Research group of academician Tang benzhong of Hong Kong University of science and technology: Exploration and application of aggregation induced luminescent materials in two-photon biomedical imaging

In recent years, fluorescence imaging has shown a wide range of applications in real-time monitoring of important biological molecules in vivo, tracking various physiological processes in cells and imaging-mediated therapy In general, living samples, especially tissues, have strong absorption, scattering and reflection of short wave length, resulting in shallow penetration depth and low imaging signal-to-noise ratio Compared with single photon fluorescence imaging, two-photon fluorescence imaging has the advantages of deep penetration depth, excellent imaging signal-to-noise ratio and high spatial-temporal resolution due to the excitation of near-infrared NIR pulse light The two-photon fluorescence imaging based on traditional fluorescent materials has achieved fruitful and impressive results However, due to the better planar π conjugation system, the aggregation fluorescence quenching (ACQ) of traditional fluorescent materials is easy to occur under high concentration or aggregation conditions, which restricts the further application of fluorescence imaging In 2001, academician Tang benzhong of Hong Kong University of science and technology put forward the phenomenon of aggregation induced luminescence (AIE), which is the opposite of ACQ effect AIE materials also show great potential applications in biomedicine In general, AIE materials with strong two-photon absorption can be obtained by introducing donor acceptor (d-π-a) or extending π conjugation system into the parent molecule of AIE In recent years, academician Tang benzhong's team has made a series of gratifying achievements in the field of two-photon imaging Tang benzhong, Professor of Hong Kong University of science and technology, academician of Chinese Academy of Sciences He received his bachelor's degree from South China University of technology in 1982 and his doctor's degree from Kyoto University in 1988 After that, he worked as a senior researcher in the Central Research Institute of Japan's Neos company and a postdoctoral researcher in the Department of chemistry and pharmacy of the University of Toronto, Canada He joined the Department of chemistry of Hong Kong University of science and technology in 1994 and was promoted to Professor in 2008 He was elected academician of the Chinese Academy of Sciences in 2009, Professor Zhang Jianquan of science at Hong Kong University of science and technology in 2013 and fellow of the Royal Society of chemistry In 2015, he was approved as a director of the Hong Kong Branch Center with the support of the National Engineering and Technology Research Center for human tissue function reconstruction of South China University of technology He is now the chief scientist of the 973 Program of the Ministry of science and technology, the project director of the basic science research center of the National Natural Science Foundation of China, the leader of the introduction and innovation research team of Guangdong Province, the director of the academic committee of the State Key Laboratory of light-emitting materials and devices of South China University of technology, and the editor in chief of materials chemistry frontiers, a joint Journal of the Chinese Chemical Society and the Royal chemical society In 2001, academician Tang benzhong and his team proposed the concept of "aggregation induced luminescence" (AIE) for the first time in the world The research in this field was listed as the top three research frontiers in the field of chemistry and materials in 2013 by Thomson Reuters science outlook As the proponent and leader of AIE concept, the team is mainly engaged in the research of AIE materials in recent years, and explores its applications in organic light-emitting devices, biological and chemical fluorescence probes, biomedical imaging and disease diagnosis and treatment So far, academician Tang benzhong has published more than 1000 academic papers, cited more than 80000 times, with an H index of 130 He has been rated as "highly cited scientist" in the fields of materials and chemical science for many times in 2013-2018 Academician Tang benzhong has won many honors and awards, such as the first prize of National Natural Science（ 2017, the first completed adult, he Liang and he Li science and Technology Progress Award (2017), 27th quarizmi International Science Award (2014), polymer Academic Report Award of American Chemical Society (2012), second prize of National Natural Science Award (2007, the fourth completed adult), Qiu Cha advanced research achievement award (2007), Wang Baoren award of polymer basic research of Chinese Chemical Society (2007) and Elsevier Award Press Feng Xinde polymer Award (2007), etc Advanced scientific research achievements (1) two photon AIE materials with various temperature control structures Figure 1 Construction of acrylonitrile AIE materials with different structures and functions, solid-state fluorescence emission and single / two photon fluorescence imaging in living cells, deep living tissues and living bodies (source: J am Chem SOC.) generally, fluorescent materials with different structures and functionalities need to be constructed by different reaction substrates through appropriate reactions Based on the control of different reaction temperatures, Tang benzhong's team reported for the first time a simple synthesis method without heavy metal catalysis, without the participation of hazardous and toxic substances, and with effective atom utilization rate, and constructed two structural controllable acrylonitrile AIE fluorescent materials (Fig 1, J am Chem SOC 2019, 141, These two AIE fluorescent materials have excellent photophysical properties, such as large Stokes shift, red light emission, high solid fluorescence quantum yield (up to 37.6%), large two-photon absorption cross section (up to 504gm), etc The potential biomedical applications in living cells, deep tissues and in vivo single / two-photon fluorescence imaging were confirmed Importantly, this work provides a new and simple synthesis strategy to construct other multi-functional two-photon fluorescence materials based on acrylonitrile Due to the novelty and importance of the research work, it was selected as the supply journal cover by the editor (II) selective illumination of subcellular organelles in living cells and deep living tissues Fig 2 Naphthalene based liposoluble AIE material and single / two-photon fluorescence imaging in living cells and deep living tissues (source: chem Mater.) lipid droplet is a kind of dynamic organelle widely existing in most cells and biological systems, which participates in many important physiological functions In recent years, it has been found that lipid droplets are closely related to obesity, diabetes, inflammation and cancer Therefore, it is very important to develop effective methods for direct and selective visualization and monitoring of lipid droplets in living cells and tissues Recently, Tang benzhong's team constructed a new type of fat soluble d-π-a AIE material based on naphthalene and used it in single / two-photon lipid droplet specific imaging at ultra-low concentration (Fig 2, chem Mater 2018, 30, 4778-4787) These AIE materials have the advantages of high solid-state fluorescence quantum yield (up to 30%), good two-photon absorption cross section (45-100 GM at 860 nm), excellent optical stability and biocompatibility It is found that this kind of AIE material can achieve the imaging of lipid droplet specific ultra-low concentration (50 nm) within 15 minutes, which is the lowest value of lipid droplet staining in living cells reported so far The calculated oil-water partition coefficient ClogP further reveals that these lipid soluble aggregation induced luminescent materials can specifically target lipid droplets In vitro and in vitro two-photon imaging of lipid droplets in living cells and liver tissues of mice has also been successfully confirmed Importantly, the selective two-photon visualization of lipid droplets has also been successfully realized for the first time in the living liver tissue of mice about 70 μ m deep This work provides an excellent candidate fluorescence probe for the study of lipid drop related physiological and pathological processes in living biological samples in the future Later, the team designed and synthesized a series of near-infrared AIE materials with d-π-a structure (ACS Nano 2018, 12, 8145-8159) by constructing push-pull electronic structure, using diphenylamine as electron donor group, introducing different strong electron absorption groups, and using electron rich carbazole as bridging group Two photon fluorescence imaging of mitochondria (77 μ m) and lipid droplets (129 μ m) in the deep tissues of mouse muscle and liver was realized under two photon excitation It is worth mentioning that these compounds can effectively produce singlet oxygen and kill cancer cells rapidly under the white light Recently, based on acrylonitrile derivatives, the team also reported two long alkyl chain substituted AIE NIR fluorescent materials CS py + so 3 − and CS py + (biomaterials 2019, 208, 72 – 82) with different charge distribution Cs-py + SO3 − and cs-py + selectively target the cell membrane and mitochondria in living cells, respectively At the same time, a variety of data results show that these two AIE materials are likely to be fixed in cells in the form of "single molecule" Moreover, CS py + successfully achieved two-photon deep fluorescence imaging (100 μ m) of mitochondria in living rat skeletal muscle tissue It is important that these two AIE materials, especially CS py + have excellent anti photobleaching ability under strong single photon and two-photon continuous irradiation (III) visualization of deep blood vessels Fig 3 Super bright red AIE material, AIE fluorescence curve and two-photon fluorescence imaging in deep blood vessels (source: chem SCI.) in general, there are two basic conditions to achieve efficient two-photon fluorescence imaging: one is strong two-photon absorption, i.e large two-photon absorption cross-section; the other is that the luminescent materials have high fluorescence quantum yield According to the calculation formula of two-photon absorption cross-section, the size of two-photon absorption cross-section is inversely proportional to the fluorescence quantum yield of the material, so it is difficult to keep the high fluorescence quantum yield while taking into account the large two-photon absorption cross-section, and there are few reports of such materials in the literature Recently, academician Tang benzhong's team prepared AIE nano materials with ultra bright red light emission and used them for high-resolution two-photon fluorescence imaging of deep blood vessels (Fig 3, chem SCI 2018, 9, 2705-2710) The water-soluble nano material has red emission (630 nm) and strong photobleaching resistance, and the quantum yield of fluorescence is as high as 34.1% At the same time, it also has strong two-photon absorption, with a two-photon absorption cross section of 310 GM Compared with single photon imaging, the AIE nanomaterials have deep two-photon fluorescence penetration and high imaging resolution in liver tissue The deep two-photon imaging of the rat's ear and brain vessels was also successfully achieved, with the penetration depth of 110 μ m and 350 μ m, respectively Importantly, for the first time, the team has achieved accurate measurement of capillary size in deep mouse ear vessels This work proposes an important strategy for building fluorescent materials with high efficiency of luminescence and strong two-photon absorption, as well as their biomedical imaging applications in deep tissue Figure 4 Niraie material and two-photon fluorescence imaging in ultra deep cerebral vessels (source: ACS Nano) has weaker tissue absorption, scattering, emission and spontaneous fluorescence than the traditional two-photon contrast agent excited by near-infrared region I (700-950 nm) and near-infrared region II (1000-1700 nm), so it is beneficial to improve the tissue penetration The team then reported that AIE nanomaterials are used for in vivo two-photon imaging of 1300 nm near-infrared region II excitation and near-infrared region I emission, with imaging depth of more than 1000 μ m in the brain of mice (Fig 4, ACS Nano 2018, 12, 7936-7945) The maximum absorption of AIE nano material is 635 nm, the emission wavelength can cover the range of 700-1200 nm, the quantum yield of fluorescence can reach 13.9%, and it has excellent optical stability, anti reactive oxygen nitrogen ability and large two-photon absorption cross section (1.22 × 103 GM) After the AIE nanomaterials were injected into the mice via the tail vein, the results of two-photon fluorescence microscopic imaging of the brain of the mice stimulated by 1300 nm femtosecond laser showed that the capillaries were 1065 μ m deep