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. Author manuscript; available in PMC: 2023 Nov 10.
Published in final edited form as: Nat Rev Methods Primers. 2022 Nov 10;2:89. doi: 10.1038/s43586-022-00168-w

Fig. 3 |. Advanced imaging techniques.

Fig. 3 |

A | Fluorescence recovery after photobleaching (FRAP) imaging involves the laser-mediated bleaching of a fluorophore in a region of interest (yellow dashed circle) followed by monitoring of fluorescence recovery into the same region of interest (black dashed circle). B | During fluorescence loss in photobleaching imaging, the fluorophore in a region of interest is continuously bleached (yellow dashed circle) while observing loss of fluorescence in adjacent/connected regions (black dashed circle). C | Photoactivation studies use caged (non-fluorescent) fluorophores, which can be uncaged and activated by a laser pulse to emit a fluorescent signal (yellow dashed circle) followed by monitoring the loss of fluorescence in the same region of interest (black dashed circle). D | During photoconversion the spectral properties of a fluorophore are permanently changed, which can, for example, allow/facilitate the tracking of individual cells in large populations. E | Examples of Förster resonance energy transfer (FRET) biosensors: intermolecular FRET from the donor fluorophore (cyan) to an unlinked receptor fluorophore (yellow) (part Ea); cleavage-dependent FRET biosensors whereby an irreversible cleavage of the substrate peptide leads to irreversible loss of/decrease in FRET (part Eb); distance-dependent FRET biosensors whereby a reversible change in the linker region leads to a reversible modification in the distance between donor and acceptor fluorophores, thus affecting FRET efficiency (part Ec); and orientation-dependent FRET biosensors whereby a reversible modification in the linker region leads to a reversible change in the alignment between donor and acceptor fluorophores, thus affecting FRET efficiency (part Ed). F | Fluorescence lifetime imaging microscopy (FLIM) is used to quantify the fluorescence lifetime, which is the time a fluorophore spends in the excited state prior to emission of a photon, an inherent feature of each fluorophore, which also depends on environmental conditions. Parts A–C and E adapted from REF.139, CC BY 3.0 (https://creativecommons.org/licenses/by/3.0/).