Near-Infrared Bioimaging Using Two-photon Fluorescent Probes

Abstract

Near-infrared (NIR) bioimaging has emerged as a transformative technology in biomedical research. Among many fluorescent probes that are suitable for NIR imaging studies, two-photon absorption (TPA) ones represent a particularly promising category, because TPA fluorescent probes can overcome the inherent limitations of one-photon absorption (OPA) counterparts. By leveraging the unique properties of two-photon absorption, TPA fluorescent probes achieve superior tissue penetration, significantly reduced photodamage, and enhanced spatial resolution. This perspective article delves into the fundamental principles, design strategies, and representative TPA probes for various imaging applications. In particular, a number of molecular fluorescent probes, ranging from organic, inorganic, and COF/MOF-based systems are highlighted to showcase the vast scope of possible TPA probe design and application scenarios. In addition, the employment of stimulated TPA probes that are responsive to different external factors, including pH, redox species, enzymes, and hypoxia, is also discussed. In the end, the future perspectives for the continuous advancement of TPA fluorescent probes in the NIR bioimaging field are presented. For instance, it is essential to transition from cellular to in vivo imaging studies to obtain more physiologically relevant insights. Additionally, the development of "dual-function" TPA probes for both disease diagnosis and therapeutic treatment is particularly promising.

Keywords: bioimaging; fluorescent probes; two‐photon absorption.

Figure 1.

(A) Wavelength-dependent ligth penetration through human skin. (B) One-photon and two-photon excited fluorescence and phosphorescence processes. S₀: singlet ground state; S₁: singlet excited state; T₁: triplet excited state; ISC: inter-system crossing; Fl: fluorescence; Ph: phosphorescence.

Figure 2.

(A-C) Dipolar, quadrupolar, and octupolar structures of TPA molecules and corresponding examples (D: electron-donating group; A: electron-accepting group; π: π-conjugated bridge). (D) Examples of designer inorganic TPA complexes where ‘R’ is an electron donating group.

Figure 3.

(A) Chemical structures of MSP-1arm, Lyso-2arm, and Mito-3arm. (B) One-photon confocal microscopy image of HeLa cells stained with Mito-3arm (λ_ex = 488 nm, λ_ex = 500–550 nm). (C) Magnified view of the dashed rectangle in (B). (D) Fluorescence intensity profile along the white line in (C). (E, F) 3D fluorescence intensity plots for the selected regions in (B). (G) Two-photon confocal microscopy image of HeLa cells stained with Mito-3arm (λ_ex = 840 nm, λ_em = 500–550 nm). (H) Enlarged view of the dashed rectangle in (G). (I) Fluorescence intensity profile along the white line in (H). (J, K) 3D fluorescence intensity plots for the selected regions in (G). (L) Z-stack confocal microscopy images of organoids stained with Mito-3arm using one-photon (488 nm) and two-photon (840 nm) excitation. Central fluorescence intensity profiles were plotted for (M) one-photon and (N) two-photon excitation. Reproduced with permission.^[33] Copyright 2024, Elsevier Inc.

Figure 4.

(A) Reaction between AzuFlu 483-BPin and ONOO⁻ or H₂O₂. (B) TPM images of RAW 264.7 macrophages stained with AzuFlu 483-BPin. (i) Control image in absence of any RNS/ROS. (ii, iii) Cells treated in the presence of RNS/ROS: (ii) H₂O₂, and (iii) ONOO⁻. λ_ex = 800 nm, λ_em = 400−600 nm. Reproduced with permission.^[34] Copyright 2019, American Chemical Society.

Figure 5.

Reaction profile between BHID-Bpin and ONOO⁻ or H₂O_2.^[35]

Figure 6.

(A) Schematic representation of the ultrafast sensing mechanism toward norepinephrine using BPS3. (B) 3D two-photon confocal fluorescence images of the hippocampus region in mouse brain labeled with BPS3 (λ_ex = 720 nm). Reproduced with permission.^[37] Copyright 2023, Springer Science Business Media, LLC, part of Springer Nature.

Figure 7.

(A) Diagram depiction of supramolecular assembly 1/CB[8]/SC4AD. (B) Mouse tissue imaging of background fluorescence interference (left), 1/CB[8]/SC4AD (middle), and merged image (right), (λ_ex = 1050 nm λ_em = 450–530 nm and 650–750 nm (green and NIR channels, respectively). (C) A549 cell imaging, incubated with 1/CB[8]/SC4AD excited at 514 and 1050 nm. (D) In vivo living mouse imaging of 1/CB[8]/SC4AD ([1/CB[8]] = 0.1 mM, λ_ex = 465 nm, λ_em = 630–670 nm). Reproduced with permission.^[38] Copyright 2022, WILEY-VCH.

Figure 8.

(A) Diagrammatic representation of molecular packing modulation through regioisomerization. (B) Chemical structures of TBP-e-TPA and TBP-b-TPA. (C) Fluorescent images of Hela cells incubated with TBP-b-TPA NPs under one-photon (λ_ex = 560 nm, λ_em = 700–800 nm) and two-photon (λ_ex=1040 nm, λ_em = 700–800 nm) excitation. (D) In vivo 2PFM imaging of TBP-b-TPA NPs stained cortical vasculature with depth profiles and 3D reconstruction from the surface to 700 μm; λ_ex = 1040 nm (50 MHz), two-photon fluorescence collected with an 800 nm short-pass filter. Reproduced with permission.^[39] Copyright 2020, WILEY-VCH. (E) Chemical structure of DPBT. (F) Molecular π–π stacking structures of DPBT. (G and H) Fluorescent images of A549 cells stained with DPBT under (G) one-photon (λ_ex = 488 nm) and (H) two-photon (λ_ex = 900 nm) excitation. (I – L) Two-photon fluorescent images of liver and mesenteric adipose tissues stained with DPBT (20 μM). Z-projected image (211 μm depth) of the excised liver tissues from (I) apolipoprotein E-deficient (ApoE^−/−) mouse, (J) normal mouse, mesenteric adipose tissues from (K) apolipoprotein E-deficient (ApoE^−/−) mouse and (L) normal mouse. Two-photon excitation wavelength: 900 nm. Green channel: λ_em = 495−540 nm. Red channel: λ_em = 575−630 nm. Reproduced with permission.^[40] Copyright 2023, WILEY-VCH.

Figure 9.

(A) Structure of [{Ru(TAP)₂}₂(tpphz)]⁴⁺. (B, C) Fluorescence images of of the Ru complex in melanoma spheroids under one-photon excitation (B, 458 nm) and two-photon excitation (C, 900 nm). Depths measured: (i) 0 μm, (ii) 60 μm, (iii) 180 μm, and (iv) 240 μm (scale bar = 100 μm). (D) Fluorescence imaging in human melanoma cells following laser excitation over a wavelength range of 840–1000 nm. Reproduced with permission.^[29] Copyright 2020, American Chemical Society.

Figure 10.

(A) Structures of pyclen-based lanthanide complexes as highly luminescent bioprobes. (B) Two-photon imaging of T24-cells stained with [EuL], [SmL] (λ_ex = 750 nm), [YbL] (λ_ex = 800 nm), [TbL], and [DyL] (λ_ex = 720 nm). Reproduced with permission.^[43] Copyright 2020, American Chemical Society.

Figure 11.

(A) Self-assembly of Pt1 for ratiometric dual emission suitable for two-photon imaging. (B) Real-time tracking of CuSO₄-induced acute inflammation in zebrafish larvae. λ_ex = 730 nm, λ_em = 445 ± 10 nm and 573 ± 10 nm. Reproduced with permission.^[44] Copyright 2021, WILEY-VCH.

Figure 12.

(A) Synthetic scheme of TPI-COF. (B) The restricted conformation of TPI-COF. (C, D) Fluorescence imaging of TPI-COF against 4T1 cancer cells via two-photon (C) and one-photon (D) excitation. Scale bar, 50 μm. (E) The two-photon fluorescence intensity of TPI-COF at different tumor tissue depth. (F) The two-photon fluorescence imaging of TPI-COF at different tumor tissue depth. Reproduced with permission.^[2a] Copyright 2020, WILEY-VCH.

Figure 13.

(A) Structure of a TPA fluorescent COF nanoprobe TpASH-NPHS. (B) Action diagram of TpASH-NPHS. (C) Cirrhotic liver model induced by 40% CCl₄ subcutaneous injection and liver tissue slices harvested at various treated periods. (D) Two-photon confocal fluorescence images of TpASH-NPHS in cirrhotic mouse liver tissues across different CCl₄-treated periods. Reproduced with permission.^[45] Copyright 2018, The Royal Society of Chemistry.

Figure 14.

(A) COF-606 chemical structure. (B) Serrated structure of COF-606. (C and D) 3D and corresponding 2D confocal fluorescence images of COF-606 nanoparticles in the tumor under (C) one-photon (560 nm) and (D) two-photon (808 nm) excitation. (E) PD-1 immunofluorescence staining in tumor tissue after COF-606 under 808 nm laser treatment. (F) Bilateral tumor model and PDT/PD-1 treatment timeline. (G) Tumor growth curves for different treatment groups: PBS, PD-1 antibody, COF-606 + 808 nm laser, and COF-606 + 808 nm laser + PD-1 antibody. Reproduced with permission.^[46] Copyright 2021, WILEY-VCH.

Figure 15.

Structures of two-photon-absorbing Cd(II)-containing MOFs. Reproduced with permission.^[48] Copyright 2023, American Chemical Society.

Figure 16.

(A) The proposed mechanism of equilibrium for BHC and its protonated and deprotonated forms.^[52] (B) Neutral non fluorescent “off” state conversion to a protonated fluorescent “on” state, together with the fluorescence images of MEF cells at different pH values. Reproduced with permission.^[53] Copyright 2020, American Chemical Society.

Figure 17.

(A) Structures of Lyso-PCE before and after protonation. (B) Two-photon fluorescence images of Lyso-PCE in HeLa cells stimulated by chloroquine. Two-photon excitation wavelength is 820 nm. (C) Relative fluorescence intensities at different time points. (D) pH values at different time points under chloroquine stimulation in cells. Reproduced with permission.^[54] Copyright 2021, Elsevier Inc.

Figure 18.

(A) Structure of Cou-Br and the proposed response mechanims toward GSH.^[57] (B) Mechanism of CPs for the activity-based fluorescence detection and intracellular imaging of GSH.^[58]

Figure 19.

Schematic diagram for the cell membrane-bound GGT probe and its enzymatic hydrolysis process (Glu: glutamic acid).^[63]

Figure 20.

(A) Schematic illustration of the action mechanism of 2-O-IrAn by photodynamic therapy and photoactivated chemotherapy. (B) Z-stack confocal microscopy images of A549 MTCS upon incubation with 2-O-IrAn and exposure to one- (405 nm) or two-photon (750 nm) light. (C) Representative images of A549 MCTS upon incubation with 2-O-IrAn and the ROS probe dichlorodihydrofluorescein diacetate and exposure to two-photon (750 nm) light. Reproduced with permission.^[64] Copyright 2022, American Chemical Society.