To unveil whether the intermolecular through-space interaction is responsible for the aggregate emission of these closed-form rhodamine derivatives, we analyzed the singlecrystal structures of the four compounds. The single crystals of BISX, ISX and MTSX were obtained by slow evaporation of their acetonitrile solutions, respectively, while the single crystal of MISX was grown from its acetonitrile-water (4:6, v/v) mixtures. For BISX crystal, except for short contacts including hydrogen bonding and C-H•••π interactions, π-π interactions at the distance of 3.359 Å were also found between the benzo-imidazole rings (Figure and). These intermolecular interactions may facilitate the restriction of molecular motion of BISX, thus inhibiting the nonradiative decay. And intermolecular π-π interactions may benefit the formation of the chromophore to induce its AIE property. Interestingly, ISX crystal, with only hydrogen bonds (2.340 Å and 2.976 Å) and C-H•••π interactions (2.837 Å and 2.806 Å) rather than π-π interactions, also exhibited similar AIE behavior like BISX (Figure and Figure ). As for MISX crystal, C-H•••π interactions with the distance of 2.892 Å were observed (Figure ). Though hydrogen bonding interactions (C-H•••O) with the distance of 2.596 Å between two isoquinolinone rings could be found, there was no observable π-π interaction. Staggered packing was observed for the two rings, similar to ISX (Figure ). MTSX crystal also possessed π-π interactions, analogous to BISX (Figure and). Therefore, comparing the packing mode of BISX and ISX crystal, and MISX and MTSX, we concluded that intermolecular through-space interaction did not necessitate the chromophore formation and the AIE properties of these rhodamine derivatives. To further verify our proposal, non-covalent interactions were calculated by reduced density gradient (RDG) method. As shown in Figure -h, substantial intermolecular through-space interactions were found for BISX and MTSX, while ISX and MISX exhibited negligible intermolecular interactions. Thus, combining the crystal structure analysis and RDG calculation results, we could conclude that intermolecular through-space interaction is not the fundamental factor in inducing AIE behavior. Since the intermolecular through-space interaction was excluded, we would like to explore other kinds of possibilities that are responsible for the chromophore formation and AIE behavior of these closed-form rhodamines. We doped BISX, ISX, MISX and MTSX into PMMA films with different ratios to restrict their molecular motion, and measured their luminescence properties subsequently (Figure and Figure ). For BISX, emission intensity of the doping system decreased with a reduced doping ratio from 5% to 0.1% (Figure ). Similar results were also found for ISX, MISX and MTSX doped in PMMA film (Figure ). This suggested that the emission of the four compounds in the isolated state was kind of forbidden even if they were confined in a rigid matrix, while the aggregation at high concentration can effectively break the transition forbidden to light up the emission. For conventional AIEgens, it is known that both rigidification in the polymeric matrix and reducing environmental temperature can promote the radiative decay rate since molecular motion is restricted, thus providing strong emission. Thus, combined with the previous cooling experimental results, we assumed that except for the RIM mechanism, there may be other reasons for the AIE properties. Although the four compounds were nonconjugated with donor (D) xanthene and acceptor (A) isoquinolinone parts isolated by an sp 3 carbon atom, intramolecular charge transfer may exist in a through-space way to narrow the band gap, facilitating the chromophore formation. This hypothesis was further supported by theoretical calculation since the highest occupied molecular orbitals (HOMOs) were distributed on the D moieties (xanthene) while the lowest unoccupied molecular orbitals (LUMOs) were generally distributed on the A moieties (isoquinolinone) as shown in Figure . Take BISX for instance, in dilute solutions, the neighboring D and A groups were almost perpendicular to each other with a dihedral angle of 89.5° in the ground state (Figure ). Thus, electron clouds of the isolated conjugation rings were not overlapped at all, which has been well recognized as a forbidden factor for electron transition. In the excited state, a relatively smaller dihedral angle of 77.73° was found for the optimized geometry which might allow the electron transition. While in the real case, such large torsion from 89.5° to 77.73° was hard to realize, and the absorbed energy would undergo a nonradiative decay pathway by structural relaxation. Thus, in the solution state, the system was almost non-emissive, most excitons will come to the ground state from the LE state rather than CT excited state, making CT become a dark state (Figure ). This diagram also well accounted for the emission behavior of BISX in the previous cooling experiments (Figure ). At room temperature, due to the flexible molecular motion, the absorbed energy was mainly dissipated via the nonradiative decay pathway, affording negligible LE emission. And the weak CT emission which is responsible for the AIE peak was ascribed to a dark state. Although reducing temperature to restrict the molecular motion benefits enhancing the LE emission at 430 nm, it affects scarcely the forbidden CT emission of the perpendicular D-A structure. However, the aggregation seems to break the transition forbidden of an isolated state since these lightless nonconjugated rhodamine single species emit brightly upon aggregation or crystallization. What happened during the aggregation? Which kind of factors breaks the transition forbidden of molecular species? To gain deep insight into these questions, theoretical calculations based on singlecrystal structures were carried out. In BISX crystals, different from its orthogonal structure in the solution state, a symmetry decreased structure with a smaller D-A dihedral angle of 83.66° within the crystal was observed. Unlike the perpendicular orthogonal structure at freedom state that exhibits almost non-overlapped HOMO and LUMO (Figure ), the symmetry decrease within the crystal structure leads to an obvious electronic structure change with more effective HOMO and LUMO overlap. The improved HOMO and LUMO overlap benefits effective CT transition to undergo radiative decay. Therefore, the aggregation helps break the transition forbidden in the solution state but facilitates the allowed CT transition in the aggregate state through structural symmetry breaking upon aggregation (Figure and). Based on the single-crystal structure, QM/MM calculations were further performed by the ONIOM model in the Gaussian 16 package (Figure ). As shown in Figure , the variation of the dihedral angle between the excited state and ground state was merely 2.45°, leading to more accessible structural relaxation to the geometry of allowed CT transition. Thus, in the aggregate state, BISX displayed CT emission at 540 nm. Compared to the high symmetric geometry in the solution state, molecules in the aggregate state were close to each other with various interactions (C-H•••π, π-π, hydrogen bonding), which caused a symmetry decreased structure with transition allowed CT emission. Therefore, AIE features of the nonconjugated molecules in our system could be understood from a new viewpoint of aggregation-induced symmetry breaking (AISB). Since symmetry breaking is common in aggregates, the highly flexible motion in dilute solutions can be considered as high symmetry of the system, while the restricted motion in the aggregate state can be taken as a decreased symmetry. Application in the detection of amines released Additionally, we checked the PL performance of these nonconjugated rhodamine derivatives in the solid state, respectively. As shown in Figure and 6b, BISX, ISX, MTSX and MISX crystals displayed bright green-blue color fluorescence with emission peaks at 475 nm, 515 nm, 478 nm, and 497 nm, respectively. Their quantum yields were measured as 64.1%, 46.0%, 69.4%, and 72.7%, which are ultrabright visible emissions for these unorthodox nonconjugated structures (Table ). Notably, we found that the bright fluorescence of BISX was quenched completely when exposed to trifluoroacetic acid (TFA). And the emission recovered when the acid-treated sample was exposed to triethylamine (TEA), as shown in Figure . We assumed introducing TFA protonated the nitrogen atom of BISX (HBISX + ), while later TEA treatment made it deprotonation.