A young researcher’s guide to three-dimensional fluorescence microscopy of living cells

Three-dimensional imaging of fast intracellular processes by fluorescence microscopy should provide decent spatial and high temporal resolution while minimizing fluorophore bleaching and cytotoxicity. We give a condensed introductory overview of three contemporary methods mostly used for imaging of living cells in 3D and compare their performance in terms of temporal and spatial resolution, imaging flexibility and specimen photodamage: point-scanning confocal microscopy, spinning-disc confocal microscopy, and lattice light-sheet microscopy. While point-scanning instruments are unsurpassed in terms of confocal performance, flexibility and configurability of their optical path, spinning-disc and lattice light-sheet optical designs excel in acquisition speed and low levels of light-inflicted specimen deterioration.