AIE Nanoprobes for Multi-Photon in Vivo Bioimaging - American

Chapter 10

AIE Nanoprobes for Multi-Photon in Vivo Bioimaging Downloaded by CORNELL UNIV on October 27, 2016 | http://pubs.acs.org Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ch010

Yalun Wang, Hequn Zhang, Nuernisha Alifu, and Jun Qian* State Key Laboratory of Modern Optical Instrumentation, Centre for Optical and Electromagnetic Research, Zhejiang University, 310058 Hangzhou, China *E-mail: [email protected]

Deep-tissue bioimaging is highly important in medical research and clinical applications. Multi-photon luminescence (MPL) imaging with excitation wavelength in the near-infrared (NIR) range is found to be an effective way to obtain large imaging depth of tissues. AIE nanoparticles with high fluorescence brightness, good biocompatibility and photobleaching resistance are ideal nanoprobes for MPL imaging. In this chapter, we introduced the concept of tissue penetration, optical tissue windows, and MPL imaging, and summarized some of the progresses in multi-photon in vivo bioimaging based on AIE nanoprobes.

1. Background 1.1. Optical Tissue Windows Bioimaging is of great significance to medical research and clinical practices (1). Since the beginning of the 20th century, various imaging techniques have been developed, and X-rays, ultrasonic imaging, magnetic resonance imaging (MRI), positron emission tomography (PET) are some of the most promising ones (2). However, there’s exposure to radiation in X-rays and PET (3, 4), and the details in ultrasonic imaging and MRI are not well recognized (5, 6). Optical imaging, with high spatial resolution, abundant spectral information and radiation free feature, is very ideal for biological applications (7). However, the depth of optical bioimaging is usually a problem, due to the limited penetration depth of visible light in biological tissues (8). © 2016 American Chemical Society Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Downloaded by CORNELL UNIV on October 27, 2016 | http://pubs.acs.org Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ch010

The attenuation of light in bioimaging is mainly from the optical scattering and absorption in tissue. Photon scattering can be scaled as λ-α, where λ is the wavelength of light and α = 0.2-4 for different types of tissues (9). Thus light with longer wavelengths would suffer less loss from scattering, and it is better for deep-tissue imaging. For light absorption in tissue, as water is the main component for nearly all the tissues, its light absorption is in dominant (10). The optimal wavelength for deep-tissue imaging is the trade-off between the tissue scattering and absorption, and these wavelengths can be classified into some optical tissue windows. Spectral rang in 750-900 nm, which is named as the first near-infrared (NIR-I) window, is very useful for deep-tissue imaging (11). The tissue scattering in this range is smaller than those in the commonly used ultraviolet (UV) and visible ranges, and the water absorption in this range is also very small. Thus, large light penetration depth can be realized in this window, and many relevant work has been performed (12–15).

Figure 1. The calculated attenuation length in mouse cortex. Dashed line, absorption length of water. Dashed-dotted line, scattering length of mouse brain cortex. Solid line, combined attenuation length. Reproduced with permission from reference (10). Copyright (2013) Nature Publishing Group. Spectral range in 1000-1700 nm, which is defined as the second near-infrared (NIR-II) window, is even better for deep-tissue imaging (16). According to the difference of water absorption, NIR-II window can be devided into NIR-IIa (1000-1400 nm) and NIR-IIb (1450-1700 nm) windows (17). In NIR-IIa window, the tissue scattering is smaller than that in visible and NIR-I ranges, and the water absorption is medium, slightly larger than those in visible and NIR-I ranges. In NIR-IIb window, the tissue scattering is the smallest. Although the water absorption is strong, the greatly reduced light scattering could compensate it. Thus, light in NIR-II window could have better light penetration capability than light in visible and NIR-I ranges. Horton et al. had calculated the attenuation of light in mouse cortex from 700-2000 nm, and found that the light near 1700 nm had the largest penetration depth, as shown in Figure1 (10). Bioimaging based on NIR-II window is very promising, and some pioneer works have been devoted on it (17–19). 246 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


1.2. Multi-Photon Imaging In a bioimaging process, light usually travels twice in the tissue. First, the excitation light penetrates into the tissue to excite the fluorophores. Second, the emitted signals come out from the tissue, and are collected by the detector. In a multi-photon luminescence (MPL) imaging process, the excitation wavelength usually locates in the aforementioned optical tissue windows, and the signal is usually collected in the visible wavelength range. Compared with NIR bioimaging, which directly collects singals in NIR range, MPL imaging has some advantages. First, much more fluorophores with visible emission can be chosen than those with NIR emission (20, 21). Second, optical detectors usually have better response in the visible range than in NIR range. Third, a better imaging resolution can be obtained from visible emitted signals, according to the Abbe’s formula ε = 0.61λ/NA (where ε is the resolution limit, λ is the wavelength, and NA is the numerical aperture of the objective lens). Besides, MPL is a typical nonlinear optical effect, and the out-of-focus signals would be greatly reduced in MPL imaging process. Two-photon luminescence (2PL) is a process in which a fluorophore molecule absorbs two photons with the same longer wavelength simultaneously, and is stimulated to the excited state. After fast relaxation, the fluorophore molecule returns to its ground state and emits a photon with shorter wavelength, as shown in Figure 2(a). The probability of two-photon absorption occurrence is characterized by two-photon absorption (2PA) cross-section (22). For convience, its unit is usually in GM, where 1 GM = 10-50cm4s. The product of 2PA cross-section and fluorescence quantum yield is defined two-photon action cross-section, which describes the two-photon brightness of a fluorophore (23).

Figure 2. A schematic illustration of multi-photon luminescence process. (a) Two-photon luminescence, (b) three-photon luminescence. In three-photon luminescence (3PL), three photons with the same longer wavelength are absorbed simultaneously by a fluorophore, and a photon with shorter wavelength is emitted, as shown in Figure 2(b). The probabitility of three-photon absorption occurrence is characterized by three-photon absorption (3PA) cross-section, and it’s usually in unit of cm6GW2 (22). As the occurrence probability of higher-order nonlinear optical process is much smaller, 2PL and 3PL are usually more feasible in bioimaging. 247 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Compared with conventional one-photon luminescence (1PL) imaging with short wavelength excitation, MPL imaging with excitation in NIR spectral window has many advantages (24–26). (1) Longer-wavelength excitation light has less tissue scattering, and much larger penetration depth could be obtained. (2) The photodamage of tissues under longer-wavelength excitation in MPL imaging, would be greatly reduced, compared with that under UV/blue wavelength exictation in 1PL imaging. (3) It’s much easier to separate the excitation light from the shor-wavelength signals in MPL imaging, and the signal-to-noise ratio would be improved accordingly. (4) The fluorescence intensity has a square or higher-order power dependence on the intensity of exicitation light, and the focusing point would be rather small. Thus, the out-of-focus fluorescence could be greatly reduced, which is very helpful to improve the imaging quality. (5) With the rather small focusing point, MPL imaging has inherent ability of sectioning, and the complicated pin-hole structure in 1PL confocal microscope could be eliminated. As the nonlinear optical coefficient of most materials is low, high instaneous power density is needed to excite enough MPL signals. To avoid photodamage to tissues, pulsed laser sources are commonly utilized in MPL imaging. In addition, due to the limitation of high excitation power density, MPL imaging is usually applicable in microscopic imaging with focusing illumination, and not so proper for macro imaging requiring wide field illumination. MPL signals are usually colleted by highly sensitive photo-multiplier tubes (PMTs) via non-descanned detection (NDD) mode. Since the first development of 2PL scanning microscope in 1990s, MPL imaging has been widely applied in cancer cell detection, neuron cell imaging, vascular imaging and so on (27–32). 1.3. AIE Nanoprobes for in Vivo MPL Imaging As the endogenous fluorescence of bio-tissues is usually very weak, exogenous fluorophore is needed to acquire good contrast in bioimaging. To date, various types of nanoprobes have been developed for 2PL imaging, including small organic dyes (33), fluorescent proteins (34), inorganic semiconductor quantum dots (35), and metal nanoparticles (36). However, the fluorescence stability of fluorescent proteins is limited (37), and there is potential toxicity in quantum dots (38). Metal nanoparticles are susceptible to photo-thermal damage (39), and common organic dyes suffer from aggregation-caused quenching (ACQ) when their concentration is high. In 2001, a new type of organic dyes with aggregation-induced emission (AIE) property was developed, and it is very promising for MPL in vivo imaging (40). First, the multi-photon absorption cross-section of AIE nanoprobes can be designed very high, which is very beneficial to MPL imaging. Second, the biocompatibility of these organic dyes is very good. Third, the fluorescence stability is greatly improved as there are many molecules inside each AIE nanoprobe. AIEgens are usually encapsulated into nanoparticles for bioimaging. One commonly used routine is the modified nanoprecipitation method, in which AIEgens are incorporated into the polyethylene glycol (PEG) matrix 248 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

(41). A micelle and silica coprotection strategy was proposed to improve the quantum yield, by providing a more hydrophobic environment for AIEgens (42). AIEgens in dimethyl sulfoxide (DMSO) can also be directly injected to form nanoaggregates spontaneously for in vivo imaging (43). In this chapter, we will summarize some progresses in MPL in vivo imaging based on AIE nanoprobes.


2. AIE Nanoprobes for in Vivo 2PL Imaging with Excitation Wavelength in NIR-I Window Compared to 1PL imaging with excitation in the ultraviolet (UV) and visible wavelength ranges, 2PL imaging with its excitation wavelength in NIR-I window (750-900 nm) has many advantages. First, it has very small water absorption, and the tissue scattering was not so distinct. Thus the penetration depth of NIR-I light in biological tissues could be greatly improved. In addition, the photodamage towards tissues would be greatly reduced, as NIR-I photons have less energy than the UV and blue photons. Furthmore, as the focusing point of excitation light is very small in 2PL imaging, the out-of-focus fluorescence can be greatly reduced, and the signal-to-noise ratio can be obviously improved. 2PL imaging with excitation wavelength in NIR-I window has been widely applied in cell, tissue, and in vivo imaging (44, 45). A mode-locked Ti:Sapphire femtosecond (fs) laser is widely used in 2PL imaging (46). It has an average output power of as high as several watts and a pulse width of ~100 fs. In addition, its output wavelength can be tuned from 700 to 1040 nm, which perfectly covered the NIR-I window (47). When fs excitation with its wavelength in NIR-I window is adopted, 2PL is the main nonlinear optical process, which has been widely applied in bioimaging. 2PL in vivo imaging based on NIR-I window and AIE nanoprobes is very proming due to the good penetration capability of the excitation light, as well as the excellent optical property of AIE nanoparticles, and there have been many relevant work on this field (43, 46, 48).

Figure 3. (a) Chemical structure and molecular geometry of BTPEBT. (b) Fluorescence spectra of BTPEBT in THF/water mixtures with water fractions from 50 to 90 vol%, excitation wavelength λex = 418 nm. Reproduced with permission from reference (46). Copyright (2013) John Wiley and Sons. 249 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


In 2013, Ding et al. contributed the first report on real-time two-photon intravital vasculature imaging with AIE nanodots (46). A type of AIEgen BTPEBT was synthesized with donor-(π-conjugated-bridge)-acceptor (D-π-A) structure, which was assumed to be benefit for large two-photon cross-section, as shown in Figure 3(a). The AIE characteristics of BTPEBT was investigated by dissolving it in different tetrahydrofuran (THF)/water mixtures. With increasing volume fraction of water from 50% to 90%, the aggregation of BTPEBT increased and the fluorescence upon excitation of 418 nm was intensified according, illustrating its AIE property, as shown in Figure 3(b). The hydrophobic BTPEBT molecules were incorporated into hydrophilic nanoparticles together with 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2000) by a modified nanoprecipitaion method, as indicated in Figure 4(a). These nanodots were found to have spherical morphology with a mean size of 29 nm on transmission electron microscopy (TEM), as shown in Figure 4(b).

Figure 4. (a) The schematic illustration of BTPEBT nanodots fabrication. (b) TEM image of BTPEBT nanodots. Reproduced with permission from reference (46). Copyright (2013) John Wiley and Sons.

Figure 5. (a) The normalized absorption and fluorescence spectra of BTPEBT nanodots, λex = 425 nm. (b) The measured 2PA cross-section of BTPEBT nanodots, QD655, and Evans Blue, λex = 800-960 nm. Reproduced with permission from reference (46). Copyright (2013) John Wiley and Sons. 250 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


These BTPEBT nanodots beared the absorption peak at 425 nm and emission peak at 547 nm, with a large Stokes shift of 122 nm, as shown in Figure 5(a). The 2PA cross-section of BTPEBT nanodots was measured via a two-photon induced fluorescence method, and it was found to be 10.2×104 GM at 810 nm, even larger than those bright quantum dots (QDs), as shown in Figure 5(b). With a high quantum yield of 62±1%, the two-photon action cross-section of BTPEBT was very high, and it was very helpful to 2PL imaging. The BTPEBT nanodots were then utilized for mouse brain imaging under an excitation of 800 nm from a Ti:Sapphire fs laser. As shown in Figure 6, the blood vessels could be visualized as deep as 400 μm, which was beyond the pia matter. From the 3D reconstruction, the mouse brain vascular system could be recognized.

Figure 6. The 2PL mouse brain imaging. (A-C) At different times. (D-I) At different depths. (J) 3D reconstruction. λex = 800 nm. Reproduced with permission from reference (46). Copyright (2013) John Wiley and Sons.

Parallelly, Wang and Qian et al. (our group) realized in vivo two-photon functional bioimaging with AIE nanodots (48). A type of AIEgen TPETPAFN (TTF) with D-π-A-π-D structure was synthesized, as shown in Figure 7(a). The TTF molecules were encapsulated by the molecules of 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-methoxy-(polyethylene glycol)-5000 (DSPE-mPEG5000) to form nanoparticles, as indicated in Figure 7(b). By varying the amount of TTF added, the diameter of TTF nanodots could be tuned. As shown in Figure 8, with 30 wt% of TTF in reactants, the mean size of TTF nanodots was 30 nm, while for TTF nanodots with 50 wt% of TTF in reactants, their mean size was 100 nm. The percentage of TTF in TTF nanodots could be defined as loading ratio. For different loading ratios, the aggregation states of TTF in nanodots would be different. The AIE property of TTF was studied by measuring the fluorescence of TTF nanodots with different loading ratios. As shown in Figure 9, when increasing the loading ratio of TTF nanodots, the fluorescence centered at 624 nm was intensified, indicating the AIE characteristics of TTF. 251 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 7. (a) The chemical structure of TTF. (b) A schematic illustration for the preparation of TTF nanodots. Reproduced with permission from reference (48). Copyright (2014) Nature Publishing Group.

Figure 8. TEM images of TTF nanodots with different TTF in reactants. (a) 30 wt% of TTF, (b) 50 wt% of TTF. Reproduced with permission from reference (48). Copyright (2014) Nature Publishing Group.

TTF nanodots were used for functional imaging of ear blood vessels under a two-photon scanning microscope. As shown in Figure 10(a)-(d), the blood vessels in mouse ear could be clearly viewed under the excitation of an 800 nm fs laser. By tracking red blood cells (RBCs) with time (49), its instantaneous velocity could be obtained, as shown in Figure 10(e). By studying the fluorescence from the blood post injection, a blood circulation half-life of about 4h was observed for TTF nanodots. 252 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 9. Absorption and fluorescence spectra of TTF nanodots with different loading ratios, λex = 365 nm. Reproduced with permission from reference (48). Copyright (2014) Nature Publishing Group.

Figure 10. (a) Bright field, (b) 1PL, (c) 2PL, (d) merged images of TTF nanodots stained mouse ear. λex = 543 nm for 1PL, λex = 800 nm for 2PL. (e) A line scan along capillaries was used to determine RBC’s instantaneous velocity (dx/dt). (f) Blood circulating kinetics of TTF nanodots in mice. Reproduced with permission from reference (48). Copyright (2014) Nature Publishing Group.

Recently, Qian et al. (our group) implemented deep-tissue in vivo neuron imaging with the 2PL of AIE nanoaggregates (43). A type of AIEgen called TPETPP was synthesized (50), and its molecular structure was shown in Figure 11(a). As shown in Figure 11(b), there was almost no fluorescence in its benign organic solvent, e.g., DMF, DMSO, while there was strong fluorescence in its solid state, indicating the AIE property of TPE-TPP. 253 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 11. (a) Chemical structure of TPE-TPP. (b) The fluorescence of TPE-TPP in DMSO, DMF, and solid state under UV irradiation. Reproduced with permission from reference (43). Copyright (2015) Optical Society of America.

The TPE-TPP nanoaggregates were formed by mixing DMSO solution of TPE-TPP with water, and their typical TEM images were shown in Figure 12(c). The absorption and fluorescence spectra of TPE-TPP were centered at 320 nm and 480 nm, with a large Stokes shift of 160 nm, as shown in Figure 12(a)-(b). Under the fs laser excitation of 740 nm, 2PL spectra of TPE-TPP was very similar to its 1PL spectra.

Figure 12. (a) Absorption and (b) Fluorescence spectra of TPE-TPP nanoaggregates, λex = 320 nm. (c) A typical TEM image of TPE-TPP nanoaggregates. Reproduced with permission from reference (43). Copyright (2015) Optical Society of America.

Neuron imaging is of great significance in neuron/brain research. TPE-TPP nanoaggregates were applied in primary neurons imaging with a two-photon fluorescence microscope. As shown in Figure 13, there was strong 2PL signals from the TPE-TPP treated primary neurons (Figure 13(a)), and it coincided very well with the bright field channel (Figure 13(b)-(c)), indicating the effective staining of TPE-TPP nanoaggregates on neurons.

254 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 13. 2PL images of TPE-TPP treated primary neurons.(a) fluorescence channel, (b) bright field channel, (c) merged. λex = 740 nm. Reproduced with permission from reference (43). Copyright (2015) Optical Society of America.

Figure 14. 2PL images of TPE-TPP stained microglia in mouse brain at different depths. (a) Top section, (b) middle section, (c) bottom section, (d) 3D reconstruction. λex = 740 nm. Reproduced with permission from reference (43). Copyright (2015) Optical Society of America. 2PL in vivo brain-microglia imaging was further conducted by microinjecting TPE-TPP into the mouse brain at a depth of 300 μm. As shown in Figure 14(a)-(c), the morphologies of the microglia at different sections of the in vivo mouse brain could be clearly discriminated. From the 3D reconstruction in Figure 14(d), the whole microglia was vividly demonstrated. Due to the resistance to photobleaching of TPE-TPP nanoaggregates, those microglia could be utilized for long-time dynamic observation. To improve the fluorescence quantum yield, Geng and Liu et al. proposed a micelle and silica coprotection strategy to synthesize PFBT-F127-SiO2 nanoparticles (42), as shown in Figure 15(a)-(b). These PFBT-F127-SiO2 255 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


nanoparticles had a high quantum yield of 75%, larger than that of PFBT-DSPE nanoparticles (40%). Also, the 2PA cross-section of PFBT-F127-SiO2 nanoparticles was large, with 1085 GM at 810 nm. These stable and biocompatible PFBT-F127-SiO2 nanoparticles were applied for 2PL imaging of mouse brain, and an imaging depth of 500 μm was obtained, as shown in Figure 15(c).

Figure 15. (a) Chemical structure of F127 and PFBT. (b) The schematic illustration of PFBT-F127-SiO2 nanoparticles fabrication. (c) 3D reconstruction of mouse brain imaging with PFBT-F127-SiO2 nanoparticles. λex = 800 nm. Reproduced with permission from reference (42). Copyright (2014) American Chemical Society. Geng and Liu et al. further applied micelle and silica coprotection method to synthesize TTF-F127-SiO2 nanoparticles (51), as shown in Figure 16(a). The TTFF127-SiO2 nanoparticles had a high quantum yield of 50%, much larger than that of TTF-F127 nanoparticles (24%). In addition, the TTF-F127-SiO2 nanoparticles had a high 2PA cross-section of 900 GM at 840 nm. TTF-F127-SiO2 nanoparticles were applied in 2PL imaging of mouse tibial muscle, and the blood vessels at a depth of 80 μm could still be visualized clearly, as shown in Figure 16(b).

Figure 16. (a) A schematic illustration of TTF-F127-SiO2 nanoparticles. (b) The z-projected image of mouse tibial mussel blood vessels stained with TTF-F127-SiO2 nanoparticles. λex = 810 nm. Reproduced with permission from reference (51). Copyright (2015) Royal Society of Chemistry. 256 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 17. (a) Chemical structure of DPP-based compounds. (b) Fluorescence spectra of DPP-2 in THF/water mixtures with different water fractions, λex = 455 nm. (c) 3D reconstruction of DDP-2 nanoparticles stained mouse ear blood vessels. λex = 800 nm. Reproduced with permission from reference (52). Copyright (2015) John Wiley and Sons. To improve the 2PA cross-section, Gao and Hua et al. synthesized DPP-2 with AIE properties (52), as shown in Figure 17(a)-(b). DPP-2 molecules were found to have a very large 2PA cross-section of 8100 GM at 800 nm, which was much larger than that of most commercial dyes (hundreds of GM). The quantum yield of DPP-2 in solid state was 11%. Furthermore, hydrophobic DPP-2 molecules were encapsulated into hydrophilic nanoparticles via a modified nanoprecipitation method, by using DSPE-PEG-Mal as the matrix, and the 2PA cross-section of DPP-2 nanoparticles reached 5.34×105 GM at 810 nm. The red emissive DPP-2 nanoparticles were applied in mouse ear imaging, and the blood vessels deep into 76 μm could be clearly viewed, as shown in Figure 17(c). Besides, Zhao et al. synthesized TPE-decorated BODIPY luminogens, PIPBT-TPE, and PITBT-TPE with large 2PA cross-sections (53, 54). They were encapsulated into nanoparticles together with DSPE-PEG and were utilized for mouse brain and ear vascular imaging. Blood vessels with good contrast could be visualized.

3. AIE Nanoprobes for in Vivo 2PL Imaging with Excitation Wavelength in NIR-IIa Window Excitation light in NIR-IIa window (1000-1400 nm) has less tissue scattering than that in conventional NIR-I window, and a deeper penetration depth can be anticipated. Wang and Cai et al. had studied the focal spots of 1040 nm and 800 nm laser beams at some representative depths of biological tissue via the Monte Carlo simulation (55). As shown in Figure 18, the 1040 nm laser beam has a better focusing than 800 nm laser beam in biological tissue, and excitation at 1040 nm would be more appropriate for deep-tissue imaging. So far, the 2PL imaging depth record (as deep as 1.6 mm in live mouse cortex) was achieved by utilizing 257 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Alexa680-Dextran (non-AIE) as the fluorophore, and the fs excitation wavelength was in NIR-IIa window (56). Besides, lower autofluorescence and better signal to background ratio of 2PL imaging could also be obtained under longer-wavelength excitation.

Figure 18. Simulation of the light intensity distribution of 1040 nm and 800 nm laser beams in biological tissue at various vertical depths. Reproduced with permission from reference (55). Copyright (2015) Optical Society of America.

There are some types of pulse laser sources in NIR-IIa window. An optical parameter oscillator (OPO, 1000-1600 nm, 76 MHz, ~200 fs), pumped by a modelocked Ti:sapphire fs laser (800 nm, 76 MHz), is an alternative (56–58). The tunable output wavelengths from 1000-1600 nm makes it very useful in NIR-II applications. The average output power of the fs OPO is not very high, about hundreds of milliwatts. A large-mode-area ytterbium-doped photonic crystal fiber (PCF) oscillator (1040 nm, 50 MHz, 150 fs) (55), with high output power of several watts, is very appropriate for the applications at 1040 nm. In addition, it has a relatively low price and is easy to operate. A Cr:forsterite fs laser (1230 nm, 110 MHz, 130 fs) is outstanding for applications at 1230 nm (59). When fs excitation with its wavelength in NIR-IIa window is adopted, 2PL will be excited from some red emitted fluorophores (55), while 3PL will be excited from some blue emitted fluorophores (60). There are not so many reports on AIE nanoprobes assisted MPL imaging in NIR-IIa window, and a lot of opportunaties still exists. Wang and Qian et al. (our group) implemented deep-tissue in vivo imaging with AIE nanodots, under the 1040 nm fs excitation (55). A red emissive AIEgen BODIPY-TPE (BT) was synthesized (61), and its chemical structure was shown in Figure 19(a), with the propeller-shaped TPE as the donor. As shown in Figure 19(b), when increasing the volume fraction of water from 65% to 95%, the fluorescence of BT in THF/water mixture near 600 nm was intensified, indicating the AIE characteristics of BT. The hydrophobic BT molecules were incorporated into hydrophilic nanodots by a modified nanoprecipitation method with DSPE-mPEG5000 as the matrix. BT nanodots had an absorption peak at 522 nm and an emission peak at 620 nm, with 258 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


a large Stokes shift of 98 nm, as shown in Figure 20(a). The 2PA cross-section of BT nanodots at 1040 nm was found to be 2.9×106 GM, and it was much larger than those at 770-860 nm, as shown in Figure 20(b).

Figure 19. (a) Chemical structure of BT. (b) Fluorescence spectra of BT in THF/water mixtures with water fractions from 65 to 95 vol%, λex = 420 nm. Reproduced with permission from reference (55). Copyright (2015) Optical Society of America.

Figure 20. (a) The absorption and fluorescence spectra of BT nanodots, λex = 420 nm. (b) The 2PA cross-section of BT nanodots at various wavelengths. Reproduced with permission from reference (55). Copyright (2015) Optical Society of America

The biodistribution and clearance of BT nanodots in mice were studied by collecting the characteristic fluorescence from the major organs at various times after the injection of nanodots. As shown in Figure 21, BT nanodots mainly accumulated in the liver, reached maxima about 12 hours post injection, and were then cleared out gradually. 259 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 21. (a) Fluorescence images of liver at various time points. (b) Fluorescence intensities of different organs at various time points. λex = 523 nm. Reproduced with permission from reference (55). Copyright (2015) Optical Society of America. The biocompatible BT nanodots were utilized for in vivo mouse brain vasculature imaging under the excitation of 1040 nm fs laser (from a large-mode-area ytterbium-doped PCF oscillator). As shown in Figure 22, the major blood vessels and small capillaries could be visualized clearly as deep as 700 μm, which was deeper than the 2PL imaging depth in most reported work performed in NIR-I window.

Figure 22. 3D reconstructed 2PL in vivo images of BT nanodots stained mouse brain blood vessels with different visual angles. λex = 1040 nm. Reproduced with permission from reference (55). Copyright (2015) Optical Society of America. There is plenty of room to achieve deeper 2PL imaging NIR-IIa window. The improvements in imaging systems and nanoprobes could result in larger imaging depth. Recently, our group found TTF nanodots had a larger 2PA cross-section than BT nanodots at 1040 nm, and it was then used for in vivo mouse brain imaging under the fs excitation at this wavelength. As shown in Figure 23, the blood vessels could be clearly viewed to a depth of 810 μm.



Figure 23. 3D reconstructed 2PL in vivo images of TTF nanodots stained mouse brain blood vessels, with different visual angles. λex = 1040 nm.

4. AIE Nanoprobes for in Vivo 3PL Imaging with Excitation Wavelength in NIR-IIb Window Excitation light in NIR-IIb window (1450-1700 nm) has the lowest tissue scattering compared with it in NIR-I and NIR-IIa windows. Although the water absorption is strong in NIR-IIb, the reduced tissue scattering can compensate it effectively. Cai et al. had studied the focal spots of 800-1680 nm laser beams at a 1.5 mm depth of biological tissue via the Monte Carlo simulation (62). As shown in Figure 24, the 1680 nm laser beam has the best focusing intensity than others, exhibiting the superiority of light excitation in the NIR-IIb window. An imaging depth of 1.3 mm (in living mouse brain) was achieved in 3PL microscopy based on Texas Red (non-AIE), where the fs excitation in NIR-IIb window was adopted (10). Besides, low autofluorescence and good contrast can be also obtained when NIR-IIb fs excitation is utilized. There are not so many types of pulsed laser sources, whose wavelengths are in NIR-IIb window. The aforementioned optical parameter oscillator (OPO, 10001600 nm, 76 MHz, ~200 fs) covers partial NIR-IIb window (58). However, the average power of the fs output is very low ( about hundreds of millwatts). A fs laser (FLCPA-01C, Calmar Laser, 1560 nm, 1 MHz, 400 fs), which takes advantage of rare-earth ion doped fiber as the active medium, can be adopted as the fs excitation source in NIR-IIb window (63). It is cost-effective, and easy to operate. Moreover, its average output power can reach one watt. Soliton self-frequency shift (SSFS) in a photonic crystal rod, which was pumped by a turnkey energetic fibre laser, could reach the optimal spectral window near 1700 nm (1675 nm, 1 MHz, 65 fs) (10). It was very helpful to deep-tissue imaging, although required lots of optical experience.



Figure 24. Simulation of the focal spots of various laser beams at a depth of 1.5 mm of biological tissue. (a) 800 nm, (b) 1040 nm, (c) 1280 nm, (d) 1440 nm, (e) 1560 nm, (f) 1680 nm. Reproduced with permission from reference (62). Copyright (2013) Electromagnetics Academy. When the fs excitation wavelength was in NIR-IIb window, 3PL is the main nonlinear optical process, and fluorophores with large 3PA coefficients are favored for bioimaging applications. There are very few reports on AIE nanoprobes assisted MPL imaging in NIR-IIb window, and the longest attenuation length of light in tissues would give those who carry out their work in NIR-IIb window a surprise. Zhu and Qian et al. (our group) proposed a new protocol to encapsulate AIE molecules into nanoparticles and implemented 3PL in vivo imaging of mouse ear with these AIE nanoparticles (64). A type of AIEgen TTF as referred was synthesized, and it was encapsulated by nano graphene oxide (NGO) to form nanoparticles, as shown in Figure 25. The stability and emission efficiency of TTFNGO NPs was improved and the size could be tuned by controlling the amount of NGO added.

Figure 25. The protocol for the synthesis of TTF-NGO nanoparticles. Reproduced with permission from reference (64). Copyright (2016) American Chemical Society. 262 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


The biocompatible TTF-NGO nanoparticles were utilized for 3PL in vivo imaging, under the excitation of a 1560 nm fs laser. As shown in Figure 26, the structure of blood vessels in the mouse ear at various vertical depths could be clearly recognized.

Figure 26. 3PL microscopic imaging of the ear blood vessels of a mouse. (a) At different depths, (b) 3D reconstruction, λex = 1560 nm. Reproduced with permission from reference (64). Copyright (2016) American Chemical Society.

Figure 27. 3PL microscopic imaging of brain blood vessels of a mouse at various depths, λex = 1560 nm. Reproduced with permission from reference (63). Copyright (2015) John Wiley and Sons. 263 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Qian et al. (our group) performed 3PL in vivo imaging of mouse brain with AIE nanodots (63). The same type of AIEgen TTF was synthesized, as shown in Figure 7(a). It was encapsulated into nanodots following the same routine, as shown in Figure 7(b). The high-order nonlinear optical effect of TTF nanodots were systematically studied and they were found to have strong 3PL under the excitation of 1560 nm. TTF nanodots were further used for 3PL imaging of mouse brain, by utilizing a 1560 nm fs laser as the excitation. As shown in Figure 27, the brain blood vessels at as deep as 550 μm can be clearly visualized. Recently, by optimizing the optical devices, e.g. using objective lens of high transmittance in NIR-IIb, we have improved the imaging depth of TTF stained mouse brain to 1000 μm. As shown in Figure 28, plenty of blood vessels and small capillaries could be vividly visualized with high contrast at various depths.

Figure 28. 3PL microscopic imaging of brain blood vessels of a mouse at various depths with improved optical devices, λex = 1560 nm.

5. Summary In this chapter, the motivation, mechanism, and some application examples of AIE nanoprobes assisted MPL in vivo imaging were introduced. Various types of AIEgens were synthesized, encapsulated into nanoparticles, and applied in in vivo imaging. The blood vessels in mouse ear and brain could be visualized clearly with a vertical depth up to hundreds of micrometers. The excitation wavelengths were classified into NIR-I, NIR-IIa, and NIR-IIb windows, according to the tissue scattering and absorption. The water absorption in NIR-I window is very small, and most MPL microscopy was carried out in this window. The tissue scattering is smaller in NIR-IIa window, and a better light penetration capability could be obtained. With the lowest tissue scattering, imaging in NIR-IIb window is very promising, although the optical devices should be specially designed. To get larger imaging depth and higher image contrast, AIEgens with large multi-photon absorption cross-section and quantum yield are highly required. With fluorophore of high brightness, fluorescence is much easier to be excited 264 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


and collected, and the deep tissues are more likely to be visulized. In addition, red and far-red emissions are beneficial to deep-tissue imaging, as fluorescence with longer wavelengths is easier to come out from the tissue to be detected. Furthermore, the compatibility and photo-stability of AIE nanoprobes are both helpful to long-term in vivo imaging. MPL imaging system still needs optimization to obtain larger imaging depth. As the scattering coefficients are not the same in different tissues, the best fs excitation wavelengths for them is also different. In addition, the factors of the multi-photon absorption cross-sections of the nanoprobes and the pulsed laser sources should also be considered when choosing the excitation. In NIR-II, the lenses and mirrors need special coatings to obtain good transmittance or reflection. The properties of the pulsed laser source, such as the output power, the repetition frequency, and the duty cycle are also very important for deep-tissue imaging (56). Nanoprobe-assisted bioimaging is a multidisciplinary research area, and it needs the effort from those who do their work on materials, optics, biology, etc. MPL in vivo imaging with AIE nanoprobes is a small and promising field of bioimaging, and it will give us much deeper images if we do deep on it.

Acknowledgments This work was supported by National Basic Research Program of China (973 Program; 2013CB834704), the National Natural Science Foundation of China (61275190), the Fundamental Research Funds for the Central Universities, and the Open Fund of the State Key Laboratory of Luminescent Materials and Devices (South China University of Technology).

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