DNA-Packing Portal and Capsid-Associated Tegument Complexes in the Tumor Herpesvirus KSHV.

Assembly of Kaposi's sarcoma-associated herpesvirus (KSHV) begins at a bacteriophage-like portal complex that nucleates formation of an icosahedral capsid with capsid-associated tegument complexes (CATCs) and facilitates translocation of an ∼150-kb dsDNA genome, followed by acquisition of a pleomorphic tegument and envelope. Because of deviation from icosahedral symmetry, KSHV portal and tegument structures have largely been obscured in previous studies. Using symmetry-relaxed cryo-EM, we determined the in situ structure of the KSHV portal and its interactions with surrounding capsid proteins, CATCs, and the terminal end of KSHV's dsDNA genome. Our atomic models of the portal and capsid/CATC, together with visualization of CATCs' variable occupancy and alternate orientation of CATC-interacting vertex triplexes, suggest a mechanism whereby the portal orchestrates procapsid formation and asymmetric long-range determination of CATC attachment during DNA packaging prior to pleomorphic tegumentation/envelopment. Structure-based mutageneses confirm that a triplex deep binding groove for CATCs is a hotspot that holds promise for antiviral development.

Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.

A positively tuned voltage indicator for extended electrical recordings in the brain.

Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.

Doping-induced assembly interface for noninvasive in vivo local and systemic immunomodulation.

Peripheral neural interfaces, potent in modulating local and systemic immune responses for disease treatment, face significant challenges due to the peripheral nerves' broad distribution in tissues like the fascia, periosteum, and skin. The incongruity between static electronic components and the dynamic, complex organization of the peripheral nervous system often leads to interface failure, stalling circuit research and clinical applications. To overcome these, we developed a self-assembling, tissue-adaptive electrode composed of a single-component cocktail nanosheet colloid, including dopants, conducting polymers, stabilizers, and an MXene catalyst. Delivered via a jet injector to designated nerve terminals, this assembly utilizes reactive oxygen species to catalytically dope poly (3,4-ethylenedioxythiophene), enhancing π-π interactions between nanosheets, and yielding a conductive, biodegradable interface. This interface effectively regulates local immune activity and promotes sensory and motor nerve functional restoration in nerve-injured mice, while engaging the vagal-adrenal axis in freely moving mice, eliciting catecholamine neurotransmitter release, and suppressing systemic cytokine storms. This innovative strategy specifically targets nerve substructures, bolstering local and systemic immune modulation, and paving the way for the development of self-adaptive dynamic neural interfaces.

Cross-modal coherent registration of whole mouse brains.

Recent whole-brain mapping projects are collecting large-scale three-dimensional images using modalities such as serial two-photon tomography, fluorescence micro-optical sectioning tomography, light-sheet fluorescence microscopy, volumetric imaging with synchronous on-the-fly scan and readout or magnetic resonance imaging. Registration of these multi-dimensional whole-brain images onto a standard atlas is essential for characterizing neuron types and constructing brain wiring diagrams. However, cross-modal image registration is challenging due to intrinsic variations of brain anatomy and artifacts resulting from different sample preparation methods and imaging modalities. We introduce a cross-modal registration method, mBrainAligner, which uses coherent landmark mapping and deep neural networks to align whole mouse brain images to the standard Allen Common Coordinate Framework atlas. We build a brain atlas for the fluorescence micro-optical sectioning tomography modality to facilitate single-cell mapping, and used our method to generate a whole-brain map of three-dimensional single-neuron morphology and neuron cell types.

Cryo-EM structures of herpes simplex virus type 1 portal vertex and packaged genome.

Herpesviruses are enveloped viruses that are prevalent in the human population and are responsible for diverse pathologies, including cold sores, birth defects and cancers. They are characterized by a highly pressurized pseudo-icosahedral capsid-with triangulation number (T) equal to 16-encapsidating a tightly packed double-stranded DNA (dsDNA) genome1-3. A key process in the herpesvirus life cycle involves the recruitment of an ATP-driven terminase to a unique portal vertex to recognize, package and cleave concatemeric dsDNA, ultimately giving rise to a pressurized, genome-containing virion4,5. Although this process has been studied in dsDNA phages6-9-with which herpesviruses bear some similarities-a lack of high-resolution in situ structures of genome-packaging machinery has prevented the elucidation of how these multi-step reactions, which require close coordination among multiple actors, occur in an integrated environment. To better define the structural basis of genome packaging and organization in herpes simplex virus type 1 (HSV-1), we developed sequential localized classification and symmetry relaxation methods to process cryo-electron microscopy (cryo-EM) images of HSV-1 virions, which enabled us to decouple and reconstruct hetero-symmetric and asymmetric elements within the pseudo-icosahedral capsid. Here we present in situ structures of the unique portal vertex, genomic termini and ordered dsDNA coils in the capsid spooled around a disordered dsDNA core. We identify tentacle-like helices and a globular complex capping the portal vertex that is not observed in phages, indicative of herpesvirus-specific adaptations in the DNA-packaging process. Finally, our atomic models of portal vertex elements reveal how the fivefold-related capsid accommodates symmetry mismatch imparted by the dodecameric portal-a longstanding mystery in icosahedral viruses-and inform possible DNA-sequence recognition and headful-sensing pathways involved in genome packaging. This work showcases how to resolve symmetry-mismatched elements in a large eukaryotic virus and provides insights into the mechanisms of herpesvirus genome packaging.

Unique dynamics and exocytosis properties of GABAergic synaptic vesicles revealed by three-dimensional single vesicle tracking.

Maintaining the balance between neuronal excitation and inhibition is essential for proper function of the central nervous system. Inhibitory synaptic transmission plays an important role in maintaining this balance. Although inhibitory transmission has higher kinetic demands compared to excitatory transmission, its properties are poorly understood. In particular, the dynamics and exocytosis of single inhibitory vesicles have not been investigated, due largely to both technical and practical limitations. Using a combination of quantum dots (QDs) conjugated to antibodies against the luminal domain of the vesicular GABA transporter to selectively label GABAergic (i.e., predominantly inhibitory) vesicles together with dual-focus imaging optics, we tracked the real-time three-dimensional position of single GABAergic vesicles up to the moment of exocytosis (i.e., fusion). Using three-dimensional trajectories, we found that GABAergic synaptic vesicles traveled a shorter distance prior to fusion and had a shorter time to fusion compared to synaptotagmin-1 (Syt1)-labeled vesicles, which were mostly from excitatory neurons. Moreover, our analysis revealed that GABAergic synaptic vesicles move more straightly to their release sites than Syt1-labeled vesicles. Finally, we found that GABAergic vesicles have a higher prevalence of kiss-and-run fusion than Syt1-labeled vesicles. These results indicate that inhibitory synaptic vesicles have a unique set of dynamics and exocytosis properties to support rapid synaptic inhibition, thereby maintaining a tightly regulated coordination between excitation and inhibition in the central nervous system.

Accelerating Neurite Growth and Directing Neuronal Connections Constrained by 3D Porous Microtubes.

Investigation of neural growth and connection is crucial in the field of neural tissue engineering. Here, using a femtosecond laser direct writing (fs-DLW) technique, we propose a directionally aligned porous microtube array as a culture system for accelerating the growth of neurons and directing the connection of neurites. These microtubes exhibited an unprecedented guidance effect toward the outgrowth of primary embryonic rat hippocampal neurons, with a wrap resembling the myelin sheaths of neurons. The speed of neurite growth inside these microtubes was significantly faster than that outside these microtubes. We also achieved selective/directing connection of neural networks inside the magnetic microtubes via precise microtube delivery to a gap between two neural clusters. This work not only proposes a powerful microtube platform for accelerated growth of neurons but also offers a new idea for constructing biological neural circuits by arranging the size, location, and pattern of microtubes.

Whole-brain mapping of efferent projections of the anterior cingulate cortex in adult male mice.

The anterior cingulate cortex (ACC) is a key cortical region that plays an important role in pain perception and emotional functions. Previous studies of the ACC projections have been collected primarily from monkeys, rabbits and rats. Due to technological advances, such as gene manipulation, recent progress has been made in our understanding of the molecular and cellular mechanisms of the ACC-related chronic pain and emotion is mainly obtained from adult mice. Few anatomic studies have examined the whole-brain projections of the ACC in adult mice. In the present study, we examined the continuous axonal outputs of the ACC in the whole brain of adult male mice. We used the virus anterograde tracing technique and an ultrahigh-speed imaging method of Volumetric Imaging with Synchronized on-the-fly-scan and Readout (VISoR). We created a three-dimensional (3D) reconstruction of mouse brains. We found that the ACC projected ipsilaterally primarily to the caudate putamen (CPu), ventral thalamic nucleus, zona incerta (ZI), periaqueductal gray (PAG), superior colliculus (SC), interpolar spinal trigeminal nucleus (Sp5I), and dorsal medullary reticular nucleus (MdD). The ACC also projected to contralateral brain regions, including the ACC, reuniens thalamic nucleus (Re), PAG, Sp5I, and MdD. Our results provide a whole-brain mapping of efferent projections from the ACC in adult male mice, and these findings are critical for future studies of the molecular and synaptic mechanisms of the ACC and its related network in mouse models of brain diseases.

Mapping thalamic-anterior cingulate monosynaptic inputs in adult mice.

The anterior cingulate cortex (ACC) is located in the frontal part of the cingulate cortex, and plays important roles in pain perception and emotion. The thalamocortical pathway is the major sensory input to the ACC. Previous studies have show that several different thalamic nuclei receive projection fibers from spinothalamic tract, that in turn send efferents to the ACC by using neural tracers and optical imaging methods. Most of these studies were performed in monkeys, cats, and rats, few studies were reported systematically in adult mice. Adult mice, especially genetically modified mice, have provided molecular and synaptic mechanisms for cortical plasticity and modulation in the ACC. In the present study, we utilized rabies virus-based retrograde tracing system to map thalamic-anterior cingulate monosynaptic inputs in adult mice. We also combined with a new high-throughput VISoR imaging technique to generate a three-dimensional whole-brain reconstruction, especially the thalamus. We found that cortical neurons in the ACC received direct projections from different sub-nuclei in the thalamus, including the anterior, ventral, medial, lateral, midline, and intralaminar thalamic nuclei. These findings provide key anatomic evidences for the connection between the thalamus and ACC.

Different functional states of fusion protein gB revealed on human cytomegalovirus by cryo electron tomography with Volta phase plate.

Human cytomegalovirus (HCMV) enters host by glycoprotein B (gB)-mediated membrane fusion upon receptor-binding to gH/gL-related complexes, causing devastating diseases such as birth defects. Although an X-ray crystal structure of the recombinant gB ectodomain at postfusion conformation is available, the structures of prefusion gB and its complex with gH/gL on the viral envelope remain elusive. Here, we demonstrate the utility of cryo electron tomography (cryoET) with energy filtering and the cutting-edge technologies of Volta phase plate (VPP) and direct electron-counting detection to capture metastable prefusion viral fusion proteins and report the structures of glycoproteins in the native environment of HCMV virions. We established the validity of our approach by obtaining cryoET in situ structures of the vesicular stomatitis virus (VSV) glycoprotein G trimer (171 kD) in prefusion and postfusion conformations, which agree with the known crystal structures of purified G trimers in both conformations. The excellent contrast afforded by these technologies has enabled us to identify gB trimers (303kD) in two distinct conformations in HCMV tomograms and obtain their in situ structures at up to 21 Å resolution through subtomographic averaging. The predominant conformation (79%), which we designate as gB prefusion conformation, fashions a globular endodomain and a Christmas tree-shaped ectodomain, while the minority conformation (21%) has a columnar tree-shaped ectodomain that matches the crystal structure of the "postfusion" gB ectodomain. We also observed prefusion gB in complex with an "L"-shaped density attributed to the gH/gL complex. Integration of these structures of HCMV glycoproteins in multiple functional states and oligomeric forms with existing biochemical data and domain organization of other class III viral fusion proteins suggests that gH/gL receptor-binding triggers conformational changes of gB endodomain, which in turn triggers two essential steps to actuate virus-cell membrane fusion: exposure of gB fusion loops and unfurling of gB ectodomain.

Differentiation and Characterization of Excitatory and Inhibitory Synapses by Cryo-electron Tomography and Correlative Microscopy.

As key functional units in neural circuits, different types of neuronal synapses play distinct roles in brain information processing, learning, and memory. Synaptic abnormalities are believed to underlie various neurological and psychiatric disorders. Here, by combining cryo-electron tomography and cryo-correlative light and electron microscopy, we distinguished intact excitatory and inhibitory synapses of cultured hippocampal neurons, and visualized the in situ 3D organization of synaptic organelles and macromolecules in their native state. Quantitative analyses of >100 synaptic tomograms reveal that excitatory synapses contain a mesh-like postsynaptic density (PSD) with thickness ranging from 20 to 50 nm. In contrast, the PSD in inhibitory synapses assumes a thin sheet-like structure ∼12 nm from the postsynaptic membrane. On the presynaptic side, spherical synaptic vesicles (SVs) of 25-60 nm diameter and discus-shaped ellipsoidal SVs of various sizes coexist in both synaptic types, with more ellipsoidal ones in inhibitory synapses. High-resolution tomograms obtained using a Volta phase plate and electron filtering and counting reveal glutamate receptor-like and GABAA receptor-like structures that interact with putative scaffolding and adhesion molecules, reflecting details of receptor anchoring and PSD organization. These results provide an updated view of the ultrastructure of excitatory and inhibitory synapses, and demonstrate the potential of our approach to gain insight into the organizational principles of cellular architecture underlying distinct synaptic functions.SIGNIFICANCE STATEMENT To understand functional properties of neuronal synapses, it is desirable to analyze their structure at molecular resolution. We have developed an integrative approach combining cryo-electron tomography and correlative fluorescence microscopy to visualize 3D ultrastructural features of intact excitatory and inhibitory synapses in their native state. Our approach shows that inhibitory synapses contain uniform thin sheet-like postsynaptic densities (PSDs), while excitatory synapses contain previously known mesh-like PSDs. We discovered "discus-shaped" ellipsoidal synaptic vesicles, and their distributions along with regular spherical vesicles in synaptic types are characterized. High-resolution tomograms further allowed identification of putative neurotransmitter receptors and their heterogeneous interaction with synaptic scaffolding proteins. The specificity and resolution of our approach enables precise in situ analysis of ultrastructural organization underlying distinct synaptic functions.

Corticosterone Signaling and a Lateral Habenula-Ventral Tegmental Area Circuit Modulate Compulsive Self-Injurious Behavior in a Rat Model.

Self-injurious behavior (SIB) is commonly observed in patients with neuropsychiatric disorders, as well as in nonclinical populations with stress-related mental-health problems. However, the exact circuitry mechanisms underlying SIB have remained poorly understood. Here, with bilateral injection of muscimol into the entopeduncular nucleus (EP), we established a rat model of SIB. Following the muscimol injection, the male rats exhibited in a dose-dependent manner stereotypic self-biting behavior that lasted for hours and often resulted in wounds of various severities. The SIB was associated with an elevated level of serum corticosterone and could be exacerbated by enhancing the corticosterone signaling and, conversely, alleviated by inhibiting the corticosterone signaling. Activity mapping using c-fos immunostaining, combined with connectivity mapping using herpes simplex virus-based anterograde tracing from the EP and pseudorabies virus-based retrograde tracing from the masseter muscle, revealed the potential involvement of many brain areas in SIB. In particular, the lateral habenula (LHb) and the ventral tegmental area (VTA), the two connected brain areas involved in stress response and reward processing, showed a significant increase in neuronal activation during SIB. Furthermore, suppressing the LHb activity or modulating the GABAergic transmission in the VTA could significantly reduce the occurrence of SIB. These results demonstrate the importance of stress hormone signaling and the LHb-VTA circuit in modulating SIB resulting from EP malfunction, and suggest potential targets for therapeutic intervention of SIB and related disorders.SIGNIFICANCE STATEMENT Self-injurious behavior (SIB) occurs in ∼4% of the general population, with substantially higher occurrence among adolescents and patients of neuropsychiatric disorders. Stress has been linked to the occurrence of SIB, yet the underlying mechanisms have remained unclear. Using a rat model of SIB induced by disruption of activity in the entopeduncular nucleus (EP), we found that the behavior is regulated by stress and linked to corticosterone signaling. Viral tracing and c-fos immunostaining revealed the involvement of various subcortical areas, especially the EP-lateral habenula (LHb)-ventral tegmental area (VTA) circuit, in SIB. Furthermore, regulating activity in the LHb or the VTA alleviates SIB. These results may have implications in the development of new strategies for treating SIB.

Electrically Controlled Neurochemical Release from Dual-Layer Conducting Polymer Films for Precise Modulation of Neural Network Activity in Rat Barrel Cortex.

Implantable microelectrode arrays (MEAs) are important tools for investigating functional neural circuits and treating neurological diseases. Precise modulation of neural activity may be achieved by controlled delivery of neurochemicals directly from coatings on MEA electrode sites. In this study, a novel dual-layer conductive polymer/acid functionalized carbon nanotube (fCNT) microelectrode coating is developed to better facilitate the loading and controlled delivery of the neurochemical 6,7-dinitroquinoxaline-2,3-dione (DNQX). The base layer coating is consisted of poly(3,4-ethylenedioxythiophene/fCNT and the top layer is consisted of polypyrrole/fCNT/DNQX. The dual-layer coating is capable of both loading and electrically releasing DNQX and the release dynamic is characterized with fluorescence microscopy and mathematical modeling. In vivo DNQX release is demonstrated in rat somatosensory cortex. Sensory-evoked neural activity is immediately (<1s) and locally (<446 µm) suppressed by electrically triggered DNQX release. Furthermore, a single DNQX-loaded, dual-layer coating is capable of inducing effective neural inhibition for at least 26 times without observable degradation in efficacy. Incorporation of the novel drug releasing coating onto individual MEA electrodes offers many advantages over alternative methods by increasing spatial-temporal precision and improving drug selection flexibility without increasing the device's size.

A pUL25 dimer interfaces the pseudorabies virus capsid and tegument.

Inside the virions of α-herpesviruses, tegument protein pUL25 anchors the tegument to capsid vertices through direct interactions with tegument proteins pUL17 and pUL36. In addition to promoting virion assembly, both pUL25 and pUL36 are critical for intracellular microtubule-dependent capsid transport. Despite these essential roles during infection, the stoichiometry and precise organization of pUL25 and pUL36 on the capsid surface remain controversial due to the insufficient resolution of existing reconstructions from cryo-electron microscopy (cryoEM). Here, we report a three-dimensional (3D) icosahedral reconstruction of pseudorabies virus (PRV), a varicellovirus of the α-herpesvirinae subfamily, obtained by electron-counting cryoEM at 4.9 Å resolution. Our reconstruction resolves a dimer of pUL25 forming a capsid-associated tegument complex with pUL36 and pUL17 through a coiled coil helix bundle, thus correcting previous misinterpretations. A comparison between reconstructions of PRV and the γ-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) reinforces their similar architectures and establishes important subfamily differences in the capsid-tegument interface.

Chemical Synthesis of K34-Ubiquitylated H2B for Nucleosome Reconstitution and Single-Particle Cryo-Electron Microscopy Structural Analysis.

Post-translational modifications (e.g., ubiquitylation) of histones play important roles in dynamic regulation of chromatin. Histone ubiquitylation has been speculated to directly influence the structure and dynamics of nucleosomes. However, structural information for ubiquitylated nucleosomes is still lacking. Here we report an alternative strategy for total chemical synthesis of homogenous histone H2B-K34-ubiquitylation (H2B-K34Ub) by using acid-cleavable auxiliary-mediated ligation of peptide hydrazides for site-specific ubiquitylation. Synthetic H2B-K34Ub was efficiently incorporated into nucleosomes and further used for single-particle cryo-electron microscopy (cryo-EM) imaging. The cryo-EM structure of the nucleosome containing H2B-K34Ub suggests that two flexible ubiquitin domains protrude between the DNA chains of the nucleosomes. The DNA chains around the H2B-K34 sites shift and provide more space for ubiquitin to protrude. These analyses indicated local and slight structural influences on the nucleosome with ubiquitylation at the H2B-K34 site.

Direct detection of optogenetically evoked oscillatory neuronal electrical activity in rats using SLOE sequence.

The direct detection of neuronal electrical activity is one of the most challenging goals in non-BOLD fMRI research. Previous work has demonstrated its feasibility in phantom and cell culture studies, but attempts in in vivo studies remain few and far between. Most recent in vivo studies used T2*-weighted sequences to directly detect neuronal electrical activity evoked by sensory stimulus. As neuronal electrical signal is usually comprised of a series of spectrally distributed oscillatory waveforms rather than being a direct current, it is most likely to be detected using oscillatory current sensitive sequences. In this study, we explored the potential of using the spin-lock oscillatory excitation (SLOE) sequence with spiral readout to directly detect optogenetically evoked oscillatory neuronal electrical activity, whose main spectral component can be manipulated artificially to match the resonance frequency of spin-lock RF field. In addition, experiments using the stimulus-induced rotary saturation (SIRS) sequence with spiral readout were also performed. Electrophysiological recording and MRI data acquisition were conducted on separate animals. Robust optogenetically evoked oscillatory LFP signals were observed and significant BOLD signals were acquired with the GE-EPI sequence before and after the whole SLOE and SIRS acquisitions, but no significant neuronal current MRI (ncMRI) signal changes were detected. These results indicate that the sensitivity of oscillatory current sensitive sequences needs to be further improved for direct detection of neuronal electrical activity.

Small quinolinium-based enzymatic probes via blue-to-red ratiometric fluorescence.

A small fluorescence ratiometric probe consisting of a single dye species, N-methyl-6-hydroxyquinolinium (MHQ), and coupled enzymatic substrates, exhibits a dramatic colour change (deep blue to red) and possesses a huge response ratio (over 2000 fold) upon specific recognition of target enzymes. Such dramatic responses are attributed to the excited-state proton transfer processes of MHQ molecules in water. Here the detection of ß-galactosidase and porcine pancreatic lipase is successfully demonstrated and this class of molecules has the potential to be developed as a "naked-eye" probe in vitro.

Versatile Room-Temperature-Phosphorescent Materials Prepared from N-Substituted Naphthalimides: Emission Enhancement and Chemical Conjugation.

Purely organic materials with room-temperature phosphorescence (RTP) are currently under intense investigation because of their potential applications in sensing, imaging, and displaying. Inspired by certain organometallic systems, where ligand-localized phosphorescence ((3) π-π*) is mediated by ligand-to-metal or metal-to-ligand charge transfer (CT) states, we now show that donor-to-acceptor CT states from the same organic molecule can also mediate π-localized RTP. In the model system of N-substituted naphthalimides (NNIs), the relatively large energy gap between the NNI-localized (1) π-π* and (3) π-π* states of the aromatic ring can be bridged by intramolecular CT states when the NNI is chemically modified with an electron donor. These NNI-based RTP materials can be easily conjugated to both synthetic and natural macromolecules, which can be used for RTP microscopy.