Bioorthogonal Turn‐On Probe Based on Aggregation‐Induced Emission Characteristics for Cancer Cell Imaging and Ablation

Bioorthogonal turn‐on probes have been widely utilized in visualizing various biological processes. Most of the currently available bioorthogonal turn‐on probes are blue or green emissive fluorophores with azide or tetrazine as functional groups. Herein, we present an alternative strategy of designing bioorthogonal turn‐on probes based on red‐emissive fluorogens with aggregation‐induced emission characteristics (AIEgens). The probe is water soluble and non‐fluorescent due to the dissipation of energy through free molecular motion of the AIEgen, but the fluorescence is immediately turned on upon click reaction with azide‐functionalized glycans on cancer cell surface. The fluorescence turn‐on is ascribed to the restriction of molecular motion of AIEgen, which populates the radiative decay channel. Moreover, the AIEgen can generate reactive oxygen species (ROS) upon visible light (λ=400–700 nm) irradiation, demonstrating its dual role as an imaging and phototherapeutic agent.     

多种超分辨荧光成像技术比较和进展评述

所见即所得是生命科学研究的中心哲学,贯穿在不断认识单个分子、分子复合体、分子动态行为和整个分子网络的历程中。活的动态的分子才是有功能的,这决定了荧光显微成像在生命科学研究中成为不可替代的工具。但是当荧光成像聚焦到分子水平的时候,所见并不能给出想要得到的。这个障碍是由于受光学衍射极限的限制,荧光显微镜无法在衍射受限的空间内分辨出目标物。超分辨荧光成像技术突破衍射极限的限制,在纳米尺度至单分子水平可视化生物分子,以前所未有的时空分辨率研究活细胞结构和动态过程,已成为生命科学研究的有力工具,并逐渐应用到材料科学、催化反应过程和光刻等领域。超分辨成像技术原理不同,其具有的技术性能各异,限制了各自特定的技术特色和应用范围。目前主流的超分辨成像技术包括3种:结构光照明显微镜技术(structured illumination microscopy, SIM)、受激发射损耗显微技术(stimulated emission depletion, STED)和单分子定位成像技术(single molecule localization microscopy, SMLM)。这些显微镜采用不同的复杂技术,但是策略却是相同和简单的,即通过牺牲时间分辨率来提升衍射受限的空间内相邻两个发光点的空间分辨。该文通过对这3种技术的原理比较和在生物研究中的应用进展介绍,明确了不同超分辨成像技术的技术优势和适用的应用方向,以方便研究者在未来研究中做合理的选择。     

Dual-Mode Superresolution Imaging Using Charge Transfer Dynamics in Semiconducting Polymer Dots

In a conjugated polymer-based single-particle heterojunction, stochastic fluctuations of the photogenerated hole population lead to spontaneous fluorescence switching. We found that 405 nm irradiation can induce charge recombination and activate the single-particle emission. Based on these phenomena, we developed a novel class of semiconducting polymer dots that can operate in two superresolution imaging modes. The spontaneous switching mode offers efficient imaging of large areas, with <10 nm localization precision, while the photoactivation/deactivation mode offers slower imaging, with further improved localization precision (ca. 1 nm), showing advantages in resolving small structures that require high spatial resolution. Superresolution imaging of microtubules and clathrin-coated pits was demonstrated, under both modes. The excellent localization precision and versatile imaging options provided by these nanoparticles offer clear advantages for imaging of various biological systems.     

Micro-Lensed Fiber Laser Desorption Mass Spectrometry Imaging Reveals Subcellular Distribution of Drugs within Single Cells

The visualization of temporal and spatial changes in the intracellular environment has great significance for chemistry and bioscience research. Mass spectrometry imaging (MSI) plays an important role because of its unique advantages, such as being label-free and high throughput, yet it is a challenge for laser-based techniques due to limited lateral resolution. Here, we develop a simple, reliable, and economic nanoscale MSI approach by introducing desorption laser with a micro-lensed fiber. Using this integrated platform, we achieved 300 nm resolution MSI and successfully visualized the distribution of various small-molecule drugs in subcellular locations. Exhaustive dynamic processes of anticancer drugs, including releasing from nanoparticle carriers entering nucleus of cells, can be readily acquired on an organelle scale. Considering the simplicity and universality of this nanoscale desorption device, it could be easily adapted to most of laser-based mass spectrometry applications.     

Dual‐Mode Superresolution Imaging Using Charge Transfer Dynamics in Semiconducting Polymer Dots

In a conjugated polymer‐based single‐particle heterojunction, stochastic fluctuations of the photogenerated hole population lead to spontaneous fluorescence switching. We found that 405 nm irradiation can induce charge recombination and activate the single‐particle emission. Based on these phenomena, we developed a novel class of semiconducting polymer dots that can operate in two superresolution imaging modes. The spontaneous switching mode offers efficient imaging of large areas, with <10 nm localization precision, while the photoactivation/deactivation mode offers slower imaging, with further improved localization precision (ca. 1 nm), showing advantages in resolving small structures that require high spatial resolution. Superresolution imaging of microtubules and clathrin‐coated pits was demonstrated, under both modes. The excellent localization precision and versatile imaging options provided by these nanoparticles offer clear advantages for imaging of various biological systems.     

Nanoprobes for super-resolution fluorescence imaging at the nanoscale

Compared with other imaging techniques,fluorescence microscopy has become an essential tool to study cell biology due to its high compatibility with living cells.Owing to the resolution limit set by the diffraction of light,fluorescence microscopy could not resolve the nanostructures in the range of<200 nm.Recently,many techniques have been emerged to overcome the diffraction barrier,providing nanometer spatial resolution.In the course of development,the progress in fluorescent probes has helped to promote the development of the high-resolution fluorescence nanoscopy.Here,we describe the contributions of the fluorescent probes to far-field super resolution imaging,focusing on concepts of the existing super-resolution nanoscopy based on the photophysics of fluorescent nanoprobes,like photoswitching,bleaching and blinking.Fluorescent probe technology is crucial in the design and implementation of super-resolution imaging methods.     

Micro‐Lensed Fiber Laser Desorption Mass Spectrometry Imaging Reveals Subcellular Distribution of Drugs within Single Cells

The visualization of temporal and spatial changes in the intracellular environment has great significance for chemistry and bioscience research. Mass spectrometry imaging (MSI) plays an important role because of its unique advantages, such as being label‐free and high throughput, yet it is a challenge for laser‐based techniques due to limited lateral resolution. Here, we develop a simple, reliable, and economic nanoscale MSI approach by introducing desorption laser with a micro‐lensed fiber. Using this integrated platform, we achieved 300 nm resolution MSI and successfully visualized the distribution of various small‐molecule drugs in subcellular locations. Exhaustive dynamic processes of anticancer drugs, including releasing from nanoparticle carriers entering nucleus of cells, can be readily acquired on an organelle scale. Considering the simplicity and universality of this nanoscale desorption device, it could be easily adapted to most of laser‐based mass spectrometry applications.     

Recent advances in molecular fluorescent probes for organic phosphate biomolecules recognition

This review provided a systematic overview of the recent researches on the small-molecule fluorescent probes for recognition various organic phosphate biomolecules (OPBs) including nucleotides, NAD(P)H, FAD/FMN and PS. The general strategies and the recognition mechanisms for these OPBs probe designs were described and emphasized to inspire the better design for fluorescent probes in the future.     

STED Imaging the Dynamics of Lysosomes by Dually Fluorogenic Si-Rhodamine

Super-resolution microscopy (SRM) imaging of the finite subcellular structures and subtle bioactivities inside organelles delivers abundant cellular information with high fidelity to unravel the intricate biological processes. An ideal fluorescent probe with precise control of fluorescence is critical in SRM technique like stimulated emission depletion (STED). Si-rhodamine was decorated with both targeting group and H⁺-receptor, affording the dually fluorogenic Si-rhodamine in which the NIR fluorescence was efficiently controlled by the coalescent of spirolactone-zwitterion equilibrium and PeT mechanism. The dually fluorogenic characters of the probe offer a perfect mutual enhancement in sensitivity, specificity and spatial resolution. Strong fluorescence only released in the existence of targeting protein at acidic lysosomal pH, ensured precisely tracking the dynamic of lysosomal structure and pH in living cells by STED.     

Quencher-Delocalized Emission Strategy of AIEgen-Based Metal–Organic Framework for Profiling of Subcellular Glutathione

Distinguishing glutathione (GSH) level in different subcellular locations is critical for studying its antioxidant function in the signaling system. However, traditional methods for imaging subcellular GSH were achieved in isolated organelles or fixed cells. In this work, we report a quencher-delocalized emission strategy for in situ profiling of GSH at different subcellular locations in living cells. A nonemissive metal–organic framework (MOF) nanoprobe was designed with AIEgen as the linker and Cu^II as the node and quencher. The AIEgen in MOF structure was lightened up with green emission in a neutral environment due to partial Cu^II delocalization by competitive binding to GSH. Meanwhile, along with the protonation of AIEgen ligand under acidic environment, the AIEgen-based MOF could be completely dissociated in the presence of GSH to yield yellow emission. The two-channel ratiometric analysis of dual-colored emission of AIEgen-based MOF allows visualization of GSH in cytoplasm and lysosome in living cells, which is also able to report the drug effects on different subcellular GSH levels.     

An Activatable AIEgen Probe for High-Fidelity Monitoring of Overexpressed Tumor Enzyme Activity and Its Application to Surgical Tumor Excision

Monitoring fluctuations in enzyme overexpression facilitates early tumor detection and excision. An AIEgen probe (DQM-ALP) for the imaging of alkaline phosphatase (ALP) activity was synthesized. The probe consists of a quinoline-malononitrile (QM) core decorated with hydrophilic phosphate groups as ALP-recognition units. The rapid liberation of DQM-OH aggregates in the presence of ALP resulted in aggregation-induced fluorescence. The up-regulation of ALP expression in tumor cells was imaged using DQM-ALP. The probe permeated into 3D cervical and liver tumor spheroids for imaging spatially heterogeneous ALP activity with high spatial resolution on a two-photon microscopy platform, providing the fluorescence-guided recognition of sub-millimeter tumorigenesis. DQM-ALP enabled differentiation between tumor and normal tissue ex vivo and in vivo, suggesting that the probe may serve as a powerful tool to assist surgeons during tumor resection.     

An Activatable AIEgen Probe for High‐Fidelity Monitoring of Overexpressed Tumor Enzyme Activity and Its Application to Surgical Tumor Excision

Monitoring fluctuations in enzyme overexpression facilitates early tumor detection and excision. An AIEgen probe (DQM‐ALP) for the imaging of alkaline phosphatase (ALP) activity was synthesized. The probe consists of a quinoline‐malononitrile (QM) core decorated with hydrophilic phosphate groups as ALP‐recognition units. The rapid liberation of DQM‐OH aggregates in the presence of ALP resulted in aggregation‐induced fluorescence. The up‐regulation of ALP expression in tumor cells was imaged using DQM‐ALP. The probe permeated into 3D cervical and liver tumor spheroids for imaging spatially heterogeneous ALP activity with high spatial resolution on a two‐photon microscopy platform, providing the fluorescence‐guided recognition of sub‐millimeter tumorigenesis. DQM‐ALP enabled differentiation between tumor and normal tissue ex vivo and in vivo, suggesting that the probe may serve as a powerful tool to assist surgeons during tumor resection.     

Pre-clinical whole-body fluorescence imaging: Review of instruments,methods and applications

Fluorescence sampling of cellular function is widely used in all aspects of biology, allowing the visualization of cellular and sub-cellular biological processes with spatial resolutions in the range from nanometers up to centimeters. Imaging of fluorescence in vivo has become the most commonly used radiological tool in all pre-clinical work. In the last decade, full-body pre-clinical imaging systems have emerged with a wide range of utilities and niche application areas. The range of fluorescent probes that can be excited in the visible to near-infrared part of the electromagnetic spectrum continues to expand, with the most value for in vivo use being beyond the 630 nm wavelength, because the absorption of light sharply decreases. Whole-body in vivo fluorescence imaging has not yet reached a state of maturity that allows its routine use in the scope of large-scale pre-clinical studies. This is in part due to an incomplete understanding of what the actual fundamental capabilities and limitations of this imaging modality are. However, progress is continuously being made in research laboratories pushing the limits of the approach to consistently improve its performance in terms of spatial resolution, sensitivity and quantification. This paper reviews this imaging technology with a particular emphasis on its potential uses and limitations, the required instrumentation, and the possible imaging geometries and applications. A detailed account of the main commercially available systems is provided as well as some perspective relating to the future of the technology development. Although the vast majority of applications of in vivo small animal imaging are based on epi-illumination planar imaging, the future success of the method relies heavily on the design of novel imaging systems based on state-of-the-art optical technology used in conjunction with high spatial resolution structural modalities such as MRI, CT or ultrasound.     

Recent Advances of AIEgens for Targeted Imaging of Subcellular Organelles

Fluorescence imaging based on luminogens with aggregation-induced emission(AIE)effect has drawn great attention in recent two decades,due to their superior advantages to overcome the technical difficulties.Thus,the AIE-active bioprobes with targeted ability at the subcellular level have been widely investigated to visualize the subcellular structures and monitor the biological processes.Considering the very rapid developments and the significance of selective imaging of subcellular structures,we summarize the recent two-year achievements about the AIEgens for targeted imaging of subcellular organelles including nuclei,membranes,lipid droplets(LDs),endoplasmic reticulum(ER),lysosomes,mitochondria and cytoplasm.The designed protocols and advantages of AIEgens,their mechanisms for targeted staining at organelles and the imaging performance are discussed.These AIE bioprobes exhibit great potentials for early diagnosis and therapeutics of diseases that related to subcellular organelles.Finally,the perspectives about AIEgens for these applications are also discussed.     

Recent advances in molecular fluorescent probes for organic phosphate biomolecules recognition

Organic phosphate biomolecules (OPBs) are indispensable components of eukaryotes and prokaryotes, such as acting as the fundamental components of cell membranes and important substrates for nucleic acids. They play pivotal roles in various biological processes, such as energy conservation, metabolism, and signal modulation. Due to the difficulty of detection caused by variety OPBs, investigation of their respective physiological effects in organisms has been restrained by the lack of efficient tools. Many small fluorescent probes have been employed for selective detection and monitoring of OPBs in vitro or in vivo due to the advantages of tailored properties, biodegradability and in situ high temporal and spatial resolution imaging. In this review, we summarize the recent advances in fluorescent probes for OPBs, such as nucleotides, NAD(P)H, FAD/FMN and PS. Importantly, we describe their identification mechanisms in detail and discuss the general strategies for these OPBs probe designs, which provide new insights and ideas for the future probe designs.     

Synthesis of D-A typed AIE luminogens in isomeric architecture and their application in latent fingerprints imaging

Among the emitters in powder dusting to visualize the latent fingerprints (LFPs), aggregation-induced emission luminogens (AIEgens) are well employed for their high brightness and resistance to photo-bleaching. However, the serious background interference and low resolution still limit their fast development. Therefore, to further enhance the signal-to-noise ratio in LFPs imaging, especially to improve the analysis for level 3 details, donor-acceptor (D-A) typed AIEgens of DTPA-2,3-P, DTPA-2,5-P and DTPA-2,6-P are designed here. It is observed that strong emission covering from 450 nm to 650 nm can be obtained for all these molecules, especially that a high PLQY value of 10.06% in solids is achieved in DTPA-2,3-P. This is much higher than that of the other two cases (0.80% and 0.51%). By utilizing the DTPA-2,3-P in powder dusting, fluorescence imaging of LFPs can be clearly captured on both smooth and rough substrates. Moreover, confocal laser scanning microscope (CLSM) enables us to achieve high-resolution LFPs imaging in both 2D and 3D views, providing more detailed information of fingerprints pores in width, distance, distribution, and shapes. The results here demonstrate that highly emissive AIEgen of DTPA-2,3-P could be an excellent candidate for the visualization of fingerprints, thus providing the potential application in criminal investigation in the future.     

DNA Nanoribbon-Templated Self-Assembly of Ultrasmall Fluorescent Copper Nanoclusters with Enhanced Luminescence

Fluorescent copper nanoclusters (CuNCs) have been widely used in chemical sensors, biological imaging, and light-emitting devices. However, individual fluorescent CuNCs have limitations in their capabilities arising from poor photostability and weak emission intensities. As one kind of aggregation-induced emission luminogen (AIEgen), the formation of aggregates with high compactness and good order can efficiently improve the emission intensity, stability, and tunability of CuNCs. Here, DNA nanoribbons, containing multiple specific binding sites, serve as a template for in situ synthesis and assembly of ultrasmall CuNCs (0.6 nm). These CuNC self-assemblies exhibit enhanced luminescence and excellent fluorescence stability because of tight and ordered arrangement through DNA nanoribbons templating. Furthermore, the stable and bright CuNC assemblies are demonstrated in the high-sensitivity detection and intracellular fluorescence imaging of biothiols.     

DNA Nanoribbon‐Templated Self‐Assembly of Ultrasmall Fluorescent Copper Nanoclusters with Enhanced Luminescence

Fluorescent copper nanoclusters (CuNCs) have been widely used in chemical sensors, biological imaging, and light‐emitting devices. However, individual fluorescent CuNCs have limitations in their capabilities arising from poor photostability and weak emission intensities. As one kind of aggregation‐induced emission luminogen (AIEgen), the formation of aggregates with high compactness and good order can efficiently improve the emission intensity, stability, and tunability of CuNCs. Here, DNA nanoribbons, containing multiple specific binding sites, serve as a template for in situ synthesis and assembly of ultrasmall CuNCs (0.6 nm). These CuNC self‐assemblies exhibit enhanced luminescence and excellent fluorescence stability because of tight and ordered arrangement through DNA nanoribbons templating. Furthermore, the stable and bright CuNC assemblies are demonstrated in the high‐sensitivity detection and intracellular fluorescence imaging of biothiols.     

Diffusion of resonance radiation in atomic vapor imaging

The effect of resonance radiation diffusion due to radiation trapping has been studied in an atomic vapor imaging filter. Using a cesium resonance fluorescence imaging monochromator, the spatial distortions due to resonance radiation trapping by Cs atoms in the pumping/imaging region have been investigated. It was found that the spatial distortions were dependent on the number density of the Cs atoms, as well as the irradiance of the signal photons at 852.12 nm (6²S_1/2→6²P^o_3/2). The pump laser (917.23 nm, 6²P^o_3/2→6²D_5/2) did not influence the radiation diffusion to the same degree as the signal beam. It is shown that there is a compromise between maximum optical density and spatial resolution. The number density of the Cs resonance fluorescence imaging monochromator was optimized to obtain the highest spatial resolution and signal-to-background ratio.     

A new sample substrate for imaging and correlating organic and trace metal composition in biological cells and tissues

Many disease processes involve alterations in the chemical makeup of tissue. Synchrotron-based infrared (IR) and X-ray fluorescence (XRF) microscopes are becoming increasingly popular tools for imaging the organic and trace metal compositions of biological materials, respectively, without the need for extrinsic labels or stains. Fourier transform infrared microspectroscopy (FTIRM) provides chemical information on the organic components of a material at a diffraction-limited spatial resolution of 2–10 μm in the mid-infrared region. The synchrotron X-ray fluorescence (SXRF) microprobe is a complementary technique used to probe trace element content in the same systems with a similar spatial resolution. However to be most beneficial, it is important to combine the results from both imaging techniques on a single sample, which requires precise overlap of the IR and X-ray images. In this work, we have developed a sample substrate containing a gold grid pattern on its surface, which can be imaged with both the IR and X-ray microscopes. The substrate consists of a low trace element glass slide that has a gold grid patterned on its surface, where the major and minor parts of the grid contain 25 and 12 nm gold, respectively. This grid pattern can be imaged with the IR microscope because the reflectivity of gold differs as a function of thickness. The pattern can also be imaged with the SXRF microprobe because the Au fluorescence intensity changes with gold thickness. The tissue sample is placed on top of the patterned substrate. The grid pattern’s IR reflectivity image and the gold SXRF image are used as fiducial markers for spatially overlapping the IR and SXRF images from the tissue. Results show that IR and X-ray images can be correlated precisely, with a spatial resolution of less than one pixel (i.e., 2–3 microns). The development of this new tool will be presented along with applications to paraffin-embedded metalloprotein crystals, Alzheimer’s disease, and hair composition.