well below that of confocal and multi-photon microscopy, it enables the study of intact organs and organisms for prolonged time periods during development. Minimal sample exposure is achieved by selectively illuminating the focal plane with a second objective orthogonal to the detection axis. The light-sheet microscope’s ability to study intact biological samples as and when they grow highlights the importance of imaging deeper into biological samples. Yet, deep-tissue microscopy is hampered by autofluorescence and the scattering of light. Direct observations are therefore limited to highly transparent and thin samples. Here, we show how autofluorescence can be eliminated effectively by relying on reversible photoswitching fluorescence while we propose a way forward to study and control light propagation in optically-thick tissues.