Fluorescence photoswitching systems using photochromic molecules, which turn on and off their fluorescence upon light irradiation, have emerged as highly promising material systems during the past two decades related to their optoelectronic applications such as high‐density optical memory, bioimaging, and super‐resolution microscopy. Single‐color fluorescence photoswitching, which provides only two different states (on/off), is limited in terms of its practical applications such as interference from autofluorescence in biological applications and limited switching states in logic gate and optical memory applications. To address such issues, studies on multicolor fluorescence photoswitching systems incorporating photochromic molecules have witnessed an explosive growth in the past decade in terms of the academic principles and technological applications. In the earlier part, this review briefly introduces the principle of fluorescence photoswitching based on the representative single‐color fluorescence photoswitching systems. Then, the review turns into the main topic of multicolor fluorescence photoswitching systems which are organized in two different subcategories of 1) color‐correlated photoswitching and 2) color‐specific photoswitching. Not only the material systems and principles of the multicolor fluorescence photoswitching, but also their important applications are described and discussed here. In the last section of this review, a brief summary and outlook on the future development are provided.

Multicolor tunable and multistate switchable organogel is reported, which consists of a cyanostilbene organogelator showing aggregation‐induced enhanced emission and a turn‐on type photochromic diarylethene dye. The mixed organogel can be reversibly switched among four different states (blue‐emitting gel, nonemissive sol, green‐emitting gel, and green‐emitting sol) modulated by a combination of orthogonal stimuli of heat and light. It is interestingly noted that this four‐state switching constitutes a combinational logic circuit consisting of two stimuli inputs and three outputs. Reversible fluorescence writing, switching, erasing, and image patterning processes on this mixture gel system are demonstrated.

Photoswitchable fluorophores—proteins and synthetic dyes—whose emission is reversibly switched on and off upon illumination, are powerful probes for bioimaging, protein tracking, and super-resolution microscopy. Compared to proteins, synthetic dyes are smaller and brighter, but their photostability and the number of achievable switching cycles in aqueous solutions are lower. Inspired by the robust photoswitching system of natural proteins, we designed a supramolecular system based on a fluorescent diarylethene ( DAE ) and cucurbit[7]uril (CB7) (denoted as DAE @CB7). In this assembly, the photoswitchable DAE molecule is encapsulated by CB7 according to the host–guest principle, so that DAE is protected from the environment and its fluorescence brightness and fatigue resistance in pure water improved. The fluorescence quantum yield (Φ fl ) increased from 0.40 to 0.63 upon CB7 complexation. The photoswitching of the DAE @CB7 complex, upon alternating UV and visible light irradiations, can be repeated 2560 times in aqueous solution before half-bleaching occurs (comparable to fatigue resistance of the reversibly photoswitchable proteins), while free DAE can be switched on and off only 80 times. By incorporation of reactive groups [maleimide and N -hydroxysuccinimidyl (NHS) ester], we prepared bioconjugates of DAE @CB7 with antibodies and demonstrated both specific labeling of intracellular proteins in cells and the reversible on/off switching of the probes in cellular environments under irradiations with 355 nm/485 nm light. The bright emission and robust photoswitching of DAE-Male3 @CB7 and DAE-NHS @CB7 complexes (without exclusion of air oxygen and addition of any stabilizing/antifading reagents) enabled confocal and super-resolution RESOLFT (reversible saturable optical fluorescence transitions) imaging with apparent 70–90 nm optical resolution.

Afterglow is superior to other optical modalities for biomedical applications in that it can exclude the autofluorescence background. Nevertheless, afterglow has rarely been applied to the high‐contrast “off‐to‐on” activatable sensing scheme because the complicated afterglow systems hamper the additional inclusion of sensory functions while preserving the afterglow luminescence. Herein, a simple formulation of a multifunctional components‐incorporated afterglow nanosensor (MANS) is developed for the superoxide‐responsive activatable afterglow imaging of cisplatin‐induced kidney injury. A multifunctional iridium complex (Ir‐OTf) is designed to recover its photoactivities (phosphorescence and the ability of singlet oxygen‐generating afterglow initiator) upon exposure to superoxide. To construct the nanoscopic afterglow detection system (MANS), Ir‐OTf is incorporated with another multifunctional molecule (rubrene) in the polymeric micellar nanoparticle, where rubrene also plays dual roles as an afterglow substrate and a luminophore. The multiple functions covered by Ir‐OTf and rubrene renders the composition of MANS quite simple, which exhibits superoxide‐responsive “off‐to‐on” activatable afterglow luminescence for periods longer than 11 min after the termination of pre‐excitation. Finally, MANS is successfully applied to the molecular imaging of cisplatin‐induced kidney injury with activatable afterglow signals responsive to pathologically overproduced superoxide in a mouse model without autofluorescence background.

Color‐specific photoswitching in dual‐color fluorescent system is important due to its potential in bioimaging and other applications. It requires elaborate manipulation of intermolecular energy transfer (ET) among two different fluorophores and one photochromic chromophore. Here, the rationally designed color‐specific photoswitching system composed of two excited‐state intramolecular proton transfer fluorophores and a color‐specific photoswitchable diarylethene in poly(methyl methacrylate) film is reported. Through their unique photophysical properties, this system demonstrates an entirely new principle of color‐specific photoswitching, which includes the frustration of ET between two fluorophores and the selective ET from only one specific fluorophore to the photochromic switch. In this novel class of smart fluorescent film, reversible photoswitching and photopatterning are successfully demonstrated.