Two-photon imaging of excitatory and inhibitory neural response to infrared neural stimulation.

Significance: Pulsed infrared neural stimulation (INS, 1875 nm) is an emerging neurostimulation technology that delivers focal pulsed heat to activate functionally specific mesoscale networks and holds promise for clinical application. However, little is known about its effect on excitatory and inhibitory cell types in cerebral cortex. Aim: Estimates of summed population neuronal response time courses provide a potential basis for neural and hemodynamic signals described in other studies. Approach: Using two-photon calcium imaging in mouse somatosensory cortex, we have examined the effect of INS pulse train application on hSyn neurons and mDlx neurons tagged with GCaMP6s. Results: We find that, in anesthetized mice, each INS pulse train reliably induces robust response in hSyn neurons exhibiting positive going responses. Surprisingly, mDlx neurons exhibit negative going responses. Quantification using the index of correlation illustrates responses are reproducible, intensity-dependent, and focal. Also, a contralateral activation is observed when INS applied. Conclusions: In sum, the population of neurons stimulated by INS includes both hSyn and mDlx neurons; within a range of stimulation intensities, this leads to overall excitation in the stimulated population, leading to the previously observed activations at distant post-synaptic sites.

Enhancing Total Optical Throughput of Microscopy with Deep Learning for Intravital Observation.

The significance of performing large-depth dynamic microscopic imaging in vivo for life science research cannot be overstated. However, the optical throughput of the microscope limits the available information per unit of time, i.e., it is difficult to obtain both high spatial and temporal resolution at once. Here, a method is proposed to construct a kind of intravital microscopy with high optical throughput, by making near-infrared-II (NIR-II, 900-1880 nm) wide-field fluorescence microscopy learn from two-photon fluorescence microscopy based on a scale-recurrent network. Using this upgraded NIR-II fluorescence microscope, vessels in the opaque brain of a rodent are reconstructed three-dimensionally. Five-fold axial and thirteen-fold lateral resolution improvements are achieved without sacrificing temporal resolution and light utilization. Also, tiny cerebral vessel dilatations in early acute respiratory failure mice are observed, with this high optical throughput NIR-II microscope at an imaging speed of 30 fps.

Single-microvessel occlusion produces lamina-specific microvascular flow vasodynamics and signs of neurodegenerative change.

Recent studies have highlighted the importance of understanding the architecture and function of microvasculature, and dysfunction of these microvessels may underlie neurodegenerative disease. Here, we utilize a high-precision ultrafast laser-induced photothrombosis (PLP) method to occlude single capillaries and then quantitatively study the effects on vasodynamics and surrounding neurons. Analysis of the microvascular architecture and hemodynamics after single-capillary occlusion reveals distinct changes upstream vs. downstream branches, which shows rapid regional flow redistribution and local downstream blood-brain barrier (BBB) leakage. Focal ischemia via capillary occlusions surrounding labeled target neurons induces dramatic and rapid lamina-specific changes in neuronal dendritic architecture. Further, we find that micro-occlusion at two different depths within the same vascular arbor results in distinct effects on flow profiles in layers 2/3 vs layer 4. The current results reveal laminar-scale regulation distinctions in microinfarct response and raise the possibility that relatively greater impacts on microvascular function contribute to cognitive decline in neurodegenerative disease.

Precision 1070 nm Ultrafast Laser-Induced Photothrombosis of Depth-Targeted Vessels In Vivo.

The cerebrovasculature plays an essential role in neurovascular and homeostatic functions in health and disease conditions. Many efforts have been made for developing vascular thrombosis methods to study vascular dysfunction in vivo, while technical challenges remain, such as accuracy and depth-selectivity to target a single vessel in the cerebral cortex. Herein, this paper first demonstrates the evaluation and quantification of the feasibility and effects of Rose Bengal (RB)-induced photothrombosis with 720-1070 nm ultrafast lasers in a raster scan. A flexible and reproducible approach is then proposed to employ a 1070 nm ultrafast laser with a spiral scan for producing RB-induced occlusion, which is described as precision ultrafast laser-induced photothrombosis (PLP). Combine with two-photon microscopy imaging, this PLP displays highly precise and fast occlusion induction of various vessel types, sizes, and depths, which enhances the precision and power of the photothrombosis protocol. Overall, the PLP method provides a real-time, practical, precise, and depth-selected single-vessel photothrombosis technology in the cerebral cortex with commercially available optical equipment, which is crucial for exploring brain vascular function with high spatial-temporal resolution in the brain.

Large-depth three-photon fluorescence microscopy imaging of cortical microvasculature on nonhuman primates with bright AIE probe In vivo.

Multiphoton microscopy has been a powerful tool in brain research, three-photon fluorescence microscopy is increasingly becoming an emerging technique for neurological research of the cortex in depth. Nonhuman primates play important roles in the study of brain science because of their neural and vascular similarity to humans. However, there are few research results of three-photon fluorescence microscopy on the brain of nonhuman primates due to the lack of optimized imaging systems and excellent fluorescent probes. Here we introduced a bright aggregation-induced emission (AIE) probe with excellent three-photon fluorescence efficiency as well as facile synthesis process and we validated its biocompatibility in the macaque monkey. We achieved a large-depth vascular imaging of approximately 1 mm in the cerebral cortex of macaque monkey with our lab-modified three-photon fluorescence microscopy system and the AIE probe. Functional measurement of blood velocity in deep cortex capillaries was also performed. Furthermore, the comparison of cortical deep vascular structure parameters across species was presented on the monkey and mouse cortex. This work is the first in vivo three-photon fluorescence microscopic imaging research on the macaque monkey cortex reaching the imaging depth of ∼1 mm with the bright AIE probe. The results demonstrate the potential of three-photon microscopy as primate-compatible method for imaging fine vascular networks and will advance our understanding of vascular function in normal and disease in humans.

Imaging the Deep Spinal Cord Microvascular Structure and Function with High-Speed NIR-II Fluorescence Microscopy.

The spinal cord (SC) is crucial for a myriad of somatosensory, autonomic signal processing, and transductions. Understanding the SC vascular structure and function thus plays an integral part in neuroscience and clinical research. However, the dense layers of myelinated ascending axons on the dorsal side inconveniently grant the SC tissue with high optical scattering property, which significantly hinders the imaging depth of the SC vasculature in vivo. Commonly used antiscattering techniques such as multiphoton fluorescence microscopy have low imaging speed and cannot capture the rapid vascular particle flow without significant motion blur. Here, advantage of the high penetration of near-infrared (NIR)-II fluorescence is taken to demonstrate a deep SC vascular structural image stack up to 350 µm, comparable to two-photon microscopy. Furthermore, the red blood cells are labelled with the clinically approved NIR dye indocyanine. The combination of a fast NIR camera and indocyanine green-red blood cells (RBCs) makes it possible to attain high-speed 100 frame-per-second NIR-II imaging to identify the corresponding changes in RBC velocity during the external hind leg stimulus. For the first time, it is established that the NIR-II region would be a promising spectral window for SC imaging. NIR-II fluorescence microscopy has excellent potential for clinical and basic science research on SC.

Intra- and Intermolecular Synergistic Engineering of Aggregation-Induced Emission Luminogens to Boost Three-Photon Absorption for Through-Skull Brain Imaging.

Three-photon fluorescence microscopic (3PFM) bioimaging is a promising imaging technique for visualizing the brain in its native environment thanks to its advantages of high spatial resolution and large imaging depth. However, developing fluorophores with strong three-photon absorption (3PA) and bright emission that meets the requirements for efficient three-photon fluorescence microscopic (3PFM) bioimaging is still challenging. Herein, four bright fluorophores with aggregation-induced emission features are facilely synthesized, and their powders exhibit high quantum yields of up to 56.4%. The intramolecular engineering of luminogens endows (E)-2-(benzo[d]thiazol-2-yl)-3-(7-(diphenylamino)-9-ethyl-9H-carbazol-2-yl)acrylonitrile (DCBT) molecules with bright near-infrared emission and large 3PA cross sections of up to 1.57 × 10-78 cm6 s2 photon-2 at 1550 nm, which is boosted by 3.6-fold to 5.61 × 10-78 cm6 s2 photon-2 in DCBT dots benefiting from the extensive intermolecular interactions in molecular stacking. DCBT dots are successfully applied for 3PFM imaging of brain vasculature on mice with a removed or intact skull, providing images with high spatial resolution, and even small capillaries can be recognized below the skull. This study will inspire more insights for developing advanced multiphoton absorbing materials for biomedical applications.

Subdural neural interfaces for long-term electrical recording, optical microscopy and magnetic resonance imaging.

Though commonly used, metal electrodes are incompatible with brain tissues, often leading to injury and failure to achieve long-term implantation. Here we report a subdural neural interface of hydrogel functioning as an ionic conductor, and elastomer as a dielectric. We demonstrate that it incurs a far less glial reaction and less cerebrovascular destruction than a metal electrode. Using a cat model, the hydrogel electrode was able to record electrical signals comparably in quality to a metal electrode. The hydrogel-elastomer neural interface also readily facilitated multimodal functions. Both the hydrogel and elastomer are transparent, enabling in vivo optical microscopy. For imaging, cerebral vessels and calcium signals were imaged using two-photon microscopy. The new electrode is compatible with magnetic resonance imaging and does not cause artifact images. Such a new multimodal neural interface could represent immediate opportunity for use in broad areas of application in neuroscience research and clinical neurology.

ACQ-to-AIE Transformation: Tuning Molecular Packing by Regioisomerization for Two-Photon NIR Bioimaging.

The traditional design strategies for highly bright solid-state luminescent materials rely on weakening the intermolecular π-π interactions, which may limit diversity when developing new materials. Herein, we propose a strategy of tuning the molecular packing mode by regioisomerization to regulate the solid-state fluorescence. TBP-e-TPA with a molecular rotor in the end position of a planar core adopts a long-range cofacial packing mode, which in the solid state is almost non-emissive. By shifting molecular rotors to the bay position, the resultant TBP-b-TPA possesses a discrete cross packing mode, giving a quantum yield of 15.6±0.2 %. These results demonstrate the relationship between the solid-state fluorescence efficiency and the molecule's packing mode. Thanks to the good photophysical properties, TBP-b-TPA nanoparticles were used for two-photon deep brain imaging. This molecular design philosophy provides a new way of designing highly bright solid-state fluorophores.

NIR-II fluorescence in vivo confocal microscopy with aggregation-induced emission dots.

Significantly reduced tissue scattering of fluorescence signals in the second near-infrared (NIR-II, 1,000-1,700 nm) spectral region offers opportunities for large-depth in vivo bioimaging. Nowadays, most reported works concerning NIR-II fluorescence in vivo bioimaging are realized by wide-field illumination and 2D-arrayed detection (e.g., via InGaAs camera), which has high temporal resolution but limited spatial resolution due to out-of-focus signals. Combining NIR-II fluorescence imaging with confocal microscopy is a good approach to achieve high-spatial resolution visualization of biosamples even at deep tissues. In this presented work, a NIR-II fluorescence confocal microscopic system was setup. By using a kind of aggregation-induced emission (AIE) dots as NIR-II fluorescent probes, 800 µm-deep 3D in vivo cerebrovascular imaging of a mouse was obtained, and the spatial resolution at 700 µm depth could reach 8.78 µm. Moreover, the time-correlated single photon counting (TCSPC) technique and femtosecond laser excitation were introduced into NIR-II fluorescence confocal microscopy, and in vivo confocal NIR-II fluorescence lifetime microscopic imaging (FLIM) of mouse cerebral vasculature was successfully realized.

Single-Molecular Near-Infrared-II Theranostic Systems: Ultrastable Aggregation-Induced Emission Nanoparticles for Long-Term Tracing and Efficient Photothermal Therapy.

Second near-infrared (NIR-II, 1000-1700 nm) fluorescence bioimaging has attracted tremendous scientific interest and already been used in many biomedical studies. However, reports on organic NIR-II fluorescent probes for in vivo photoinduced imaging and simultaneous therapy, as well as the long-term tracing of specific biological objects, are still very rare. Herein we designed a single-molecular and NIR-II-emissive theranostic system by encapsulating a kind of aggregation-induced emission luminogen (AIEgen, named BPN-BBTD) with amphiphilic polymer. The ultra-stable BPN-BBTD nanoparticles were employed for the NIR-II fluorescence imaging and photothermal therapy of bladder tumors in vivo. The 785 nm excitation triggered photothermal therapy could completely eradicate the subcutaneous tumor and inhibit the growth of orthotopic tumors. Furthermore, BPN-BBTD nanoparticles were capable of monitoring subcutaneous and orthotopic tumors for a long time (32 days). Single-molecular and NIR-II-emitted aggregation-induced emission nanoparticles hold potential for the diagnosis, precise treatment, and metastasis monitoring of tumors in the future.

Tunable Aggregation-Induced Emission Nanoparticles by Varying Isolation Groups in Perylene Diimide Derivatives and Application in Three-Photon Fluorescence Bioimaging.

The development of fluorogens with deep-red emission is one of the hottest topics of investigation in the field of bio/chemosensors and bioimaging. Herein, the tunable fluorescence of perylene diimide (PDI) derivatives was achieved by the incorporation of varied isolation groups linked on the PDI core. With the enlarged sizes of isolation groups, the conversion from aggregation caused quenching to aggregation-induced emission was obtained in their fluorescence variations from solutions to nanoparticles, as the result of the efficient inhibition of π-π stacking by the larger isolation groups. Accordingly, DCzPDI bearing 1,3-di(9H-carbazol-9-yl)benzene as the biggest isolation group exhibited the bright deep-red emission in the aggregated state with a quantum yield of 12.3%. Combined with the three-photon excited fluorescence microscopy (3PFM) technology, through-skull 3PFM imaging of mouse cerebral vasculature can be realized by DCzPDI nanoparticles with good biocompatibility, and the penetration depth can be as deep as 450 µm.

Aggregation-Induced Emission Luminogen with Near-Infrared-II Excitation and Near-Infrared-I Emission for Ultradeep Intravital Two-Photon Microscopy.

Currently, a serious problem obstructing the large-scale clinical applications of fluorescence technique is the shallow penetration depth. Two-photon fluorescence microscopic imaging with excitation in the longer-wavelength near-infrared (NIR) region (>1100 nm) and emission in the NIR-I region (650-950 nm) is a good choice to realize deep-tissue and high-resolution imaging. Here, we report ultradeep two-photon fluorescence bioimaging with 1300 nm NIR-II excitation and NIR-I emission (peak ∼810 nm) based on a NIR aggregation-induced emission luminogen (AIEgen). The crab-shaped AIEgen possesses a planar core structure and several twisting phenyl/naphthyl rotators, affording both high fluorescence quantum yield and efficient two-photon activity. The organic AIE dots show high stability, good biocompatibility, and a large two-photon absorption cross section of 1.22 × 103 GM. Under 1300 nm NIR-II excitation, in vivo two-photon fluorescence microscopic imaging helps to reconstruct the 3D vasculature with a high spatial resolution of sub-3.5 µm beyond the white matter (>840 µm) and even to the hippocampus (>960 µm) and visualize small vessels of ∼5 µm as deep as 1065 µm in mouse brain, which is among the largest penetration depths and best spatial resolution of in vivo two-photon imaging. Rational comparison with the AIE dots manifests that two-photon imaging outperforms the one-photon mode for high-resolution deep imaging. This work will inspire more sight and insight into the development of efficient NIR fluorophores for deep-tissue biomedical imaging.

Real-Time and High-Resolution Bioimaging with Bright Aggregation-Induced Emission Dots in Short-Wave Infrared Region.

Fluorescence imaging in the spectral region beyond the conventional near-infrared biological window (700-900 nm) can theoretically afford high resolution and deep tissue penetration. Although some efforts have been devoted to developing a short-wave infrared (SWIR; 900-1700 nm) imaging modality in the past decade, long-wavelength biomedical imaging is still suboptimal owing to the unsatisfactory materials properties of SWIR fluorophores. Taking advantage of organic dots based on an aggregation-induced emission luminogen (AIEgen), herein microscopic vasculature imaging of brain and tumor is reported in living mice in the SWIR spectral region. The long-wavelength emission of AIE dots with certain brightness facilitates resolving brain capillaries with high spatial resolution (≈3 µm) and deep penetration (800 µm). Owning to the deep penetration depth and real-time imaging capability, in vivo SWIR microscopic angiography exhibits superior resolution in monitoring blood-brain barrier damage in mouse brain, and visualizing enhanced permeability and retention effect in tumor sites. Furthermore, the AIE dots show good biocompatibility, and no noticeable abnormalities, inflammations or lesions are observed in the main organs of the mice. This work will inspire new insights on development of advanced SWIR techniques for biomedical imaging.

Biocompatible aggregation-induced emission nanoparticles with red emission for in vivo three-photon brain vascular imaging.

Three-photon luminescence (3PL) imaging with near-infrared (NIR) excitation is quite promising for its deep penetration, high resolution, and good signal-to-noise ratio (SNR). In this report, a type of red emissive fluorophore TPEPT with aggregation-induced emission (AIE) properties was synthesized, and it was found to possess a large three-photon absorption (3PA) cross-section of 6.33 × 10-78 cm6 s2 under 1550 nm femtosecond laser excitation. TPEPT was then encapsulated with mPEG5000-DSPE to form AIE nanoparticles, and the chemical stability, optical properties and toxicity were studied afterwards. TPEPT nanoparticles were then applied for 3PL in vivo vascular imaging of mouse brain under 1550 nm fs laser excitation, and a fine three-dimensional (3D) reconstruction with a depth of 500 µm was achieved.

Stable and Size-Tunable Aggregation-Induced Emission Nanoparticles Encapsulated with Nanographene Oxide and Applications in Three-Photon Fluorescence Bioimaging.

Organic fluorescent dyes with high quantum yield are widely applied in bioimaging and biosensing. However, most of them suffer from a severe effect called aggregation-caused quenching (ACQ), which means that their fluorescence is quenched at high molecular concentrations or in the aggregation state. Aggregation-induced emission (AIE) is a diametrically opposite phenomenon to ACQ, and luminogens with this feature can effectively solve this problem. Graphene oxide has been utilized as a quencher for many fluorescent dyes, based on which biosensing can be achieved. However, using graphene oxide as a surface modification agent of fluorescent nanoparticles is seldom reported. In this article, we used nanographene oxide (NGO) to encapsulate fluorescent nanoparticles, which consisted of a type of AIE dye named TPE-TPA-FN (TTF). NGO significantly improved the stability of nanoparticles in aqueous dispersion. In addition, this method could control the size of nanoparticles' flexibly as well as increase their emission efficiency. We then used the NGO-modified TTF nanoparticles to achieve three-photon fluorescence bioimaging. The architecture of ear blood vessels in mice and the distribution of nanoparticles in zebrafish could be observed clearly. Furthermore, we extended this method to other AIE luminogens and showed it was widely feasible.

High-order non-linear optical effects in organic luminogens with aggregation-induced emission.

2,3-bis(4-(phenyl(4-(1,2,2-triphenylvinyl)phenyl)amino)phenyl)fumaronitrile (TTF) shows unique aggregation-induced emission (AIE) characteristics. Under the excitation of a 1560 nm femtosecond laser, simultaneous three-photon-excited luminescence (3PL) and third-harmonic-generation signals can be observed from its nanoaggregate and the solid state. TTF is further encapsulated with DSPE-mPEG (a type of amphiphilic polymer) to form AIE-active nanoparticles. 3PL brain imaging of mice is achieved based on the nanoparticles.