Molecular regioisomerism: an advantageous strategy for optimizing two-photon absorption performance of organic chromophores

Abstract

Two-photon absorbing fluorophores have emerged as powerful imaging agents, offering advantages such as high spatial resolution, deep light penetration, minimal photobleaching, minor photodamage, and low autofluorescence. However, existing two-photon absorbing fluorophores still face the limitation of a small two-photon absorption cross-section. The conventional approaches toward fluorophores with a large two-photon absorption cross-section involve enhancing intramolecular charge transfer, extending the π-conjugation length, increasing the number of π-conjugation paths, improving coplanarity, etc. These approaches are promising but hindered by synthesis complexities, large molecular weight (low membrane permeability), poor solubility, low photostability and aggregation-caused quenching. Herein, we summarize an emerging strategy, namely molecular regioisomerism, which could improve the two-photon absorption performance through adjusting molecular symmetries, molecular π-conjugations, molecular orbital distributions, molecular dipoles, and/or intermolecular interactions. This review can guide the design and synthesis of regioisomers of organic chromophores with good two-photon absorption performance, as well as deepen the research on the structure–property relationship of the regioisomers.

---

1. Introduction

Two-photon absorption (2PA) enables access to a given excited state by using photons of half the energy (or twice the wavelength) of the corresponding one-photon absorption.¹ 2PA has several notable advantages: (i) 2PA has high spatial resolution. Two-photon excitation is a nonlinear optical process that is quadratically dependent on the laser intensity. The excitation is confined at the focal point of the laser beam; a relatively small volume is localized in both the transverse plane and along the laser beam; (ii) 2PA has deep penetration. The near-infrared excitation wavelength used in 2PA coincides with the biological transparency window (700–1000 nm), resulting in a 10 to 100-fold increase in the light penetration depth; (iii) 2PA has minimal photobleaching. The lack of out-of-focus excitation in 2PA reduces photobleaching to samples, which is crucial for long-term imaging; (iv) 2PA has minor photodamage. Compared to high energy UV and visible light irradiation, low energy near-infrared light irradiation causes less photodamage to biological tissue; (v) 2PA has low autofluorescence. Compared to short-wavelength irradiation, long-wavelength irradiation reduces autofluorescence from biological species that interfere with the fluorescence signal, thereby enhancing the contrast of the imaging.^2–12

The two-photon absorbability can be quantitatively represented by the 2PA cross-section, commonly denoted by the symbol δ₂. Theoretically, the molecular 1PA cross-sections (A) are approximately 10⁻¹⁷ to 10⁻¹⁶ cm², and the molecular 2PA cross-sections (δ₂) are approximately 10⁻⁵⁰ to 10⁻⁴⁸ cm⁴ s (i.e., A²Δτ, Δτ = 10⁻¹⁶ s).¹³ In practice, most of the measured values of δ₂ are in the range of 10⁻⁵¹ to 10⁻⁴⁶ cm⁴ s. The unit of δ₂ is GM (the name abbreviation of Göppert-Mayer, 1 GM = 10⁻⁵⁰ cm⁴ s). It is evident that, for the two-photon technology to realize its full potential, the design and synthesis of highly active molecules with large δ₂ will play a pivotal role. Therefore, a continuing need is to establish and fine tune the structure–property relationships for a large number of molecules with systematically varied molecular structure factors and precisely reproducible characterization of 2PA properties.¹⁴ In terms of low cost, molecular diversity, structure tailorability, flexible processability and good biocompatibility, organic materials are better alternatives for 2PA materials.¹⁵

1.1. Conventional strategies for optimizing 2PA performance

So far, a number of key organic molecular features that have a strong correlation with the two-photon absorptivity have been identified, such as the intramolecular charge-transfer (ICT) process, conjugation extent (i.e., path length and the number of paths), coplanarity, diradical character, etc. Specifically, strong ICT can be achieved by creating dipolar structures and using side groups with strong electron-donating or electron-accepting ability. Increasing the π-conjugation length can be obtained by using ethylene, ethynylene or phenylene units as the π-conjugated bridge. Increasing the number of π-conjugation paths can be derived by creating quadrupolar or octupolar structures. Maintaining a planar structure can be acquired by using rigidified structures.^16–20 Stable diradicaloids have been demonstrated including quinoidal polycyclic aromatic hydrocarbons, quinoidal oligothiophenes, and graphene fragments. Besides, donor–acceptor conjugated polymers and diketopyrrolopyrrole derivatives, generally known as closed-shell species, could also exhibit the intermediate open-shell character.^21,22 In the past five years, some new progress has been made in this area. In 2021, Ishii and coworkers investigated the substituent effect on the 2PA properties of diphenylacetylene with R groups at the para-position (compounds 1–9). 2PA spectra of compounds 1–9 with stronger electron-donating (ED) or electron-withdrawing (EW) groups displayed more significant red-shifts and larger δ₂ values (Fig. 1a and Table 1).²³ In 2020, Allen and coworkers investigated the π-conjugation length effect on the 2PA properties of indacene-cored quadrupolar A–π–D–π–A chromophores 10–12 and thieno[2,3-b]thiophene-cored quadrupolar A–π–D–π–A chromophores 13–15. Increasing the core size (i.e., π-conjugation length) resulted in larger δ₂ values (Fig. 1b and Table 1).²⁴ In 2022, Wang and coworkers theoretically studied the effect of increasing the number of branches (i.e., conjugation paths) on the 2PA properties of phenothiazine derivatives (compounds 16 and 17). Increasing the number of oxadiazole branches significantly promoted the red shift of 2PA peaks and enhanced the δ₂ values (Fig. 1c and Table 1).²⁵ In 2025, our group studied the coplanarity effect on the 2PA properties of perylene diimide (PDI) derivatives (compounds 18–20). The fusion of the bay substituent with the perylene core improved the coplanarity of the molecule and thus enhanced the δ₂ values (Fig. 1d and Table 1).²⁶ In 2025, Oka and coworkers studied the 2PA properties of croconaine dyes with phenyl-substituted chalcogenopyrylium components (O, S, and Se). They found that their diradical characteristics were enhanced by replacing the chalcogen atom in the chalcogenopyrylium ring from O to S, to Se (compounds 21–23). And the enhanced diradical character lowered the transition energy for 2PA, thus increasing the δ₂ values (Fig. 1e and Table 1).²⁷

Fig. 1 (a) The molecular structures, two-photon absorption spectra and one-photon absorption spectra (black dashed lines, top and right axes) of compounds 1–9. Reproduced from ref. 23 with permission from American Chemical Society, copyright 2021. (b) The molecular structures of indacene-cored quadrupolar chromophores 10–12 and thieno[2,3-b]thiophene-cored quadrupolar chromophores 13–15. (c) The molecular structures of the strip structure molecule 16 and trigonal structure molecule 17. (d) The molecular structures of compounds 18–20. (e) The molecular structures of compounds 21–23.

Table 1 Summary of the linear absorption (λ^max_abs and ε^max, when available) and nonlinear absorption (λ^max₂ and δ^max₂) for the reported 2PA dyes 1–23 in ref. 23–27

Dye	Solvent	λ ^maxabs (nm)	ε ^max (10^4 L mol^−1 cm^−1)	λ ^max2 (nm)	δ ^max2 (GM)	Method	Ref.
1	MeOH	325	3.6	514	44	OPPAS	23
2	MeOH	305	2.9	480	40	OPPAS	23
3	MeOH	300	3.0	474	38	OPPAS	23
4	MeOH	296	2.5	472	30	OPPAS	23
5	MeOH	294	2.6	470	27	OPPAS	23
6	MeOH	301	3.4	480	37	OPPAS	23
7	MeOH	312	3.0	484	45	OPPAS	23
8	MeOH	312	3.4	484	49	OPPAS	23
9	MeOH	325	2.3	524	41	OPPAS	23
10	CHCl3	665	18.9	1200	230	fs Z-scan	24
11	CHCl3	691	21.0	1200	1000	fs Z-scan	24
12	CHCl3	710	21.7	1200	7300	fs Z-scan	24
13	CHCl3	763	18.0	1200	2600	fs Z-scan	24
14	CHCl3	790	21.0	1300	4900	fs Z-scan	24
15	CHCl3	799	20.0	1300	5300	fs Z-scan	24
16	—	280	—	418	1427	TD-DFT	25
17	—	350	—	530	3149	TD-DFT	25
18	THF	498	7.76	1000	338	fs 2PEF	26
19	THF	552	6.12	1100	1370	fs 2PEF	26
20	THF	502	6.33	1100	366	fs 2PEF	26
21	CHCl3	955	33.0	1255	1008	fs Z-scan	27
22	CHCl3	1047	18.3	1395	1011	fs Z-scan	27
23	CHCl3	1096	16.4	1445	1177	fs Z-scan	27

---

1.2. The influence of molecular regioisomerism on photophysical properties

The above-mentioned strategies can efficiently increase the δ₂ values but may also introduce other issues. For instance, enhancing ICT may cause a small energy gap, which is detrimental to obtaining a high Φ_F value. Extending the π-conjugation extent may cause poor solubility and photostability, large molecular weight and low membrane permeability. Excessive planarity can lead to strong intermolecular interactions, such as π–π stacking, which may result in aggregation-caused quenching. Increasing the diradical properties may reduce the stability.^28–35 Alternatively, molecular regioisomerism can result in distinct molecular symmetries, molecular π-conjugations, molecular orbital distributions, molecular dipoles, and/or intermolecular interactions, thereby exerting substantial influences on the optical properties of the molecules.^36,37 Regioisomers are structural isomers sharing the same chemical formula but differing in the spatial arrangements of functional groups or substituents within the molecule. These arrangement variations arise from the groups occupying different positions across the molecular skeleton, leading to regioisomerism.^38,39 In the past three years, the influence of molecular regioisomerism on photophysical properties has been reported successively. In 2023, Nakka and coworkers designed and synthesized isomeric organic dyes bearing phenylethynyl (π-spacer), rhodanine-3-acetic acid (acceptor) and carbazole (donor) units. In dye 24, the rhodanine-3-acetic acid unit was positioned at the phenylethynyl side of the carbazole/phenylethynyl pair, whereas in dye 25, the rhodanine-3-acetic acid unit was positioned at the carbazole side of the carbazole/phenylethynyl pair. The molar extinction coefficient (ε) of dye 24 was higher than that of dye 25, suggesting that the strength of ICT for dye 24 is stronger than that of dye 25 (Fig. 2a and Table 2).⁴⁰ In 2023, Cai and coworkers synthesized chromene derivatives incorporated with tosyl amide and regiostructure-dependent substituents (Fig. 2b). A regiostructure-dependent fluorescence property was observed, in that the six-substituted series (compounds 26–29) showed significantly stronger fluorescence at longer wavelengths than the seven-substituted series (compounds 30–33). The six-substituted products had a weaker molecular motion and stronger charge transfer (CT) effect than the seven-substituted ones.⁴¹ In 2023, Tian and coworkers designed and synthesized three regioisomers with an ortho-/meta-/para-substituted electron-withdrawing group (pyridinium conjugated acrylonitrile) on the phenyl unit of tetraphenylethylene (compounds 34–36). The maximum absorption peak (λ^max_abs) of the para-substituted product 36 red-shifted significantly because its strong ICT process leading to a lower energy gap (Fig. 2c and Table 2).⁴² In 2023, Li and coworkers discovered a not-previously reported ring-flipping isomerization based on benzothiazolopyridine derivatives 37 and 38 (Fig. 2d). The significant difference in the Φ_F values for the two isomers was elucidated from the effective and ineffective intramolecular motions (Table 2).⁴³ In 2024, Zhang and coworkers reported that the positional isomerism of bithiazole core D–A–D quadrupolar molecules (compounds 39 and 40) resulted in enhanced molecular planarity and red-shifted spectra (Fig. 2e and Table 2).⁴⁴ In 2024, Cai and coworkers synthesized coumarin derivatives with triphenylamine substituted on the ortho- or para-position (compounds 41 and 42). They found that the ortho-substituted product showed stronger emission in the aggregate states due to the restriction of molecular motion (Fig. 2f and Table 2).⁴⁵

Fig. 2 (a) The molecular structures of compounds 24 and 25. (b) The molecular structures of compounds 26–33. (c) The molecular structures of compounds 34–36. (d) The molecular structures of compounds 37 and 38. (e) The molecular structures of compounds 39 and 40. (f) The molecular structures of compounds 41 and 42.

Table 2 Summary of the linear absorption (λ^max_abs, ε^max, λ^max_em and Φ_F, when available) for the reported dyes 24–60 in ref. 40–50

Dye	Solvent	λ ^maxabs (nm)	ε ^max (10^4 L mol^−1 cm^−1)	λ ^maxem (nm)	Φ F (%)	Ref.
24	CH2Cl2	430	2.9	—	—	40
25	CH2Cl2	448	2.6	—	—	40
34	THF	320	—	—	0.9	42
35	THF	348	—	430	0.4	42
36	THF	425	—	612	0.4	42
37	In the PMMA film	426	—	550	1.9	43
38	In the PMMA film	411	—	510	34.2	43
39	DMSO	491	4.6	—	—	44
40	DMSO	603	2.9	—	—	44
41	THF/H2O (fw = 90%)	—	—	550	9.5	45
42	THF/H2O (fw = 90%)	—	—	600	1.1	45
43	CH2Cl2	344	—	415	15	46
44	CH2Cl2	353	—	422	36	46
45	CH2Cl2	357	—	438	36	46
46	CH2Cl2	371	—	454	49	46
47	NPs in water	760	9.25	942	5.6	47
48	NPs in water	716	—	941	8.7	47
49	NPs in water	718	—	786	2.9	47
50	NPs in water	721	—	939	2.8	47
51	NPs in water	698	—	938	5.3	47
52	NPs in water	694	—	728	2.2	47
53	PhMe	382	3.66	425	0.1	48
54	PhMe	383	4.40	421	15	48
55	PhMe	371	3.35	629	2	48
56	PhMe	465	3.9	572	93	48
57	CH2Cl2	633	—	663	82.9	49
58	CH2Cl2	633	—	663	71	49
59	PhMe	401	6.17	495	45	50
60	PhMe	343	6.66	472	15	50

---

In 2024, Cuadrado and coworkers functionalized the 3,8,13-(compounds 43 and 44) or 2,7,12-position (compounds 45 and 46) of the triindole core with the 2-thienyl or 2-benzothienyl group (Fig. 3a). Whether for the 3,8,13- or 2,7,12-substitued products, the alteration of the substituent from the 2-thienyl group to the 2-benzothienyl group all increased the Φ_F values in CH₂Cl₂. More importantly, the transition from the 3,8,13- to the 2,7,12-substituted product improved the Φ_F values from 0.15 to 0.36 for the 2-thienyl substituted product and from 0.36 to 0.49 for the 2-benzothienyl substituted product (Table 2).⁴⁶ In 2025, Li and coworkers designed and synthesized indacenodithiophene derivatives with phenyls respectively located at the outside (o-series) and inside (i-series) of the side chain (Fig. 3b). The o-series fluorophores (compounds 47–49) displayed significantly higher near-infrared (NIR) fluorescence intensity than the i-series counterparts (compounds 50–52), possibly attributed to the phenyl group at the end of the side chain restricting the movement of the alkyl chain in the o-series (Table 2).⁴⁷ In 2025, Górski and coworkers designed and synthesized dibenzoxazepine modified pyrrolopyrrole derivatives with bromic (compounds 53 and 54) or nitro groups (compounds 55 and 56) (Fig. 3c). The dyes possessing bromic or nitro groups at various positions exhibited markedly different λ^max_abs and Φ_F values (Table 2). The key difference lied in relative energies of dark and bright excited states.⁴⁸ In 2025, Wang and coworkers designed and synthesized meta/para regioisomeric xanthene dyes by incorporating a conjugated tetraphenylethylene (TPE) unit (compounds 57 and 58, Fig. 3d). The meta-substituted product outperformed the para-substituted product in reactive oxygen species (ROS) generation (22.7% vs. 11.7%) and anticancer activity. The meta-substituted product exhibited a narrower ΔE_ST, further improving ISC efficiency (Table 2).⁴⁹ In 2025, Shen and coworkers designed and synthesized D–A–D structured cyanostilbene derivatives with triphenylamine and pyrene as the donors and cyanostilbene as the acceptor. Linear-shaped or curved-shaped molecules were obtained by adjusting the connecting positions of the phenyl rings (para or meta) of the cyanostilbene moiety with the donor units (compounds 59 and 60, Fig. 3e). The Φ_F values of the curve-shaped molecules are smaller than that of the linear-shaped molecules, indicating that the intramolecular motion can more easily occur in the curve-shaped molecules (Table 2).⁵⁰

Fig. 3 (a) The molecular structures of compounds 43–46. (b) The molecular structures of o-series derivatives 47–49 and i-series derivatives 50–52. (c) The molecular structures of compounds 53–56. (d) The molecular structures of compounds 57 and 58. (e) The molecular structures of compounds 59 and 60.

It is worth mentioning that the molecular regioisomerism can also affect the 2PA properties. From a theoretical perspective, isomer positioning may result in important modulations of the twist angle of the molecular backbone or the effective distance between the electron–donor and electron–acceptor groups, thus altering the transition dipole moments.^51–53 The reported examples are mainly concentrated on a few organic chromophores. For instance, in 2020, Li and coworkers realized ACQ-to-AIE transformation by shifting triphenylamine from the end to the bay position of the dithienobenzophenazine (TBP) core (compounds 61 and 62) (Fig. 4a). In the aggregate state, the Φ_F values of the end-decorated product 61 is only 0.2%, while that of the bay-decorated product 62 is 15.6%. Only the bay-decorated product exhibited a sizeable δ₂ value of 608 GM at 880 nm and 207 GM at 1040 nm (Table 3).⁵⁴ In 2020, Jia and coworkers designed and synthesized chalcone derivatives, of which a pair of regioisomer (compounds 63 and 64) exhibited slightly different 2PA characteristics due to the slightly different degree of ICT (Fig. 4b and Table 3).⁵⁵ The contributions of our research group in this field mainly focus on the study of the 2PA properties of bay- and ortho-substituted perylene diimides, which will be discussed in detail in Section 2.4. The categories of the reported organic chromophores mainly include nile red, coumarin, benzothiazole, perylene diimide, triphenylamine, dicyanobenzene and dithienylethene. This review will focus on the first seven categories of organic chromophores, emphasizing the structural characteristics of the regioisomers, the influence of molecular regioisomerism on the 2PA performance of organic chromophores (such as δ₂ and Φ_F values), and the intrinsic structure–property relationships. Finally, it will also briefly discuss the application of these regioisomeric organic chromophores in the field of biological imaging.

Fig. 4 (a) The molecular structures of compounds 61 and 62. (b) The molecular structures of compounds 63 and 64.

Table 3 Summary of the linear absorption (λ^max_abs, ε^max, λ^max_em and Φ_F, when available) and nonlinear absorption (λ^max₂ and δ^max₂, when available) for the reported dyes 61–64 in ref. 54 and 55

Dye	Solvent	λ ^maxabs (nm)	ε ^max (10^4 L mol^−1 cm^−1)	λ ^maxem (nm)	Φ F (%)	λ ^max2 (nm)	δ ^max2 (GM)	Method	Ref.
61	THF	513	—	670	0.2 (solid-state)	—	—	—	54
62	THF	556	—	690	15.6 (solid-state)	880	608	fs 2PEF	54
63	DMSO	447	2.14	598	—	580	1930	fs Z-scan	55
64	DMSO	447	1.86	598	—	580	1737	fs Z-scan	55

---

2. The molecular regioisomerism strategy for optimizing 2PA performance

At present, there are a few examples that the 2PA performances are significantly affected by the regioisomerism. The reported organic chromophores include nile red, coumarin, benzothiazole, perylene diimide, triphenylamine, dicyanobenzene and dithienylethene.

2.1 Nile red derivatives

Nile red (9-diethylamino-5H-benzo[a]phenoxazin-5-one), featuring an electron-donating carbonyl group at the 5-position and an electron-withdrawing diethylamino group at the 9-position, is an important fluorescent dye renowned for its significant solvatochromism and large Stokes shift. The benzo[a]phenoxazine core provide various positions for the substituents, including 1-, 2-, 3-positions, etc.^56,57 In 2020, Hornum and coworkers compared how the installation of a strongly electron-withdrawing CF₃ substituent at different positions affected the 2PA properties of the nile red (NR) chromophore (Fig. 5a). They found that the δ^max₂ value of the 2-substituted NR (compound 65) was 232 GM (λ^max₂ = 1057 nm, in methanol), while those of 4-substituted NR (compound 66) and 3-substituted NR (compound 67) were 183 GM (λ^max₂ = 1055 nm, in methanol) and 123 GM (λ^max₂ = 1065 nm, in methanol), respectively (Table 4).⁵⁸ In 2021, their group designed and synthesized 2-, or 3- or 1-hydroxy substituted NR derivatives (compounds 68–70, Fig. 5b). The 1-hydroxy derivative (compound 70) was able to form a hydrogen bond to the nitrogen atom of the oxazine ring. Increasing the solvent polarity greatly shifted the one-photon absorption and fluorescence emission maxima to longer wavelengths for the three regioisomers 68, 69 and 70 (Fig. 5c and e). The Φ_F values of compounds 68, 69 and 70 all decreased with the solvent polarity increasing. Polar environments might foster solute relaxation into the nonemissive TICT state. The 1-hydroxy derivative (compound 70) stood out as essentially nonemissive, which indicated that the intramolecular hydrogen bond might facilitate nonradiative decay pathways, possibly by excited state intramolecular proton transfer (ESIPT) (Fig. 5f). The δ₂ values of compounds 68, 69 and 70 also varied with the solvent. In chloroform, compound 68 had the largest δ^max₂ value of 170 GM (λ^max₂ = 878 nm), while those of compounds 69 and 70 were 51 (λ^max₂ = 877 nm) and 48 GM (λ^max₂ = 870 nm) (Fig. 5g and Table 4).⁵⁹

Fig. 5 (a) The molecular structures of compounds 65–67. (b) The molecular structures of compounds 68–70. (c)–(e) One-photon absorption and fluorescence emission spectra of compounds 68, 69 and 70 in PhMe, CHCl₃ and MeOH. (f) The Φ_F values of compounds 68, 69 and 70 in PhMe, CHCl₃ and MeOH. (g) The δ^max₂ values of compounds 68, 69 and 70 in PhMe, CHCl₃ and MeOH. Reproduced from ref. 59 with permission from American Chemical Society, copyright 2021.

Table 4 Summary of the linear absorption (λ^max_abs, ε^max, λ^max_em and Φ_F) and nonlinear absorption (λ^max₂ and δ^max₂) for the reported dyes 65–70 in ref. 58 and 59

Dye	Solvent	λ ^maxabs (nm)	ε ^max (10^4 L mol^−1 cm^−1)	λ ^maxem (nm)	Φ F (%)	λ ^max2 (nm)	δ ^max2 (GM)	Method	Ref.
65	MeOH	565	3.64	638	35	1057	232	fs 2PEF	58
66	MeOH	554	3.69	631	43	1055	183	fs 2PEF	58
67	MeOH	569	2.08	632	45	1065	123	fs 2PEF	58
68	CHCl3	530	3.61	603	44	878	170	fs 2PEF	59
69	CHCl3	539	2.90	611	50	877	51	fs 2PEF	59
70	CHCl3	554	3.52	608	0.6	870	48	fs 2PEF	59

---

2.2. Coumarin derivatives

Coumarins are a large family of oxygen-rich heterocyclic compounds consisting of a benzene ring fused to the α-pyrone ring. And coumarin is also one of the most attractive organic fluorophores due to its diverse structures, broad spectral range, high fluorescence quantum yields, excellent photostability, and favourable solubility.⁶⁰ A particularly important class of coumarin derivatives are donor–acceptor systems possessing an electron-donating substituent at position 7 and an electron-withdrawing one at position 3. In 2021, Górski and coworkers designed and synthesized bis-coumarins 71 and 72 possessing an electron-rich pyrrolo[3,2-b]pyrrole bridging unit (Fig. 6a). In this work, the pyrrolo[3,2-b]pyrrole group was chosen for the electron-donating substituent and the CO₂Me group for the electron-withdrawing substituent. Meanwhile, the position of the pyrrolo[3,2-b]pyrrole group was also switched from 7 to 6 for comparison. Their photophysical parameters were highly dependent on the linking position between both chromophores. In PhMe, compound 71 exhibited intense UV light absorption (λ_abs = 358 nm), whereas in the low-energy part of the absorption spectrum a weak, broad band (λ^max_abs = 439 nm) could be observed. Compound 72 showed strong absorption of yellow light (λ^max_abs = 486 nm) in PhMe. No solvatochromism was observed in their absorption spectra of these centrosymmetric dyes; however, a significant drop of absorption coefficient was observed when the polarity of the solvent increased. Solvatofluorochromism was observed in the fluorescence emission spectra of compound 72 because of excited-state symmetry-breaking (Fig. 6b and c). It was worth mentioned that changing the substitution position on the coumarin subunit from 6 to 7 resulted in a 650-fold increase in the Φ_F values from 0.06% to 39% in toluene. At the same time, the δ^max₂ value of compound 71 was ≤10 GM (λ^max₂ = 840 nm), while that of compound 72 was 850 GM (λ^max₂ = 840 nm) in MeTHF (Fig. 6d and e). The frontier orbitals indicated that the S₀ → S₁ transitions in compounds 71 and 72 were ICT transitions from the electron-rich pyrrolo[3,2-b]pyrrole core to the two electron-accepting coumarin subunits, with a lesser degree of charge transfer in compound 72 (Fig. 6f and Table 5).⁶¹

Fig. 6 (a) The molecular structures of compounds 71 and 72. (b) and (c) Absorption (solid line) and normalized fluorescence (dotted line) spectra of compounds 71 and 72 measured in three different solvents. (d) and (e) Two-photon absorption spectra of compounds 71 (red circles) and 72 (blue squares) both in MeTHF. (f) HOMO and LUMO of compounds 71 and 72. Reproduced from ref. 61 with permission from American Chemical Society, copyright 2021.

Table 5 Summary of the linear absorption (λ^max_abs, λ^max_em and Φ_F, when available) and nonlinear absorption (λ^max₂ and δ^max₂) for the reported dyes 71 and 72 in ref. 61

Dye	λ ^maxabs (nm)(solvent)	λ ^maxem (nm) (solvent)	Φ F (%)	λ ^max2 (nm) (solvent)	δ ^max2 (GM)	Method	Ref.
71	439 (PhMe, MeTHF)	641 (PhMe)	0.06 (PhMe)	840 (MeTHF)	≤10	fs Z-scan	61
72	486 (PhMe), 485 (MeTHF)	574 (PhMe), 608 (MeTHF)	39 (PhMe), 30 (MeTHF)	840 (MeTHF)	850	fs Z-scan	61

---

2.3. Benzothiazole derivatives

Benzothiazole is a class of electron-withdrawing heterocyclic compounds containing nitrogen and sulfur.⁶² In 2021, Shu and coworkers designed and synthesized a series of benzothiazole-pyridinium salt derivatives, including the regioisomers with pyridine nitrogen at the meta or para position, named compounds 73 and 74 (Fig. 7a). The Φ_F value of compound 73 in DMF was 23%, which was almost twice that of compound 74 (12%) in DMF. Perhaps, the reason was that compound 73had a stronger π–π conjugative effect than compound 74 due to the closer distance between the pyridine N and the styryl group. The δ^max₂ values of compounds 73 and 74 were 621.66 and 1036.78 GM (λ^max₂ = 770 nm, in DMF), respectively. The possible reason was that compound 74 had better ICT ability than compound 73 (Fig. 7b). The cytotoxicity of compounds 73 and 74 presented a dose-dependent relationship with the cell viability. When the concentration of compounds 73 and 74 was increased to 40 µM, the cell viability dropped to about 65–70% (Fig. 7c). Bio-imaging studies indicated that compounds 73 and 74 could be effectively internalized by HeLa cells. Compound 74 could aggregate in the nuclear membrane, while compound 73 could penetrate the nuclear membrane and mainly concentrated in the nucleus. The possible reason was that the nature of the regioisomers influenced their particle shape and size in aggregates in the cell, thus led to the different stain cell ability (Fig. 7d and Table 6).⁶³

Fig. 7 (a) The molecular structures of compounds 73 and 74. (b) The Φ_F and δ^max₂ values of compounds 73 and 74 in DMF. (c) MTT assay of HeLa cells treated with compounds 73 and 74 at different concentrations for 24 h. (d) One- and two-photon fluorescence microscopy images of HeLa cells stained with compounds 73 and 74. Reproduced from ref. 63 with permission from Elsevier, copyright 2021.

Table 6 Summary of the linear absorption (λ^max_abs, λ^max_em and Φ_F) and nonlinear absorption (λ^max₂ and δ^max₂) for the reported dyes 73 and 74 in ref. 63

Dye	Solvent	λ ^maxabs (nm)	λ ^maxem (nm)	Φ F (%)	λ ^max2 (nm)	δ ^max2 (GM)	Method	Ref.
73	DMF	356	464	23	770	622	fs 2PEF	63
74	DMF	358	503	12	770	1037	fs 2PEF	63

---

2.4. Perylene diimide derivatives

Perylene-3,4,9,10-tetracarboxylic diimide, also referred to as perylene diimides (PDIs), contains a rigid π-conjugated core composed of two naphthalene half units, providing three distinct regions for chemical modification, the bay (1,6,7,12), ortho (2,5,8,11) and imide positions.^64–66 The substitution at the bay-position causes a severe distortion to the perylene core, while the substitution at the ortho-position retains the planarity of the perylene core. In 2021, our group designed and synthesized two positional isomers (compounds 75 and 76) by substituting benzanthrone at the bay- or ortho-position of PDI (Fig. 8a). The one-photon and two-photon excited fluorescence experiments suggested that compound 75 was one-photon and two-photon excited aggregation-caused quenching (ACQ) molecules, while compound 76 was one photon and two-photon excited aggregation-induced emission (AIE) molecules (Fig. 8b, c and e, f). It was probably because that the intramolecular rotation (non-radiative process) of compound 76 is more active than that of compound 75, which could be effectively suppressed by molecular aggregation. The Φ_F values of compounds 75 and 76 in THF were 46% and 0.35%, respectively. The δ^max₂ value of compound 76 is 1104 GM (λ^max₂ = 1100 nm, in THF), while that of compound 75 is 260 GM (λ^max₂ = 850 nm, in THF) (Fig. 8d and Table 7). The ICT process is more dominant in compound 76 than that in compound 75.⁶⁷

Fig. 8 (a) The molecular structures of compounds 75 and 76. (b) and (c) The fluorescence emission spectra of compounds 75 and 76 in the THF/water mixture with different water fractions (f_w). The insets show the photographs of FL images (taken under 365 nm UV light) for the corresponding mixtures. (d) The Φ_F and δ^max₂ values of compounds 75 and 76 in THF. (e) and (f) Two-photon excited FL spectra of compounds 75 and 76 in the THF/water mixture with different f_w. Reproduced from ref. 67 with permission from Royal Society of Chemistry, copyright 2021.

Table 7 Summary of the linear absorption (λ^max_abs, λ^max_em and Φ_F) and nonlinear absorption (λ^max₂ and δ^max₂) for the reported dyes 75–80 in ref. 67–69

Dye	Solvent	λ ^maxabs (nm)	λ ^maxem (nm)	Φ F (%)	λ ^max2 (nm)	δ ^max2 (GM)	Method	Ref.
75	THF	525	632	46	850	260	fs 2PEF	67
76	THF	524	575	0.35	1100	1104	fs 2PEF	67
77	THF	526	573	0.9	1100	969.1	fs 2PEF	68
78	THF	525	574	0.1	1100	4786.5	fs 2PEF	68
79	THF	530	567	54	800	134	fs 2PEF	69
80	THF	524	535	70	800	179	fs 2PEF	69

---

In 2022, our group designed and synthesized two regioisomers (compounds 77 and 78) with AIE properties based on PDI and anthracene moieties (Fig. 9a). Femtosecond transient absorption study on their aggregated state indicated that the increase of the Φ_F values of aggregated molecules was due to the decrease in the amplitude of intramolecular rotation (P3). The rapid Φ_F value growth of compound 78 within the aggregates was due to its much quicker rotation constraint (Fig. 9b and c) than that of compound 77. In THF, the Φ_F values of compounds 77 and 78 were 0.9% and 0.1%, respectively, and the δ^max₂ values of compounds 77 and 78 were 969.1 and 4786.5 GM (λ^max₂ = 1100 nm), respectively (Fig. 9d and Table 7). The potential energy surface (PES) scanning results indicated that the intramolecular rotation process was easier for compound 78 because it had no energy barrier compared to compound 77 (Fig. 9e and f). The significant enhancement of the δ^max₂ value for compound 78 could be attributed to the large π-extensively conjugation due to the planar perylene core.⁶⁸

Fig. 9 (a) The molecular structures of compounds 77 and 78. (b) and (c) The obtained amplitudes of the internal conversion (P1), ICT (P2) and intramolecular rotation (P3) for compounds 77 and 78 in THF/H20 with different f_w. (d) The Φ_F and δ^max₂ values of compounds 77 and 78 in THF. (e) and (f) The potential energy curves of compounds 77 and 78 in S₀ and S₁ as a function of the dihedral angle between the perylene and ANT planes. Reproduced from ref. 68 with permission from Royal Society of Chemistry, copyright 2022.

In 2023, our group designed and synthesized regioisomeric PDI derivatives (compounds 79 and 80) with the electron-withdrawing anthraquinone group substituted at the bay- or ortho-position (Fig. 10a). In THF, the Φ_F values of compounds 79 and 80 were 54% and 70%, respectively, and the δ^max₂ values of compounds 79 and 80 were 134 and 179 GM (λ^max₂ = 800 nm), respectively (Fig. 10d and Table 7). Banking on their bright emission, cellular imaging studies were conducted. The results showed that both compounds 79 and 80 had good biocompatibility (Fig. 10b and c) and lysosome-imaging capability (Fig. 10e).⁶⁹

Fig. 10 (a) The molecular structures of compounds 79 and 80. (b) and (c) Cell viability of HeLa cells incubated with different concentrations of compounds 79 and 80 for 8 h. (d) The Φ_F and δ^max₂ values of compounds 79 and 80 in THF. (e) Confocal images of HeLa cells co-stained with compounds 79 and 80 (25 µM) and LTB (25 µM). Reproduced from ref. 69 with permission from Royal Society of Chemistry, copyright 2023.

2.5. Triphenylamine derivatives

Possessing three aromatic phenyl groups and a lone pair of electrons on the central nitrogen atom, triphenylamine exhibits strong electron-donating ability and excellent light-harvesting capabilities.⁷⁰ In 2022, Klikar and coworkers designed and synthesized triphenylamine-based regioisomers bearing peripheral electron-withdrawing diazine units (pyridazine, pyrimidine, and pyrazine moieties). The diazine ring can provide six various attaching positions, namely pyridazin-3-yl, pyridazin-4-yl, pyrimidin-2-yl, pyrimidin-4-yl, pyrimidin-5-yl, and pyrazin-2-yl. These fluorophores had varied Φ_F and δ^max₂ values.⁷¹ In 2022, Zhu and coworkers designed and synthesized two regioisomeric D–A–A-conjugated fluorescent dyes (compounds 81 and 82) by changing the relative position of the 2,1,3-benzothiadiazole unit in the skeleton of triphenylamine–acrylonitrile dyad. The 2,1,3-benzothiadiazole unit was either separated from the triphenylamine unit by the phenyl-substituted acrylonitrile unit, or directly attached to the triphenylamine and the phenyl-substituted acrylonitrile units (Fig. 11a). Compared to compound 81 (λ^max_abs = 402 nm), compound 82 displayed a red-shifted λ^max_abs (488 nm) in hexane (Fig. 11b). This indicated better π-conjugation in compound 82 and its narrower energy band gap. In hexane, the λ^max_em values of compounds 81 and 82 were 468 and 558 nm, respectively. Notably, compound 81 possessed a low Φ_F value of 1.5% in hexane, while compound 82 exhibited strong fluorescence with a Φ_F value of 93%. This suggested that compound 81 was much more prone to molecular motions in solution than compound 82. The δ^max₂ value of compound 82 was 253 GM (λ^max₂ = 820 nm, in hexane), while the δ^max₂ value of compound 81 could not be obtained by the two-photon excited fluorescence method due to the weak fluorescence signal (Table 8). With the increasing of solvent polarity, gradually red-shifted λ^max_em and decreased fluorescence intensity were observed for compounds 81 and 82 (Fig. 11c and d). This indicated typical twisted intramolecular charge transfer (TICT) features for their flexible twisted D–A–A structures. Both compounds 81 and 82 exhibited AIE properties, and the α_AIE (fluorescence intensity I/I₀) of compound 82 (about 220) was much larger than that of compound 81 (about 40) (Fig. 11e). Cell imaging experiments indicated that these dyes and Lipi-Deep Red showed remarkably similar staining patterns. The Pearson correlation coefficients of compounds 81 and 82 with Lipi-Deep Red were computed as 0.87 and 0.86, respectively (Fig. 11f). These fluorescent dyes could serve as excellent lipid droplet trackers for potential disease-related biomedical applications.⁷²

Fig. 11 (a) The molecular structures of compounds 81 and 82. (b) Normalized absorption spectra of compounds 81 and 82 in hexane. (c)–(d) Fluorescence spectra of compounds 81 and 82 in various solvents. (e) α_AIE of compounds 81 and 82 in the DMSO/water mixtures. The insets show fluorescence photographs of compounds 81 and 82 in DMSO/water mixtures with 99.5% water fractions taken under 365 nm UV irradiation. (f) CLSM images of HeLa cells incubated with compounds 81 and 82 (5 µM) and Lipi-Deep Red (100 nM). PCC = Pearson's correlation coefficient. The scale bar represents 20 µm. Reproduced from ref. 72 with permission from American Chemical Society, copyright 2022.

Table 8 Summary of the linear absorption (λ^max_abs, ε^max, λ^max_em and Φ_F, when available) and nonlinear absorption (λ^max₂ and δ^max₂, when available) for the reported dyes 81–85 in ref. 72 and 73

Dye	Solvent	λ ^maxabs (nm)	ε ^max (10^4 L mol^−1 cm^−1)	λ ^maxem (nm)	Φ F (%)	λ ^max2 (nm)	δ ^max2 (GM)	Method	Ref.
81	Hexane	402	—	468	1.5	—	—	—	72
82	Hexane	488	—	558	93	820	253	fs 2PEF	72
83	PhMe	381	7.79	418	88	750	210	fs 2PEF	73
84	PhMe	376	7.58	410	76	750	207	fs 2PEF	73
85	PhMe	387	1.58	423	78	750	30	fs 2PEF	73

---

In 2025, Fecková and coworkers designed and synthesized tripodal D–(π–A)₃ chromophores bearing electron withdrawing SF₅-group(s) at different peripheral positions (para or meta) of triphenylamine (compounds 83–85), of which compounds 83 and 84 were a pair of regioisomers (Fig. 12a). Compound 83 showed a slightly red-shifted λ^max_abs and λ^max_em values compared to compound 84 (Fig. 12b). With the solvent polarity increasing from toluene (TOL) to acetone (ACT), the fluorescence lifetime of compound 83 decreased more significantly than that of compound 84, which related to a stronger ICT towards the para-placed SF₅-group (Fig. 12c). For the same reason, the δ^max₂ values of compound 83 is slightly larger than that of compound 84 in toluene. In more polar solvent, the difference in δ^max₂ values is becoming increasingly evident (Fig. 12d and Table 8).⁷³

Fig. 12 (a) The molecular structures of the compounds 83–85. (b) Normalized absorption (solid lines) and emission spectra (dashed lines) of chromophores 83–85 in toluene. (c) Fluorescence dynamics in the ns timescale for 83–85 in TOL, THF and ACT, exc.: 400 nm. (d) 2PA spectra for compounds 83–85 in TOL, THF and ACT. Reproduced from ref. 73 with permission from Royal Society of Chemistry, copyright 2025.

2.6. Dicyanobenzene derivatives

Dicyanobenzenes represent a class of cyclic nitrile-bearing molecular motifs. The three dicyanobenzene regioisomers, namely, 1,2-dicyanobenzene (ortho-isomer), 1,3-dicyanobenzene (meta-isomer), and 1,4-dicyanobenzene (para-isomer), have been utilized to explore the influence of electron-withdrawing substitution in the chemistry and structure of the phenyl ring. The ortho- and meta-isomers have relatively large dipole moments, while the para-isomer does not possess a permanent dipole moment due to its symmetry.⁷⁴ In 2023, Chatterjee and coworkers designed and synthesized three regioisomers (compounds 86–88) based on diphenylamine donors and dicyanobenzene acceptors (Fig. 13a). The emergence of the high-energy absorption band (<430 nm) could be attributed to the molecular π–π* transition and the low-energy absorption band (>430 nm) to the charge transfer (CT) transition from the peripheral diphenylamine donor moieties to the core acceptor moiety. With an increasing in the solvent polarity, the absorption band showed no obvious shift, while the fluorescence emission band showed red-shift. The extent of red-shift in the emission maxima was most pronounced in compound 86, suggesting the highest dipole moment of compound 86 in the excited state (Fig. 13b–g). The two-photon cell imaging showed that these fluorophores could effectively illuminate cells, mainly the cytoplasmic part (Fig. 13h).⁷⁵

Fig. 13 (a) The molecular structures of compounds 86–88. (b), (d) and (f) Absorption spectra of compounds 86–88 in solvents of different polarities (10 µM concentration). (c), (e) and (g) Fluorescence emission spectra of compounds 86–88 in solvents of different polarities (10 µM concentration). (h) Two-photon microscopy image (λ^max₂ = 800 nm and collection window is from 530 nm to 620 nm for compound 86, 510 nm to 580 nm for compound 87, and 550 to 630 for compound 88) of MCF-7 cells. Reproduced from ref. 75 with permission from Royal Society of Chemistry, copyright 2023.

In 2025, Mageswar and coworkers designed and synthesized three fluorophores (compounds 89–91) utilizing terephthalonitrile (1,4-dicyanobenzene) as the electron acceptor and diphenylcarbazole and triarylamine as electron donors, of which compounds 90 and 91 were a pair of regioisomers (Fig. 14a). The absorption bands below 400 nm were attributed to the typical π–π* transition, while the broad bands in the 400 to 500 nm range were associated with the CT transition. In toluene, compound 89 exhibited strong greenish-yellow emission centered at 554 nm. Compared to compound 89, compound 91 showed a red-shifted fluorescence emission maximum at 567 nm owing to its electron-rich naphthalene ring connected through the β-position, whereas compound 90 showed blue-shifted fluorescence emission maximum at 548 nm due to the connection via the electron-deficient α-position of naphthalene (Fig. 14b). Compounds 89, 90, and 91 demonstrated δ^max₂ values of 81 GM at 860 nm, 131 GM at 850 nm, and 143 GM at 850 nm, respectively (Fig. 14c–e and Table 9).⁷⁶

Fig. 14 (a) The molecular structures of compounds 89–91. (b) The normalized ultraviolet-visible absorption spectra and fluorescence emission spectra of compounds 89–91 in toluene solution. (c)–(e) 2PA spectra of compounds 89–91 in toluene at 10⁻⁴ mol L⁻¹ concentration (1PA spectra are shown in black dashed line). Reproduced from ref. 76 with permission from John Wiley and Sons, copyright 2024.

Table 9 Summary of the linear absorption (λ^max_abs, λ^max_em and Φ_F) and nonlinear absorption (λ^max₂ and δ^max₂) for the reported dyes 89–91 in ref. 76

Dye	Solvent	λ ^maxabs (nm)	λ ^maxem (nm)	Φ F (%)	λ ^max2 (nm)	δ ^max2 (GM)	Method	Ref.
89	PhMe	459	554	82	860	81	fs 2PEF	76
90	PhMe	469	548	78	850	131	fs 2PEF	76
91	PhMe	474	567	74	850	143	fs 2PEF	76

---

2.7 Dithienylethene derivatives

Dithienylethenes (DTEs) are a class of well-known fluorophores. The previous works were mainly focused on the photocyclization process, in which DTEs can be reversibly transformed from a flexible ring-open isomer to a rigid ring-closed isomer.⁷⁷ In 2022 and 2025, Barale and coworkers designed and synthesized regioisomers based on DTEs including bis-(2-pyrenyl) DTE (compound 92) and bis-(1-pyrenyl) DTE (compound 93) (Fig. 15a). The two regioisomers were obtained by connecting different positions (1- or 2-position) of the pyrene substituent with dithienylethenes. The λ^max_abs and λ^max_em values for compound 92 were 378 and 490 nm (in CH₂Cl₂), respectively; while those for compound 93 were 422 and 515 nm (in CH₂Cl₂), respectively. The red-shift in the absorption and emission spectra indicated that the 1-connection of pyrene enhanced the electronic communication between pyrene and DTE. The Φ_F values of compound 92 and compound 93 were 5% and 25% (in CH₂Cl₂), respectively (Fig. 15b). Functionalization of pyrene at the 1-position provides intense electronic delocalization throughout the entire internal arm, thereby establishing a pathway for radiative deactivation. The δ^max₂ values of compound 92 and compound 93 were 90 and 51 GM (λ^max₂ = 700 nm, in CH₂Cl₂), respectively (Fig. 15c and d and (Table 10).).^78,79

Fig. 15 (a) The molecular structures of compounds 92 and 93. (b) The Φ_F values of compounds 92 and 93 in CH₂Cl₂. (c) and (d) Two photon absorption cross section values vs. excitation wavelength in dichloromethane for compounds 92 and 93; the error on calculated values is estimated as ±15%. Reproduced from ref. 78 with permission from American Chemical Society, copyright 2022. Reproduced from ref. 79 with permission from Elsevier, copyright 2025.

Table 10 Summary of the linear absorption (λ^max_abs, ε^max, λ^max_em and Φ_F) and nonlinear absorption (λ^max₂ and δ^max₂) for the reported dyes 92 and 93 in ref. 78 and 79

Dye	Solvent	λ ^maxabs (nm)	ε ^max (10^4 L mol^−1 cm^−1)	λ ^maxem (nm)	Φ F (%)	λ ^max2 (nm)	δ ^max2 (GM)	Method	Ref.
92	CH2Cl2	378	2.79	490	5	700	90	fs 2PEF	78
93	CH2Cl2	422	3.28	515	25	700	51	fs 2PEF	79

---

3. Conclusions

In this review, firstly, we discussed the recent progress on optimizing the 2PA performance of organic chromophores through conventional strategies in the last five years, such as enhancing intramolecular charge transfer, extending the π-conjugation length, increasing the number of π-conjugation paths, and improving coplanarity. Secondly, we discussed the progress on tuning the photophysical properties of organic chromophores through the strategy of molecular regioisomerism in the past three years. These photophysical properties generally include a molar extinction coefficient, maximum absorption wavelength, fluorescence emission intensity, fluorescence emission wavelength, fluorescence quantum yield, reactive oxygen species generation, etc. Thirdly, we focused on discussing the structural characteristics of seven categories of regioisomeric organic chromophores and their two-photon absorption properties. The seven categories of organic chromophores include nile red, coumarin, benzothiazole, perylene diimide, triphenylamine, dicyanobenzene and dithienylethene. Compared to the conventional strategy for improving the 2PA performance of organic chromophores, molecular regioisomerism can regulate the symmetry, π-conjugation, orbital distribution, dipole and intermolecular interactions of a molecule, ultimately optimizing the 2PA performance of the molecule without changing the molecular weight. In the future, the possibility of designing and synthesizing various regioisomers of organic chromophores will be further explored. Research on optimizing the two-photon absorption performance of organic chromophores through the regioisomerism strategy will also be further deepened.

Conflicts of interest

There are no conflicts to declare.

Data availability

No primary research results, software or code have been included and no new data were generated or analysed as part of this review. All results, discussions, and conclusions presented herein are based exclusively on previously published studies. The relevant sources are fully cited in the reference list to ensure transparency and reproducibility of the information discussed.

Acknowledgements

L. X. acknowledges financial support from the National Natural Science Foundation of China (52073167) and the Southwest Petroleum University Scientific Research Foundation for Talents. G. N. acknowledges the funding support from the National Natural Science Foundation of China (22478033).

References

1. M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu and C. Xu, Design of Organic Molecules with Large Two-Photon Absorption Cross Sections, Science, 1998, 281, 1653–1656.
2. M. Pawlicki, H. A. Collins, R. G. Denning and H. L. Anderson, Two-Photon Absorption and the Design of Two-Photon Dyes, Angew. Chem., Int. Ed., 2009, 48, 3244–3266.
3. T. Zhao, L. Li, S. Li, X.-F. Jiang, C. Jiang, N. Zhou, N. Gao and Q.-H. Xu, Gold nanorod-enhanced two-photon excitation fluorescence of conjugated oligomers for two-photon imaging guided photodynamic therapy, J. Mater. Chem. C, 2019, 7, 14693–14700.
4. L. Wu, J. Liu, P. Li, B. Tang and T. D. James, Two-photon small-molecule fluorescence-based agents for sensing, imaging, and therapy within biological systems, Chem. Soc. Rev., 2021, 50, 702–734.
5. L. Xu, H. Zhu, G. Long, J. Zhao, D. Li, R. Ganguly, Y. Li, Q.-H. Xu and Q. Zhang, 4-Diphenylamino-phenyl substituted pyrazine: nonlinear optical switching by protonation, J. Mater. Chem. C, 2015, 3, 9191–9196.
6. C.-L. Sun, S.-K. Lv, Y.-P. Liu, Q. Liao, H.-L. Zhang, H. Fu and J. Yao, Benzoindolic squaraine dyes with a large two-photon absorption cross-section, J. Mater. Chem. C, 2017, 5, 1224–1230.
7. Z.-H. Wu, Z.-T. Huang, R.-X. Guo, C.-L. Sun, L.-C. Chen, B. Sun, Z.-F. Shi, X. Shao, H. Li and H.-L. Zhang, 4,5,9,10-Pyrene Diimides: A Family of Aromatic Diimides Exhibiting High Electron Mobility and Two-Photon Excited Emission, Angew. Chem., Int. Ed., 2017, 56, 13031–13035.
8. X. Liang and Q. Zhang, Recent progress on intramolecular charge-transfer compounds as photoelectric active materials, Sci. China Mater., 2017, 60, 1093–1101.
9. C.-L. Sun, J. Li, X.-Z. Wang, R. Shen, S. Liu, J.-Q. Jiang, T. Li, Q.-W. Song, Q. Liao, H.-B. Fu, J.-N. Yao and H.-L. Zhang, Rational Design of Organic Probes for Turn-On Two-Photon Excited Fluorescence Imaging and Photodynamic Therapy, Chem, 2019, 5, 600–616.
10. L. Xu, J. Zhang, L. Yin, X. Long, W. Zhang and Q. Zhang, Recent progress in efficient organic two-photon dyes for fluorescence imaging and photodynamic therapy, J. Mater. Chem. C, 2020, 8, 6342–6349.
11. Q.-S. Zhang, X.-D. Zhang, J.-Y. Zhuang and M. Pan, Highly two-photon and X-ray excited long-persistent luminescence in a crystalline host-guest aggregate, Aggregate, 2024, 5, e456.
12. Z. Chen, Z. Zhang, F. Zeng and S. Wu, Visualizing Detection of Diabetic Liver Injury by a Biomarker-Activatable Probe via NIR-II Fluorescence Imaging, Chem. Biomed. Imaging, 2023, 1, 716–724.
13. C. Xu, W. Zipfel, J. B. Shear, R. M. Williams and W. W. Webb, Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy, Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 10763–10768.
14. G. S. He, L.-S. Tan, Q. Zheng and P. N. Prasad, Multiphoton Absorbing Materials:  Molecular Designs, Characterizations, and Applications, Chem. Rev., 2008, 108, 1245–1330.
15. L. Xu, W. Lin, B. Huang, J. Zhang, X. Long, W. Zhang and Q. Zhang, The design strategies and applications for organic multi-branched two-photon absorption chromophores with novel cores and branches: a recent review, J. Mater. Chem. C, 2021, 9, 1520–1536.
16. C. Han, B. K. Kundu, R. Chen, Pragti, P. Srivastava, C. G. Elles and Y. Sun, Near-Infrared Light-Driven Condensation Using Branched Two-Photon-Absorbing Organic Photocatalysts with Viscosity-Dependent Properties, J. Am. Chem. Soc., 2025, 147, 20525–20533.
17. Pragti, B. K. Kundu, R. Chen, J. Diao and Y. Sun, Near-Infrared Bioimaging Using Two-photon Fluorescent Probes, Adv. Healthcare Mater., 2025, 14, 2403272.
18. B. Kumar Kundu, N. Bashar, P. Srivastava, C. G. Elles and Y. Sun, Organic Two-Photon-Absorbing Photosensitizers Can Overcome Competing Light Absorption in Organic Photocatalysis, Chem. – Eur. J., 2024, 30, e202402856.
19. C. Han, B. K. Kundu, Y. Liang and Y. Sun, Near-Infrared Light-Driven Photocatalysis with an Emphasis on Two-Photon Excitation: Concepts, Materials, and Applications, Adv. Mater., 2024, 36, 2307759.
20. B. K. Kundu, C. Han, P. Srivastava, S. Nagar, K. E. White, J. A. Krause, C. G. Elles and Y. Sun, Trifluoromethylative Bifunctionalization of Alkenes via a Bibenzothiazole-Derived Photocatalyst under Both Visible- and Near-Infrared-Light Irradiation, ACS Catal., 2023, 13, 8119–8127.
21. K. Kamada, S.-I. Fuku-en, S. Minamide, K. Ohta, R. Kishi, M. Nakano, H. Matsuzaki, H. Okamoto, H. Higashikawa, K. Inoue, S. Kojima and Y. Yamamoto, Impact of Diradical Character on Two-Photon Absorption: Bis(acridine) Dimers Synthesized from an Allenic Precursor, J. Am. Chem. Soc., 2013, 135, 232–241.
22. T. Maeda, T. Oka, D. Sakamaki, H. Fujiwara, N. Suzuki, S. Yagi, T. Konishi and K. Kamada, Unveiling a new aspect of oxocarbons: open-shell character of 4- and 5-membered oxocarbon derivatives showing near-infrared absorption, Chem. Sci., 2023, 14, 1978–1985.
23. T. Ishii, T. Isozaki, S. Kinoshita, R. Takeuchi, W. Kashihara and T. Suzuki, A Substituent Effect on Two-Photon Absorption of Diphenylacetylene Derivatives with an Electron-Donating/Withdrawing Group, J. Phys. Chem. A, 2021, 125, 1688–1695.
24. T. G. Allen, S. Benis, N. Munera, J. Zhang, S. Dai, T. Li, B. Jia, W. Wang, S. Barlow, D. J. Hagan, E. W. Van Stryland, X. Zhan, J. W. Perry and S. R. Marder, Highly Conjugated, Fused-Ring, Quadrupolar Organic Chromophores with Large Two-Photon Absorption Cross-Sections in the Near-Infrared, J. Phys. Chem. A, 2020, 124, 4367–4378.
25. X. Wang, D. Wang, J. Li, M. Zhang, D. Kang and P. Song, Super-Exchange Charge Transfer in One-Photon and Two-Photon Absorption of Multibranched Compounds, ACS Omega, 2022, 7, 9743–9753.
26. L. Xu, J. Liu, X. Long, J. Qing, Z. Yu, B. Huang, J. Wu, S. Liu, L. Liu, Y. Wu, Z. Deng, X. Liu, X.-F. Jiang and J. Wang, Improving two-photon active cross-section value of perylene diimide derivatives through intramolecular photocyclization, J. Mater. Chem. C, 2025, 13, 20769–20779.
27. T. Oka, T. Maeda, D. Sakamaki, H. Fujiwara, N. Suzuki, S. Kodama, S. Yagi, L. Mauri and K. Kamada, Croconaine dyes with intermediate diradical character exhibiting intense one- and two-photon absorption in the short-wavelength infrared region, J. Mater. Chem. C, 2025, 13, 11970–11978.
28. X. Dong, C.-Z. Wang, X. Ran, J.-Y. Cao, Z. Wang and T. Zhang, Substituent effects on the emission quantum efficiency in pyrene-based luminogens featured two-photon absorption: A theoretical and experimental study, Dyes Pigm., 2024, 222, 111867.
29. Q. Li and Z. Li, Miracles of molecular uniting, Sci. China Mater., 2020, 63, 177–184.
30. J. Yang, M. Fang and Z. Li, Organic luminescent materials: The concentration on aggregates from aggregation-induced emission, Aggregate, 2020, 1, 6–18.
31. Y. Fan, Q. Li and Z. Li, Organic luminogens bearing alkyl substituents: design flexibility, adjustable molecular packing, and optimized performance, Mater. Chem. Front., 2021, 5, 1525–1540.
32. Q. Liao, Q. Li and Z. Li, The Key Role of Molecular Packing in Luminescence Property: From Adjacent Molecules to Molecular Aggregates, Adv. Mater., 2025, 37, 2306617.
33. L. H. Zucolotto Cocca, J. V. P. Valverde, C. M. Leite, N. S. Moreno, A. L. Neto, A. G. Macedo, S. Pratavieira, D. L. Silva, P. C. Rodrigues, V. Zucolotto, C. R. Mendonça and L. De Boni, Advancements in organic fluorescent materials: unveiling the potential of peripheral group modification in dithienyl-diketopyrrolopyrrole derivatives for one- and two-photon bioimaging, J. Mater. Chem. B, 2025, 13, 1013–1023.
34. K. Fuchs, N. Oberhof, G. Sauter, A. Pollien, K. Brödner, F. Rominger, J. Freudenberg, A. Dreuw, P. Tegeder and U. H. F. Bunz, Azaacene Diradicals Based on Non-Kekulé Meta-Quinodimethane with Large Two-Photon Cross-Sections in the Infrared Spectral Region, Angew. Chem., Int. Ed., 2024, 63, e202406384.
35. D. Chen, Y. Xu, Y. Wang, X. Li, D. Yin and L. Yan, Diradicaloid-Loaded Polypeptide Nanoparticles for Two-Photon NIR Phototheranostics, ACS Appl. Mater. Interfaces, 2024, 16, 59907–59920.
36. Y. Hu, X. Ding, J. Feng, Y. Yan, C. Shao, X. Yu, J. Shao, Y. Ji, Y. Li, W. Huang and Y. Li, Molecular Regioisomerism of Benzobisthiazole-Based Conjugated Polymers Promotes Photocatalytic Hydrogen Production, Adv. Funct. Mater., 2024, 34, 2400946.
37. Q. Lu, G. K. Kole, A. Friedrich, K. Müller-Buschbaum, Z. Liu, X. Yu and T. B. Marder, Comparison Study of the Site-Effect on Regioisomeric Pyridyl–Pyrene Conjugates: Synthesis, Structures, and Photophysical Properties, J. Org. Chem., 2020, 85, 4256–4266.
38. C. Verma, A. Singh, P. Singh, K. Yop Rhee and A. Alfantazi, Regioisomeric effect of heteroatoms and functional groups of organic ligands: Impacts on coordination bonding and corrosion protection performance, Coord. Chem. Rev., 2024, 515, 215966.
39. L. Holzhauer, C. Francener, D. Elsing, A. Danos, O. Fuhr, N. Jung, W. Wenzel, A. P. Monkman, S. Bräse, M. Kozlowska and E. V. Puttock, Steering the luminescence of donor–acceptor materials by regioisomerism of triazole linkers, Mater. Chem. Front., 2025 10.1039/d5qm00572.
40. N. Nakka, D. Kushavah, S. Ghosh and S. K. Pal, Photophysical, electrochemical and electron donating properties of rhodanine-3-acetic acid-linked structural isomers, Chem. Phys., 2023, 566, 111793.
41. X.-M. Cai, Y. Lin, J. Zhang, Y. Li, Z. Tang, X. Zhang, Y. Jia, W. Wang, S. Huang, P. Alam, Z. Zhao and B. Z. Tang, Chromene-based BioAIEgens: ‘in-water’ synthesis, regiostructure-dependent fluorescence and ER-specific imaging, Natl. Sci. Rev., 2023, 10, nwad233.
42. X. Tian, H. Wang, S. Cao, Y. Liu, F. Meng, X. Zheng and G. Niu, Cationic tetraphenylethylene-based AIE-active acrylonitriles: investigating the regioisomeric effect, mechanochromism, and wash-free bioimaging, J. Mater. Chem. C, 2023, 11, 5987–5994.
43. X. Li, W. Li, X. Liu, M. Zhang, E. Y. Yu, A. W. K. Law, X. Ou, J. Zhang, H. H. Y. Sung, X. Tan, J. Sun, J. W. Y. Lam, Z. Guo, B. Z. Tang and A. Photoactivatable Luminescent Motif, through Ring-Flipping Isomerization for Multiple Photopatterning, J. Am. Chem. Soc., 2023, 145, 26645–26656.
44. P. Zhang, Q. Shen, J. Yang, Z. Zhao, A. Gao, S. Chen, Y. Zhang, L. Meng and D. Dang, Shuttle-like nanoassemblies by isomeric photosensitizers to enhance ROS generation and tumor penetration for photodynamic therapy, Matter, 2024, 7, 4342–4355.
45. X.-M. Cai, S. Li, W.-J. Wang, Y. Lin, W. Zhong, Y. Yang, F. E. Kühn, Y. Li, Z. Zhao and B. Z. Tang, Natural Acceptor of Coumarin-Isomerized Red-Emissive BioAIEgen for Monitoring Cu2+ Concentration in Live Cells via FLIM, Adv. Sci., 2024, 11, 2307078.
46. A. Cuadrado, R. Bujaldón, C. Fabregat, J. Puigdollers and D. Velasco, Confronting positions: para- vs. meta-functionalization in triindole for p-type air-stable OTFTs, Org. Electron., 2024, 128, 107020.
47. C. Li, M. Yao, G. Jiang, L. Feng, Y. Wu, R. Sha, Y. Li, B. Z. Tang and J. Wang, Side Chain Phenyl Isomerization-Induced Spatial Conjugation for Achieving Efficient Near-Infrared II Phototheranostic Agents, Angew. Chem., Int. Ed., 2025, 64, e202419785.
48. K. Górski, S. Shelton, J. Lingagouder, P. Data, D. Jacquemin and D. T. Gryko, 1,4-Dihydropyrrolo[3,2-b]pyrrole modified with dibenzoxazepine: a highly efficient core for charge-transfer-based OLED emitters, Chem. Sci., 2025, 16, 5223–5233.
49. X. Wang, X. Han, X. Tian, H. Lee, C. Xiang, C. Wang, L. Luo, H.-Y. Hu, G. Niu and J. Yoon, Regioisomeric Engineering of Sterically Hindered Bright Near-Infrared Paraptosis Agents for Chemo-Photodynamic Therapy, J. Am. Chem. Soc., 2025, 147, 27068–27080.
50. H. Shen, X. Fu, Y. Wang, Q. Shi, X. Xu, L. Li, Q. Li, J. Yang and C. Wang, Molecular conformation and cyano regio-isomerization effects to the piezochromic properties of cyanostilbene-bridged donor-acceptor-donor structured organic luminogens, J. Mol. Struct., 2025, 1327, 141198.
51. C. Katan, M. Blanchard-Desce and S. Tretiak, Position Isomerism on One and Two Photon Absorption in Multibranched Chromophores: A TDDFT Investigation, J. Chem. Theory Comput., 2010, 6, 3410–3426.
52. S. S. Rajput, R. Zaleśny and M. M. Alam, Chromophore Planarity, –BH Bridge Effect, and Two-Photon Activity: Bi- and Ter-Phenyl Derivatives as a Case Study, J. Phys. Chem. A, 2023, 127, 7928–7936.
53. S. Sirimatayanant and T. Andruniów, Tuning Two-Photon Absorption in Rhodopsin Chromophore via Backbone Modification: The Story Told by CC2 and TD-DFT, J. Chem. Theory Comput., 2024, 20, 8118–8126.
54. Y. Li, S. Liu, H. Ni, H. Zhang, H. Zhang, C. Chuah, C. Ma, K. S. Wong, J. W. Y. Lam, R. T. K. Kwok, J. Qian, X. Lu and B. Z. Tang, ACQ-to-AIE Transformation: Tuning Molecular Packing by Regioisomerization for Two-Photon NIR Bioimaging, Angew. Chem., Int. Ed., 2020, 59, 12822–12826.
55. J. Jia, X. Zhang, Y. Wang, Y. Shi, J. Sun, J. Yang and Y. Song, Enhanced Two-Photon Absorption of Cross-Conjugated Chalcone Derivatives: Modulation of the Effective π-Conjugated Structure, J. Phys. Chem. A, 2020, 124, 10808–10816.
56. M. W. Mulberg, M. Hornum, P. Reinholdt, B. B. Jensen, M. Szomek, J. R. Brewer, D. Wüstner, J. Kongsted and P. Nielsen, Facile Suzuki Coupling Strategy toward New Nile Red Derivatives with Improved Two-Photon Brightness, Eur. J. Org. Chem., 2023, e202300238.
57. C. Gajo, D. Shchepanovska, J. F. Jones, G. Karras, P. Malakar, G. M. Greetham, O. A. Hawkins, C. J. C. Jordan, B. F. E. Curchod and T. A. A. Oliver, Nile Red Fluorescence: Where's the Twist?, J. Phys. Chem. B, 2024, 128, 11768–11775.
58. M. Hornum, P. Reinholdt, J. K. Zaręba, B. B. Jensen, D. Wüstner, M. Samoć, P. Nielsen and J. Kongsted, One- and two-photon solvatochromism of the fluorescent dye Nile Red and its CF3, F and Br-substituted analogues, Photochem. Photobiol. Sci., 2020, 19, 1382–1391.
59. M. Hornum, M. W. Mulberg, M. Szomek, P. Reinholdt, J. R. Brewer, D. Wüstner, J. Kongsted and P. Nielsen, Substituted 9-Diethylaminobenzo[a]phenoxazin-5-ones (Nile Red Analogues): Synthesis and Photophysical Properties, J. Org. Chem., 2021, 86, 1471–1488.
60. Q. Xie, C. Liao, H. Liu, S. Wang and X. Li, Oxygen heterocyclic coumarin-based hybridized local and charge-transfer deep-blue emitters for solution-processed organic light-emitting diodes, J. Mater. Chem. C, 2024, 12, 11085–11093.
61. K. Górski, I. Deperasińska, G. V. Baryshnikov, S. Ozaki, K. Kamada, H. Ågren and D. T. Gryko, Quadrupolar Dyes Based on Highly Polarized Coumarins, Org. Lett., 2021, 23, 6770–6774.
62. D. Yang, S. Li, X. Wu, W. Wang, Z. Cai and C. Ma, Synthesis, Optical Properties, and Applications of Luminescent Benzothiazole: Base Promoted Intramolecular C–S Bond Formation, J. Org. Chem., 2023, 88, 11581–11589.
63. D. Shu, D. Wang, M. Li, H. Cai, L. Kong, Y. Tian, X. Gan and H. Zhou, Small molecules based Benzothiazole-pyridinium salts with different anions: Two-photon fluorescence regulation and difference in cell imaging application, Dyes Pigm., 2021, 194, 109639.
64. L. Zong, H. Zhang, Y. Li, Y. Gong, D. Li, J. Wang, Z. Wang, Y. Xie, M. Han, Q. Peng, X. Li, J. Dong, J. Qian, Q. Li and Z. Li, Tunable Aggregation-Induced Emission Nanoparticles by Varying Isolation Groups in Perylene Diimide Derivatives and Application in Three-Photon Fluorescence Bioimaging, ACS Nano, 2018, 12, 9532–9540.
65. A. J. Riives, Z. Huang, N. T. Anderson and P. H. Dinolfo, 1,7-, 1,6-, and 1,6,7-Derivatives of dodecylthio perylene diimides: Synthesis, characterization, and comparison of electrochemical and optical properties, J. Photochem. Photobiol., A, 2023, 437, 114441.
66. D. Li, W. Zhao, G. Li, Y. Xu, L. Xu and B. Tang, S/Se-Annulated Star-Shaped Perylene Diimides (PDIs) with Large Two-Photon Absorption (TPA) Cross-Section in NIR-I Region, Chem. – Eur. J., 2025, 31, e202403510.
67. X. Long, J. Wu, S. Yang, Z. Deng, Y. Zheng, W. Zhang, X.-F. Jiang, F. Lu, M.-D. Li and L. Xu, Discovery of and insights into one-photon and two-photon excited ACQ-to-AIE conversion via positional isomerization, J. Mater. Chem. C, 2021, 9, 11679–11689.
68. L. Xu, X. Long, F. Kang, Z. Deng, J. He, S. Yang, J. Wu, L. Yin, X.-F. Jiang, F. Lu, M.-D. Li and Q. Zhang, Simultaneously enhancing aggregation-induced emission and boosting two-photon absorption of perylene diimides through regioisomerization, J. Mater. Chem. C, 2022, 10, 7039–7048.
69. L. Xu, X. Long, J. He, L. Liu, F. Kang, Z. Deng, J. Wu, X.-F. Jiang, J. Wang and Q. Zhang, Aggregation effects on the one- and two-photon excited fluorescence performance of regioisomeric anthraquinone-substituted perylenediimide, J. Mater. Chem. C, 2023, 11, 8037–8044.
70. S. Li, J. Lin, X. Chen, W. Liu, Z. Xie, C. Zeng, J. Wang, Y. Min and X. Feng, Pyrene-Based Triphenylamine Light-Harvesting Materials with Related Structure–Property Relationships, J. Org. Chem., 2025, 90, 6702–6712.
71. M. Klikar, D. Georgiou, I. Polyzos, M. Fakis, Z. Růžičková, O. Pytela and F. Bureš, Triphenylamine-based fluorophores bearing peripheral diazine regioisomers. Synthesis, characterization, photophysics and two-photon absorption, Dyes Pigm., 2022, 201, 110230.
72. X. Zhu, L. Feng, S. Cao, J. Wang and G. Niu, Donor–Acceptor–Acceptor-Conjugated Dual-State Emissive Acrylonitriles: Investigating the Effect of Acceptor Unit Order and Biological Imaging, Org. Lett., 2022, 24, 8305–8309.
73. M. Fecková, M. Klikar, C. Vourdaki, I. Georgoulis, O. Pytela, S. Achelle, Z. Růžičková, M. Fakis, P. Beier and F. Bureš, Selective employment of electronic effects of the pentafluorosulfanyl group across linear and tripodal push–pull chromophores with two-photon absorption, Mater. Adv., 2025, 6, 5713–5725.
74. K. T. Workman, R. A. Firth and W. K. Gichuhi, From Benzonitrile to Dicyanobenzenes: The Effect of an Additional CN Group on the Thermochemistry and Negative Ion Photoelectron Spectra of Dicyanobenzene Radical Anions, J. Phys. Chem. A, 2023, 127, 181–194.
75. A. Chatterjee, J. Chatterjee, S. Sappati, R. Tanwar, M. D. Ambhore, H. Arfin, R. M. Umesh, M. Lahiri, P. Mandal and P. Hazra, Engineering TADF, mechanochromism, and second harmonic up-conversion properties in regioisomeric substitution space, Chem. Sci., 2023, 14, 13832–13841.
76. G. V. Mageswari, Y. Chitose, Y. Tsuchiya, J.-H. Lin and C. Adachi, Rational Molecular Design for Balanced Locally Excited and Charge- Transfer Nature for Two-Photon Absorption Phenomenon and Highly Efficient TADF-Based OLEDs, Angew. Chem., Int. Ed., 2025, 64, e202420417.
77. H. Zhang, Y. Qi, X. Zhao, M. Li, R. Wang, H. Cheng, Z. Li, H. Guo and Z. Li, Dithienylethene-Bridged Fluoroquinolone Derivatives for Imaging-Guided Reversible Control of Antibacterial Activity, J. Org. Chem., 2022, 87, 7446–7455.
78. M. Barale, M. Escadeillas, G. Taupier, Y. Molard, C. Orione, E. Caytan, R. Métivier and J. Boixel, Nondestructive All-Optical Readout through Photoswitching of Intramolecular Excimer Emission, J. Phys. Chem. Lett., 2022, 13, 10936–10942.
79. M. Barale, I. Turcas, V. Gousseau, M. Escadeillas, E. Caytan, G. Taupier, Y. Molard, A. Fihey and J. Boixel, Photo-modulation of the two-photon excited fluorescence of dithienylethene bis-(1-pyrenyl) compounds: An experimental and theoretical study, Dyes Pigm., 2025, 232, 112473.

---