Aggregation-Induced Fluorogens in Bio-Detection, Tumor Imaging, and Therapy: A Review

Abstract

Open AccessCCS ChemistryMINI REVIEW1 Feb 2022Aggregation-Induced Fluorogens in Bio-Detection, Tumor Imaging, and Therapy: A Review Yekkuni L. Balachandran and Xingyu Jiang Yekkuni L. Balachandran Shenzhen Key Laboratory of Smart Healthcare Engineering, Department of Biomedical Engineering, Southern University of Science and Technology, Nanshan District, Shenzhen, Guangdong 518055 Google Scholar More articles by this author and Xingyu Jiang *Corresponding author: E-mail Address: [email protected] Shenzhen Key Laboratory of Smart Healthcare Engineering, Department of Biomedical Engineering, Southern University of Science and Technology, Nanshan District, Shenzhen, Guangdong 518055 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202101307 SectionsAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Aggregation-induced emission (AIE) fluorogens produce strong, stable fluorescence upon aggregation, thus enabling a new frontier in bio-detection, cell imaging, and other biomedical applications. In this review, we summarize the recent developments of AIE fluorogen design strategy, bio-detection, cell imaging, tumor imaging, vascular imaging, and image-guided tumor therapy. We highlight a section on the microfluidic approach to synthesize AIE nanoparticles (NPs) and current challenges and potentials of AIE NPs. Download figure Download PowerPoint Introduction Noninvasive fluorescence imaging in biomedicine has undergone rapid growth to obtain dynamic information about living cells or organisms.1,2 Fluorescence imaging provides real-time visualization of live cells, increases image sensitivity, improves temporal–spatial resolution, and so forth.3,4 Several well-defined fluorophores of various origins such as organic molecules, quantum dots (QDs), up-conversion nanoparticles (NPs), and fluorescent proteins are synthesized to emit superior and stable fluorescence.5,6 Among them, organic fluorophores are the most studied because of their strong fluorescence, simple preparation method, and satisfactory biocompatibility.7 However, most organic fluorophores suffer from poor solubility in physiological solutions by forming small aggregates or clusters,8,9 which cause partial or complete aggregation-caused quenching (ACQ) due to the π–π stacking.10,11 The ACQ effects restrict fluorophore transformation into NPs because increasing the doping number can provoke fluorescence quenching. Moreover, the fluorophores for cell imaging should be cautiously used (optimum AIEgen concentration) to minimize the intracellular aggregation to prevent compromising fluorescence.12 Furthermore, using small quantities of organic fluorophores produces unsatisfactory fluorescence, poor photostability, and photobleaching.13 Aggregation-induced emission (AIE) fluorogens are a new class of fluorogens that produce strong and stable fluorescence in the aggregation state. AIE fluorophores are nonemissive as single molecules but produce strong fluorescence in an “abnormal” aggregate or cluster state.14,15 The rotor structure of the AIE fluorogens (AIEgens) activates the nonradiative decay pathway to restrict the intermolecular motion to yield bright and stable emissions in the aggregated state.16 Furthermore, the nonplanar conformation of AIEgens prevents π–π stacking, causing strong emission in the aggregate or solid state.17 The AIEgens exhibits a high signal-to-noise ratio (10–40 times brighter than the organic fluorescent molecules), high sensitivity, increased quantum efficiency, anti-photobleaching capability, good photophysical stability, and excellent cytocompatibility.14,15 Tuning the emission characteristics is crucial for fluorescence-based applications. AIEgens produce emission in the visible spectral range,18 and further deep emission in the near-infrared (NIR) window I (700–1000 nm)19,20 and NIR-II window (1000–1500 nm)21,22 to provide high-quality images, deep penetration, and limitation of the photon absorption and scattering. Recently, AIEgens capable of two-photon fluorescence have been explored considerably due to their advantages of longer excitation wavelength, minimal autofluorescence, poor photobleaching, and higher three-dimensional (3D) resolution with deep tissue imaging.23,24 The AIEgens are used in diverse biological applications to detect various biomolecules (amino acids, carbohydrates, DNA or RNA, and proteins or enzymes),25,26 monitor biological processes (protein fibrillation, cell apoptosis, osteogenic differentiation, autophagy or mitophagy, and bacterial labeling and ablation),27 and label cellular organelle.14,15 Due to their exceptional optical properties, AIEgens serve as potential materials of choice for tumor imaging applications.28 Furthermore, the photosensitization and photothermal conversion efficiency of AIEgens broadens the application in tumor diagnosis and image-guided therapy29 such as photodynamic therapy (PDT), photothermal therapy (PTT), and photoacoustic (PA) imaging.30 Herein, we summarize the AIEgens and advances in their biological applications. First, we discuss the design and synthesis of AIE probes and then focus on the advances in applications, such as detection, imaging, and image-guided therapeutics. Next, we focus on the microfluidics approach to prepare AIEgens. We briefly discuss AIEgen fabrication by microfluidics technology and different microfluidic chips (straight and spiral) to prepare various AIEgen compounds. The microfluidic technique manipulates the reaction in a micrometer scale to continuously produce uniform sized nanomaterials in a straightforward approach without laborious postsynthetic processing steps.31–33 The microfluidic technique enables tasks such as generating size-controlled nanomaterials,31 fabricating core–shell materials,34 encapsulating drug molecules,35 and functionalizing the nanomaterial with ligand molecules.35,36 Furthermore, the microfluidic technique promotes continuous large-scale production of AIE nanomaterials for clinical translation and applications. Finally, we discuss the prospective possibilities of AIE platforms for clinical translation and different biomedical applications. AIEgens Phenomenon and Design Strategies Of the various theories proposed for AIE,37,38 the restriction of intramolecular motion (RIM) is a widely accepted phenomenon.14,37,39,40 The RIM mechanism combines restriction of intramolecular rotation (RIR)40 and restriction of intramolecular vibration (RIV) theories (Scheme 1).41 The AIE single molecules are unrestricted and rotate (Scheme 1a) and vibrate freely (Scheme 1b) to activate the nonradiative decay, thereby becoming nonemissive.38 However, when aggregated, the intramolecular rotation and vibrations are restricted, blocking the nonradiative decay channels, thus enabling the radiative decay processes to induce the emission.38 Scheme 1 | RIM phenomenon of AIE emission. (a) The RIR theory proposes, when the AIEgen is in a single molecular state, the intermolecular rotation (e.g., TPE) activates the nonradiative decay pathway resulting in no fluorescence. However, when aggregated, the restricted intramolecular rotation activates radiative decay in excited states to initiate the fluorescence. (b) The RIV theory proposes the free intramolecular vibration of the AIEgens [e.g., 10,10′,11,11′-tetrahydro-5,5′-bidibenzo[a,d][7]-annulenylidene (THBA)] enables the nonradiative decay of the excited state, resulting in no fluorescence, and upon aggregation, the vibration is completely halted, which initiates fluorescence via the radiative decay process. Comprehensively, the AIE phenomena RIR and RIV merge into the RIM theory (RIM = RIR + RIV). Reproduced with permission from ref 38. Copyright 2018 ACS. Download figure Download PowerPoint The excitation and emission of AIEgens are influenced by the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy gaps of the ground and excited state.42,43 Reducing the HOMO–LUMO gap by elevating the HOMO and lowering the LUMO energy level by expanding the π-conjugation length can bring about the emission.42,43 Constructing the low bandgap AIEgens by conjugating the electron donor (D) (to elevate HOMO level) and acceptor (A) (to lower the LUMO level) produces AIE with negligible ACQ.42,43 By enhancing the D–A conjugation, several AIEgens are constructed. Sometimes the electron donation and acceptance characteristics are improved by inserting spacer or bridging molecules or modifying the D or A molecule.38,39 Scheme 2 shows examples of D–A strengthened AIE probes. Acceptor core units such as benzothiazole ( 1),44 cyano ( 2),45 and 9,10-anthraquinone ( 3)46 are conjugated with different electron donor units to lower the bandgap to realize the AIE. Scheme 2 | AIEgen molecular probes. (1) Benzothiazole, (2) cyano, and (3) 9,10-anthraquinone acceptor centric AIEgens are conjugated with AIE moieties to obtain AIE emission. The TPE central fluorogens such as (4) anthraquinone, (5) benzoselenodiazole, and (6) hemicyanine dye molecule impart the needed stable emission. ACQ converted AIEgens formed by conjugating TPE with IR-780: (7) and (8). Download figure Download PowerPoint In contrast, the electron donor core probe units are conjugated with different acceptor molecules to afford a series of new AIEgens, whose spectra range from visible to infrared.18 Especially, the tetraphenylethene (TPE) AIEgens exhibit excellent AIE characteristics.18 Introducing the anthraquinone acceptor into the TPE produced the emission in the infrared region ( 4).19 The conjugation of TPE with the benzoselenodiazole dye induces the AIE emission by taking advantage of the polarization effect of selenium and a strong D–π–A electronic property ( 5).20 The HOMO and LUMO electron density of the TPE-hemicyanine molecules have charge-transfer characteristics with a low bandgap to shift the optical emission ( 6).18 Occasionally, changing the ACQ to AIE has been realized for stable emission.47 The red emissive fluorophores are converted from ACQ to AIE by conjugating with AIE molecules to induce AIE emission. Introducing the TPE with the IR-780 core dye exhibits a typical D–A molecular structure favoring “off–on” fluorescence ( 7 and 8).47 The design strategies are further improved to fine-tune the emission to an NIR region43,48 and enable two-photon imaging.23,49 Because of low photon scattering and phototoxicity, negligible autofluorescence,22,23,50 and deep tissue penetration, the NIR and two-photon capable AIEgens effectively improve biomedical applications.47,48,50,51 The NIR capable AIE molecules are constructed by improving the D–A–D effect by installing substituents with strong electron donors and acceptors to elevate the HOMO level and lower the LUMO level.43 Furthermore, the quantum efficiency of the molecules are boosted by careful design strategies. As the emission wavelength shifts to the red region, the photoluminescence quantum yield (PLQY) decreases due to vibrational overlap of the ground and excited states. Suppressing the twisted intramolecular charge transfer (TICT) and simultaneously increasing the radiative decay and stimulating the AIE feature by preventing close intermolecular interactions cause higher fluorescence quantum efficiency.43 Two-photon capable materials are characterized by the fluorogens possessing a large two-photon absorption cross-section (σ2PA).52–54 The intramolecular charge transfer (ICT) between the terminal donor group and the π-bridge molecule correlates with the cross-section (δ) of the fluorogen, and the magnitude of δ is influenced by the ICT of the molecular structure.52,54 Modification of the molecular structure through extending the conjugation length of the π-system increases the δ of the fluorogens to enable two-photon characteristics.52,55 Therefore, the improvement of σ2PA relies on the strong electron donor and acceptor groups as well as the length of the π-bridge. The NIR building block, benzo[1,2-c:4,5-c′]bis([1,2,5] thiadiazole) (BBTD)56,57 acceptor molecule is widely employed to design NIR AIEgens. By either modifying the acceptor or donor unit, the emission wavelength can be tuned to the NIR range. For example, replacing the sulfur group in the BBTD acceptor unit with selenium shifts the emission maxima from 810 ( 9) to 897 nm ( 11).51 On the other side, strengthening the electron-donor molecules (triphenylamine, thiophene, and alkoxy chains) lowers the bandgap to achieve NIR emission.58 Introducing a strong electron-donating moiety, for example, N,N-diphenyl-4-(1,2,2-triphenylvinyl)aniline and AIE unit (TPE) with the central BBTD core, yields emission ca. 975 nm ( 11).58 Furthermore, inserting a spacer between the central BBTD and modifying the TPE with diethylamine units contributes to electron-donating capability to further shift the emission to 1034 nm ( 12).59 Scheme 3 shows the examples of NIR AIEgens ( 13 and 14)21,60 developed by substituting the donor and acceptor molecules. Similarly, by rigidifying the D–A geometry, various two-photon capable AIEgens were developed with strong and stable fluorescence ( 15 and 16).24,61 Scheme 3 | Structure of NIR and two-photon capable AIEgens. Replacing the sulfur of electron acceptor BBTD (9) with selenium (10) shifts emission to the NIR-II region. Substitution on the electron donor (AIE molecule) (11 and 12) shifts the absorption-maximum to the NIR-II region. When the electron acceptor and donor are modified, the emission-maximum shifts toward the NIR-II region (13 and 14) and enables two-photon capabilities (15 and 16). Download figure Download PowerPoint Applications of AIEgens The distinctive luminescent features of AIE bio-probes have become essential in biomedicine applications.15,16,27,43 The characteristics of AIEgens have been improved for broader biomedical applications.16,43 For example, understanding the mechanism and optimizing the design strategy improves the fluorescence (bio-imaging), photosensitization, and photothermal characteristics of AIEgens.15,16 Furthermore, introducing responsiveness to external stimuli broadens the scope and specificity of AIEgens in biomedical applications.62,63 In particular, an AIEgen response to the external pH is beneficial for tumor applications because of the difference in the tumor microenvironment pH (pH 6.5–5.5).64 The pH-responsive AIEgen platform (such as drug molecules grafted onto pH-responsive polymers with AIE characteristics65,66) induces fluorescence imaging capabilities and delivers therapeutic molecules to eliminate the tumor upon a trigger by the acidic tumor environment.67,68 Such pH-responsive AIE theranostic systems specifically tag the tumor and deliver drug molecules, thereby avoiding damage to the surrounding normal cells.69 Because of the advancements in bioimaging and image-guided theranostic applications, the characteristics of AIEgens are integrated for multifunction biomedical applications.16,43 In the following section, we summarize the major properties of AIEgens in bio-detection, imaging (cellular, vascular, and tumoral) and image-guided therapy. AIEgens Application in Bio-Detection Fluorescent AIE molecular probes have become important as detection sensors due to their noninvasive and real-time visualization features. AIEgens are used to monitor the biomolecules of interest in solutions or more complex biological cell and tissue environments.70,71 Our group has developed various AIE probes for the rapid and efficient identification of biomolecules [e.g., casein and hydrogen sulfide (H2S)]25,26 and bacteria.27 We have developed the TPE-based AIE probe, sodium 1,2-bis[4-(3-sulfonatopropoxyl)phenyl]-1,2-diphenylethene (BSPOTPE), for detecting casein in milk to evaluate the quality of milk powder (Figure 1a).25 The hydrophobic caves in the casein micelle captures BSPOTPE molecules and the interaction induces BSPOTPE aggregation, thereby enabling fluorescence enhancement by AIE (Figures 1a and 1b). Figure 1 | AIEgen enabled bio-detection. (a) Casein detection using AIE BSPOTPE probe. (b) Confocal image of casein micelles and casein micelle–BSPOTPE complex inducing fluorescence. (c) TPE-indo AIE probe detection of H2S. The nucleophilic addition between TPE-indo and H2S weakens the aggregation and fluorescence. (d) In vivo detection of H2S in HeLa, HUVEC, and MCF-7 cells using TPE-indo AIEgens. (d1) Cells incubated with TPE-indo for 30 min, (d2) cells incubated with NaHS, (d3) cells incubated with NaHS for 60 min, and (d4) bright-field images of the same field of cells. Reproduced with permission from ref 25. Copyright Royal Society of Chemistry 2012; Reproduced with permission from ref 26. Copyright Springer Nature 2015. Download figure Download PowerPoint Our group developed TPE-indolium (TPE-indo) AIEgens to detect H2S in live cells (Figures 1c and 1d).26 H2S is recognized as the third endogenous gasotransmitter, and abnormal levels of H2S are linked to various physiologic effects in health.72,73 H2S has been reported to regulate blood pressure, inflammation, neurodegenerative diseases, and metabolic disorders, including obesity and diabetes.26,72,73 The H2S in the solution and mitochondria of the cells is monitored using TPE-indo AIEgens comprised of the florescent TPE moiety (fluorogen unit) and a H2S reactive positive-charged indolium moiety.26 The TPE-indo interacts with H2S via a specific nucleophilic reaction with CN− on the indolium group. The aggregated TPE-indo probe changes drastically upon interaction with HS−, thereby weakening aggregation and influencing the fluorescence (Figure 1c). With the fluorescence quenching of TPE-indo NPs, the effective visual detection of H2S in the solution and cells is achieved (Figure 1d). Rapid identification of bacteria has become increasingly important in clinical diagnosis and the food industry to monitor the outbreaks of pathogens, trends in infection, and contamination in food products.74,75 Especially in clinical diagnosis, rapid and sensitive detection of bacteria provide evidence of disease origin and guidance for antibiotic treatment. Furthermore, differentiating the clinical drug resistant bacteria gives an advantage for treating the pathogen with a suitable antibiotic. Our group has developed a rapid AIE-based assay to detect and differentiate multidrug-resistant bacteria from normal bacteria (Figure 2).27 Derivatives of TPE AIEgens with different functional groups (Figure 2a) selectively identify eight different kinds of bacteria, including multidrug-resistant bacteria such as methicillin-resistant Staphyloccocus aureus (MRSA), Staphylococcus epidermidis, and Bacillus subtilis; and multidrug-resistant (MDR) Escherichia coli, Escherichia coli, Klebsiella Pneumonia, and Salmonella choleraesuis. The mixing of bacteria and AIE probes differentiates the bacteria simultaneously by the fluorescent intensity data in automated flow cytometry without any washing step. The TPE AIEgens interact with bacterial protoplasm to form aggregates that produce fluorescence, and such fluorescence was unique for each bacteria (Figure 2b). Figure 2 | Detection and differentiation of bacteria. (a) AIEgen probes used for differentiating various bacteria. (b) The design of a sensor array based on AIE probes to differentiate bacteria, in which A1–A5 denote five different AIE probes and B1–B8 represent eight different types of bacteria. Reproduced with permission from ref 27. Copyright 2014 Wiley-VCH GmbH. Download figure Download PowerPoint Tumor Cell and Mitochondria Imaging Real-time imaging of cellular activity is crucial for precisely assessing cell health, which forms the basic structural and functional unit of any organism.37 The function of cellular organelles is important for the diverse biochemical processes and physiological pathways, and studying the abnormities of cellular organelles is vital because the organelles serve as an important biomarker for cell and tissue status and many diseases.76 NIR AIE probes are designed to target specifically the cellular membrane, mitochondria, lysozyme, and nucleus to induce fluorescence.77 Furthermore, the wash-free capability of AIEgens allows cell visualization for a longer duration with extended photostability and antibleaching.78 Of all cellular organelles, the mitochondria has been extensively studied because abnormalities in mitochondria are closely related to the cell and tissue functions and homeostasis.79,80 Dysfunction in mitochondrial morphology is detected in various human pathologies, such as cancer, neurodegeneration, ageing, diabetes, obesity, and cardiomyopathies.79,80 In tumorigenesis, the mitochondria are involved in alteration of fuel utilization, cell death susceptibility, oxidative stress, and allows survival during starvation and resistance toward treatments during chemotherapeutic or targeted cancer treatments.81,82 Furthermore, dysfunction in the mitochondria-regulated apoptosis pathway accelerates tumorigenesis.82 Understanding the mitochondria effectively favors in designing of novel drug molecules to improve cancer therapeutics.83 Mitochondrial imaging is primarily performed using commercial, small organic fluorescent molecules, and such organic fluorophores have limitations including poor cellular uptake, the ACQ effect, and low photostability. The use of AIEgens overcomes these limitations to provide excellent fluorescence and better photostability. Our group has designed a series of one or two-photon multicolor fluorescent AIEgens to target and image mitochondria.61 TPE-based nitrogen (N)-containing heterocycles are designed and synthesized by coupling pyridine (Py), quinoline (Quino), or acridine (Acr) (Figure 3a). The emission shifts toward the longer region in accordance with the number of the phenyl groups in the heterocyclic conjugates (Figure 3b). Because of cationic moieties, TPE AIEgens can aggregate into nanosized dots of 20–40 nm without any need for polymer or cell-penetrating peptide encapsulation (Figure 3c). The TPE-dots possess both one- and two-photon fluorescent capabilities. The two-photon fluorescence upon NIR excitation enables low photobleaching, low background signal, and low phototoxicity and emits light only when it is in the focus position in bioimaging. Furthermore, TPE-dots are wash-free probes78 favoring long-duration imaging of live cells and making the staining process more convenient and economical. The TPE-dots produced strong fluorescence from the mitochondrial regions of HeLa cells (Figure 3d). The MitoTracker Red verified the fluorescence signal of mitochondria, and co-staining experiments showed perfect overlap between TPE-dot and MitoTracker Red FM (Figure 3d). Furthermore, the TPE-dots spontaneously produced both one- and two-photon fluorescence (Figure 3e). The TPE-dots targeting mitochondria are not limited to HeLa cells; the mitochondria in cell types such as HUVEC (HUVEC = human umbilical vein endothelial cells) and NIH 3T3 (NIH 3T3 = National Institutes of Health Swiss mouse embryo fibroblasts/3-day transfer, inoculum 3 × 105 cells) are also efficiently targeted by TPE-dots, indicating the capability of imaging mitochondria in vast cell types. Figure 3 | TPE-dots targeting mitochondria. (a) Chemical structure of TPE-Py, TPE-Quino and TPE-Acr. (b1) Absorbance and (b2) emission spectra of TPE molecules. (c) TEM image of TPE-Acr, TPE-Py, and TPE-Quino dots. (d) One-photon image of HeLa cells after incubation with TPE-dots or MitoTracker red. TPE-dots and MitoTracker red merged images. (e) One- and two-photon fluorescence images of HeLa cells tracking mitochondria. Reproduced with permission from ref 61. Copyright Royal Society of Chemistry 2017. Download figure Download PowerPoint In Vivo Tumor and Tumor Vascular Imaging of AIEgens Effective real-time monitoring of in vivo tumor is necessary to evaluate and intervene with a therapeutic plan.84 Fluorescence imaging, being an indispensable and versatile tool, allows highly sensitive, non-invasive imaging in real-time and for long-term diagnosis.24,57,85 Short-wavelength light is easily absorbed by water in biological tissues, affording undesirable shallow depth bioimaging, and influenced by Rayleigh scattering effect. In contrast, imaging in the NIR range produces low scattering, deeper penetration depth, high fluorescence quantum yield, and reduced phototoxicity.19,21,22,50 AIEgens with NIR emission distinctly portray the tumor mass over the surrounding normal tissues with high sensitivity, deep tissue penetration, and enhanced contrast resolution. Despite the efforts made, off-targeting is of great concern preventing the needed accumulation of AIE NPs for the contrast-enhanced image of the tumor mass. We have recently developed an AIE QD with a NIR-II system that targets tumor in higher numbers evading the liver.31 The NIR-II AIEgen 4,7-(bis4-(4-octylthiophen-2-yl)-N,N-diphenylaniline)benzo [1,2-c:4,5-c′]bis ([1,2,5]thiadiazole) (TTB) (Figure 4a) is made into quantum-sized dots using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 (DSPE-PEG2k) as a surfactant. The transmission electron microscopy (TEM) determined core size of TTB QDs measured <10 nm (Figure 4b). Due to the quantum size, the TTB QDs can internalize more efficiently in cells without any modification with membrane penetrating peptides. The TTB-exposed in vivo tumor produced a much brighter NIR-II fluorescence (Figure 4c). Polyethylene glycol (PEG) coating of TTB QD extends the circulation time in blood86 and, hence, the possibility of higher tumor targeting for bright NIR-II fluorescence. To calculate the off-targeting efficacy, two different sizes of TTB NPs were compared. The TTB QD evaded liver accumulation more than the larger sized 25 nm TTB dots (>25 nm) (Figure 4c).31,87 The tumor and liver mean NIR-II fluorescence intensity ratio from TTB QDs-treated mice was two to three times that of TTB dots (Figure 4d). Furthermore, conjugating with trans-activator of transcription (TAT) membrane penetrating peptide (RKKRRQRRRC) failed to improve tumor accumulation (Figures 4e and 4f). Together, TTB QD improved tumor accumulation to produce brighter NIR-II fluorescence and reduced the off-target accumulation in the liver more than the bigger sized dots and TAT surface-modified TTB QD. Figure 4 | TTB-QDs NIR-II fluorescence imaging of tumor. (a) TTB NIR-II AIE molecule and DSPE-PEG capped TTB QDs. (b) TEM image of TTB QDs. (c) Left side and ventral views of NIR-II tumor (green circle) and liver (yellow ellipse) fluorescent image acquired at different time intervals after exposure to TTB QDs and TTB dots. (d) The tumor (T) to liver (L) mean ratio of fluorescence intensity. (e) The difference in NIR-II fluorescence of TAT capped and uncapped TTB QDs or TTB dots exposed tumor. The yellow line outlines the mice body, and the green circle highlights tumor. (f) NIR-II fluorescence intensity of the tumor treated with TTB QDs or TTB dots with and without conjugation of TAT peptide. Reproduced with permission from ref 31. Copyright 2020 Wiley-VCH GmbH. Download figure Download PowerPoint Vascular imaging in tumors has gained as much consideration as solid tumor imaging.88 In general, the vascular networks play an important role in regulating the delivery of nutrients and oxygen; meanwhile, the tumor vascular network allows the cancer cells to disseminate and influences the response of the tumor cells to therapy.89 Tumor vascular networks are typically sinusoidal, exhibit discontinuous basement membranes, and lack tight endothelial cell junctions making them highly permeable.89 Imaging the tumor vasculature heterogeneity is indispensable to understand the tumor angiogenesis mechanism that is strongly affected by the microenvironment of cancer tissues.89 The NIR AIEgens, due to their deep tissue penetration, provide a superior image of the blood vessels and branching structures than the other imaging modes.90 Our group designed lipid AIE NPs to image tumor vasculature at deeper regions.24 The two-phot