Intact Scalp Research Articles

Advanced deep-tissue imaging and manipulation enabled by biliverdin reductase knockout.

We developed near-infrared (NIR) photoacoustic and fluorescence probes, as well as optogenetic tools from bacteriophytochromes, and enhanced their performance using biliverdin reductase-A knock-out model (Blvra-/-). Blvra-/- elevates endogenous heme-derived biliverdin chromophore for bacteriophytochrome-derived NIR constructs. Consequently, light-controlled transcription with IsPadC-based optogenetic tool improved up to 25-fold compared to wild-type cells, with 100-fold activation in Blvra-/- neurons. In vivo , light-induced insulin production in Blvra-/- reduced blood glucose in diabetes by ∼60%, indicating high potential for optogenetic therapy. Using 3D photoacoustic, ultrasound, and two-photon fluorescence imaging, we overcame depth limitations of recording NIR probes. We achieved simultaneous photoacoustic imaging of DrBphP in neurons and super-resolution ultrasound localization microscopy of blood vessels ∼7 mm deep in the brain, with intact scalp and skull. Two-photon microscopy provided cell-level resolution of miRFP720-expressing neurons ∼2.2 mm deep. Blvra-/- significantly enhances efficacy of biliverdin-dependent NIR systems, making it promising platform for interrogation and manipulation of biological processes.

Bone flap binding and transposition: a method for bone reconstruction in cranial burst fractures and early-stage growing skull fractures.

To introduce a method of cranial bone reconstruction for cranial burst fractures and early-stage growing skull fractures, named bone flap binding and transposition. Cranial burst fractures, severe head injuries predominantly observed in infants, are characterized by widely diastatic skull fractures coupled with acute extracranial cerebral herniation beneath an intact scalp through ruptured dura mater. These injuries can develop into growing skull fractures. This study included two cases to illustrate the procedure, with a particular focus on the bone steps in managing these conditions. The medical history, clinical presentation, surgical procedures, and postoperative follow-up were retrospectively studied. The details of the surgical procedure were described. The method of bone reconstruction, named bone flap binding and transposition, was applied after the lacerated dural repair. Two bone pieces were combined to eliminate the diastatic bone defect and then fixed by an absorbable cranial fixation clip and bound by sutures. The combined bone flap was repositioned into the bone window, completely covering the area of the original dural laceration. Subsequently, the bone defect was transferred to the area of normal dura. The postoperative courses for the two infants were uneventful. Follow-up CT scans revealed new bone formation at the previous bone defect and no progressive growing skull fracture. The major cranial defects had disappeared, leaving only small residual defects at the corners of the skull bone window, which required further recovery and did not affect the solidity of the skull. Bone flap binding and transposition provide a straightforward, cost-effective, and reliable method for cranial bone reconstruction of cranial burst fractures and early-stage growing skull fractures. This method has taken full advantage of the small infant's dura osteogenic potential without the need for artificial or metallic bone repair materials. The effectiveness of the method needs further validation with more cases in the future.

Deep-brain imaging with 3D integrated photoacoustic tomography and ultrasound localization microscopy

Photoacoustic computed tomography (PACT) is a proven technology for imaging hemodynamics in deep brain of small animal models. PACT is inherently compatible with ultrasound (US) imaging, providing complementary contrast mechanisms. While PACT can quantify the brain’s oxygen saturation of hemoglobin (sO2), US imaging can probe the blood flow based on the Doppler effect. Furthermore, by tracking gas-filled microbubbles, ultrasound localization microscopy (ULM) can map the blood flow velocity with sub-diffraction spatial resolution. In this work, we present a 3D deep-brain imaging system that seamlessly integrates PACT and ULM into a single device, 3D-PAULM. Using a low ultrasound frequency of 4 MHz, 3D-PAULM is capable of imaging the whole-brain hemodynamic functions with intact scalp and skull in a totally non-invasive manner. Using 3D-PAULM, we studied the mouse brain functions with ischemic stroke. Multi-spectral PACT, US B-mode imaging, microbubble-enhanced power Doppler (PD), and ULM were performed on the same mouse brain with intrinsic image co-registration. From the multi-modality measurements, we future quantified blood perfusion, sO2, vessel density, and flow velocity of the mouse brain, showing stroke-induced ischemia, hypoxia, and reduced blood flow. We expect that 3D-PAULM can find broad applications in studying deep brain functions on small animal models.

Signatures of migraine aura in high-density-EEG

ObjectiveCortical spreading depolarization is highly conserved among the species. It is easily detectable in direct cortical surface recordings and has been recorded in the cortex of humans with severe neurological disease. It is considered the pathophysiological correlate of human migraine aura, but direct electrophysiological evidence is still missing. As signatures of cortical spreading depolarization have been recognized in scalp EEG, we investigated typical spontaneous migraine aura, using full band high-density EEG (HD-EEG). MethodsIn this prospective study, patients with migraine with aura were investigated during spontaneous migraine aura and interictally. Time compressed HD-EEG were analyzed for the presence of cortical spreading depolarization characterized by (a) slow potential changes below 0.05 Hz, (b) suppression of faster activity from 0.5 Hz − 45 Hz (c) spreading of these changes to neighboring regions during the aura phase. Further, topographical changes in alpha-power spectral density (8–14 Hz) during aura were analyzed. ResultsIn total, 26 HD-EEGs were recorded in patients with migraine with aura, thereof 10 HD-EEGs during aura. Eight HD-EEGs were recorded in the same subject. During aura, no slow potentials were recorded, but alpha-power was significantly decreased in parieto-occipito-temporal location on the hemisphere contralateral to visual aura, lasting into the headache phase. Interictal alpha-power in patients with migraine with aura did not differ significantly from age- and sex-matched healthy controls. ConclusionsUnequivocal signatures of spreading depolarization were not recorded with EEG on the intact scalp in migraine. The decrease in alpha-power contralateral to predominant visual symptoms is consistent with focal depression of spontaneous brain activity as a consequence of cortical spreading depolarization but is not specific thereof. SignificanceCortical spreading depolarization is relevant in migraine, other paroxysmal neurological disorders and neurointensive care.

Acoustic hologram-induced virtual in vivo enhanced waveguide (AH-VIEW).

Optical imaging and phototherapy in deep tissues face notable challenges due to light scattering. We use encoded acoustic holograms to generate three-dimensional acoustic fields within the target medium, enabling instantaneous and robust modulation of the volumetric refractive index, thereby noninvasively controlling the trajectory of light. Through this approach, we achieved a remarkable 24.3% increase in tissue heating rate in vitro photothermal effect tests on porcine skin. In vivo photoacoustic imaging of mouse brain vasculature exhibits an improved signal-to-noise ratio through the intact scalp and skull. These findings demonstrate that our strategy can effectively suppress light scattering in complex biological tissues by inducing low-angle scattering, achieving an effective depth reaching the millimeter scale. The versatility of this strategy extends its potential applications to neuroscience, lithography, and additive manufacturing.

Non-invasive Deep-Brain Imaging with 3D Integrated Photoacoustic Tomography and Ultrasound Localization Microscopy (3D-PAULM).

Photoacoustic computed tomography (PACT) is a proven technology for imaging hemodynamics in deep brain of small animal models. PACT is inherently compatible with ultrasound (US) imaging, providing complementary contrast mechanisms. While PACT can quantify the brain's oxygen saturation of hemoglobin (sO2), US imaging can probe the blood flow based on the Doppler effect. Further, by tracking gas-filled microbubbles, ultrasound localization microscopy (ULM) can map the blood flow velocity with sub-diffraction spatial resolution. In this work, we present a 3D deep-brain imaging system that seamlessly integrates PACT and ULM into a single device, 3D-PAULM. Using a low ultrasound frequency of 4 MHz, 3D-PAULM is capable of imaging the brain hemodynamic functions with intact scalp and skull in a totally non-invasive manner. Using 3D-PAULM, we studied the mouse brain functions with ischemic stroke. Multi-spectral PACT, US B-mode imaging, microbubble-enhanced power Doppler (PD), and ULM were performed on the same mouse brain with intrinsic image co-registration. From the multi-modality measurements, we further quantified blood perfusion, sO2, vessel density, and flow velocity of the mouse brain, showing stroke-induced ischemia, hypoxia, and reduced blood flow. We expect that 3D-PAULM can find broad applications in studying deep brain functions on small animal models.

Tunable and Enhanced NIR-II Luminescence from Heavily Doped Rare-Earth Nanoparticles for In Vivo Bioimaging

The last decade has witnessed the booming development of optical imaging in the second near-infrared (NIR-II, 1000-1700 nm) window for disease screening and image-guided surgical interventions, due to the merits of multi-color observations and high spatio-temporal resolution in deep tissue. Therefore, bright and multispectral NIR-II probes are required and play a key role. Here, we report the synthesis of a set of bright rare-earth based NIR-II downshifting nanoparticles (DSNPs) with hexagonal phase (β phase). As compared with the widely reported DSNPs (β-NaYF4@NaYF4:20Yb/(0.5-2)A@NaYF4; A = Ho, Pr, Tm or Er) previously, we reveal that the concentrations of both sensitizers and activators can be further highly doped, not limited by the concentration quenching effect. Our results demonstrate that the optimized formula in the heavily doped DSNPs (β-NaYF4@NaYbF4:A@NaYF4, A = 20Ho, 3Pr, 4Tm or 10Er) leads to 1.2- to 4.2-folds NIR-II luminescence enhancement. Especially for the heavily Er-doped DSNPs with long-wavelength photons extending to the NIR-IIb window (1500-1700 nm), we can further boost their luminescence through introducing a beneficial cross-relaxation and host matrix with higher phonon energy (cubic phase NaYF4@NaYbF4:10Er/5Ce@NaYF4), leading to a total of ∼11.4-fold enhancement. The resulting biocompatible, bright NIR-II emitting DSNPs enable us to in vivo monitor the cerebral vessels through the intact scalp and skull, as well as two-color dynamic tumor imaging with high spatial resolution. This work suggests the potential of the heavily doped DSNPs for multiplexed imaging in cerebrovascular abnormalities toward the diagnosis and therapy of the cerebral diseases.

Diffuse optical localization imaging for noninvasive deep brain microangiography in the NIR-II window

Fluorescence microscopy is a powerful enabling tool for biological discovery, albeit its effective penetration depth and resolving capacity are limited due to intense light scattering in living tissues. The recently introduced short-wave infrared cameras and contrast agents featuring fluorescence emission in the second near-infrared (NIR-II) window have extended the achievable penetration to about 2 mm. However, the effective spatial resolution progressively deteriorates with depth due to photon diffusion. Here we introduce diffuse optical localization imaging (DOLI) to enable super-resolution deep-tissue fluorescence microscopy beyond the limits imposed by light diffusion. The method is based on localization of flowing microdroplets encapsulating lead sulfide (PbS)-based quantum dots in a sequence of epi-fluorescence images acquired in the NIR-II spectral window. Experiments performed in tissue mimicking phantoms indicate that high-resolution detection of fluorescent particles can be preserved over 4 mm depth range, while in vivo microangiography of murine cerebral vasculature can be accomplished through intact scalp and skull. The method further enables retrieving depth information from planar fluorescence image recordings by exploiting the localized spot size. DOLI operates in a resolution-depth regime previously inaccessible with optical methods, thus massively enhancing the applicability of fluorescence-based imaging techniques.

Behavioral and Histopathological Impairments Caused by Topical Exposure of the Rat Brain to Mild-Impulse Laser-Induced Shock Waves: Impulse Dependency.

Although an enormous number of animal studies on blast-induced traumatic brain injury (bTBI) have been conducted, there still remain many uncertain issues in its neuropathology and mechanisms. This is partially due to the complex and hence difficult experimental environment settings, e.g., to minimize the effects of blast winds (tertiary mechanism) and to separate the effects of brain exposure and torso exposure. Since a laser-induced shock wave (LISW) is free from dynamic pressure and its energy is spatially well confined, the effects of pure shock wave exposure (primary mechanism) solely on the brain can be examined by using an LISW. In this study, we applied a set of four LISWs in the impulse range of 15–71 Pa·s to the rat brain through the intact scalp and skull; the interval between each exposure was ~5 s. For the rats, we conducted locomotor activity, elevated plus maze and forced swimming tests. Axonal injury in the brain was also examined by histological analysis using Bodian silver staining. Only the rats with exposure at higher impulses of 54 and 71 Pa·s showed significantly lower spontaneous movements at 1 and 2 days post-exposure by the locomotor activity test, but after 3 days post-exposure, they had recovered. At 7 days post-exposure, however, these rats (54 and 71 Pa·s) showed significantly higher levels of anxiety-related and depression-like behaviors by the elevated plus maze test and forced swimming test, respectively. To the best of the authors' knowledge, there have been few studies in which a rat model showed both anxiety-related and depression-like behaviors caused by blast or shock wave exposure. At that time point (7 days post-exposure), histological analysis showed significant decreases in axonal density in the cingulum bundle and corpus callosum in impulse-dependent manners; axons in the cingulum bundle were found to be more affected by a shock wave. Correlation analysis showed a statistically significant correlation between the depression like-behavior and axonal density reduction in the cingulum bundle. The results demonstrated the dependence of behavior deficits and axonal injury on the shock wave impulse loaded on the brain.

Bioinspired nanoantennas for opsin sensitization in optogenetic applications: a theoretical investigation

Opsins with high sensitivity are desired to reduce dependence on optical fibers and enable deep-brain optogenetic stimulation through the intact scalp and skull, while minimizing brain tissue heating and the associated biasing of neural activity. While optimized opsin engineering has produced ultrasensitive and red-shifted opsins suitable for transcranial optogenetic stimulation, further improvements in sensitivity are throttled by biological limitations. Nanostructures are capable of generating near-field intensity enhancements of over 104, but thus far nanomaterials have not been applied to amplify local light intensity for optogenetic applications. In this manuscript, we propose the use of bowtie nanoantennas for local enhancement of 470 nm light to sensitize channelrhodopsin (ChR2) to low light intensities. We begin with a comparison of the near-field intensity enhancement offered by different metals at 470 nm, before selecting aluminum as the optimal material. Next, we tune the geometric parameters of aluminum bowtie nanoantennas to maximize the intensity enhancement at 470 nm. We further optimize enhancement by constructing bowtie nanoantenna arrays inspired by patterns occurring in biology, obtaining intensity enhancements up to a factor of 5000. Monte Carlo simulations suggest that transcranial 470 nm illumination of only 50 mW mm−2 is capable of stimulating bowtie-sensitized ChR2 in the deep brain (∼5 mm) in mice, enabling minimally invasive deep-brain stimulation with opsins found in the traditional optogenetic toolbox. This computation-guided optical antenna engineering approach opens opportunities for designing multifunctional materials for enhancing the efficiency of optogenetic neuromodulation, optical neural activity imaging, and highly localized electrical microstimulation in the brain.

Implementation of a six-around-one optical probe based on diffuse light spectroscopy for study of cerebral properties in a murine mouse model of autism spectrum disorder.

Light reflectance spectroscopy (LRS) is a multispectral technique, sensitive to the absorption and scattering properties of biological molecules in tissues. It is used as a noninvasive tool to extract quantitative physiological information from human tissues and organs. A near-infrared LRS based on a single optical probe was used to monitor changes in optical and hemodynamic parameters in a mouse model of autism. A murine model of autism induced by developmental exposure to valproic acid (VPA) was used. Since autism could be attributed to neuroanatomical changes, we hypothesize that these changes can be detected using the LRS because spectral properties depend on both molecular composition and structural changes. The fiber-optic probe in the setup consisted of seven small optical fibers: six fibers for illumination placed in a circular manner around a central single collection fiber. Overall, measurements demonstrate changes in diffused reflectance spectra, cerebral optical tissue properties (absorption and scattering), and chromophore levels. Furthermore, we were able to identify differences between male and female groups. Finally, the effectiveness of S-Adenosylmethionine as a drug therapy was studied and found to improve the hemodynamic outcome. For the first time, to the best of our knowledge, the LRS is utilized to study variations in brain parameters in the VPA autism model mice through an intact scalp.

Constitutional Isomerization Enables Bright NIR‐II AIEgen for Brain‐Inflammation Imaging

AbstractThe shortage of high quantum yield (QY) organic fluorophores in the second near‐infrared window (NIR‐II) has become a bottleneck in bioimaging field. Now, a simple strategy is proposed to address this: constitutional isomerization on the basis of the molecular design philosophy of aggregation‐induced emission. With the combination of backbone distortion and rotor twisting, the resultant NIR‐II fluorophore 2TT‐oC6B displays an emission peak at 1030 nm and a QY of 11% in nanoparticles, one of the highest reported so far. Control molecules confirm that the distorted backbone and twisted rotors play equally important roles in determining the fluorescence properties of the NIR‐II fluorophores. To allow for the targeting ability to reach deeply located diseases, neutrophils (NEs) are used to penetrate the brain tissues and accumulate in the inflammation site. Herein, it is shown that NEs carrying 2TT‐oC6B nanoparticles can penetrate the blood‐brain‐barrier and visualize the deeply located inflammation through an intact scalp and skull. Notably, the bright 2TT‐oC6B contributes to a significantly enhanced signal‐to‐background ratio of 30.6 in the brain inflammation site.

Sono-optogenetics facilitated by a circulation-delivered rechargeable light source for minimally invasive optogenetics.

Optogenetics, which uses visible light to control the cells genetically modified with light-gated ion channels, is a powerful tool for precise deconstruction of neural circuitry with neuron-subtype specificity. However, due to limited tissue penetration of visible light, invasive craniotomy and intracranial implantation of tethered optical fibers are usually required for in vivo optogenetic modulation. Here we report mechanoluminescent nanoparticles that can act as local light sources in the brain when triggered by brain-penetrant focused ultrasound (FUS) through intact scalp and skull. Mechanoluminescent nanoparticles can be delivered into the blood circulation via i.v. injection, recharged by 400-nm photoexcitation light in superficial blood vessels during circulation, and turned on by FUS to emit 470-nm light repetitively in the intact brain for optogenetic stimulation. Unlike the conventional "outside-in" approaches of optogenetics with fiber implantation, our method provides an "inside-out" approach to deliver nanoscopic light emitters via the intrinsic circulatory system and switch them on and off at any time and location of interest in the brain without extravasation through a minimally invasive ultrasound interface.

Transcranial Recording of Electrophysiological Neural Activity in the Rodent Brain in vivo Using Functional Photoacoustic Imaging of Near-Infrared Voltage-Sensitive Dye.

Minimally-invasive monitoring of electrophysiological neural activities in real-time—that enables quantification of neural functions without a need for invasive craniotomy and the longer time constants of fMRI and PET—presents a very challenging yet significant task for neuroimaging. In this paper, we present in vivo functional PA (fPA) imaging of chemoconvulsant rat seizure model with intact scalp using a fluorescence quenching-based cyanine voltage-sensitive dye (VSD) characterized by a lipid vesicle model mimicking different levels of membrane potential variation. The framework also involves use of a near-infrared VSD delivered through the blood-brain barrier (BBB), opened by pharmacological modulation of adenosine receptor signaling. Our normalized time-frequency analysis presented in vivo VSD response in the seizure group significantly distinguishable from those of the control groups at sub-mm spatial resolution. Electroencephalogram (EEG) recording confirmed the changes of severity and frequency of brain activities, induced by chemoconvulsant seizures of the rat brain. The findings demonstrate that the near-infrared fPA VSD imaging is a promising tool for in vivo recording of brain activities through intact scalp, which would pave a way to its future translation in real time human brain imaging.

A differential role for human hippocampus in novelty and contextual processing: Implications for P300.

The role of the hippocampus in P300 has long been debated. Here, we present a theoretical framework that elucidates hippocampal contributions to scalp P300 based on intracranial and lesion research combined with emerging evidence on the role of the hippocampus in rapid statistical learning, memory, and novelty processing. The P300 has been divided in two subcomponents: a fronto-central P3a related to novelty and distractor processing, and a parietal P3b related to target detection. Interest in a role for hippocampus in scalp P300 was sparked by P3-like ERPs measured intracranially in human hippocampus. Subsequent medial temporal lobe lesion studies show intact scalp P3b, indicating that the hippocampus is not critical for P3b. This contrasts with the scalp P3a, which was significantly diminished in human patients with lesions in the posterior hippocampus. This suggests a differential role for hippocampus in P3a and P3b. Our framework purports that the hippocampus plays a central role in distractor processing that leads to P3a generation in cortical regions. We also propose that the hippocampus is involved at the end of the cognitive episode for both P3a and P3b implementing contextual updating. P3-like ERPs measured in hippocampus may reflect input signals from cortical regions implementing updates based on the outcome of cognitive processes underlying scalp P3, enabling a model update of the environment facilitated by the hippocampus. Overall, this framework proposes an active role for the hippocampus in novelty processing leading up to P3a generation, followed by contextual updating of the outcome of both scalp P3a and P3b.

Noninvasive noncontact speckle contrast diffuse correlation tomography of cerebral blood flow in rats

Continuous and longitudinal imaging of cerebral blood flow (CBF) variations provide vital information to investigate pathophysiology and interventions for a variety of neurological and cerebral diseases. An innovative noncontact speckle contrast diffuse correlation tomography (scDCT) system was downscaled and adapted for noninvasive imaging of CBF distributions in rat brain through intact scalp and skull. Algorithms for 2D mapping and 3D image reconstruction of CBF distributions were developed and optimized. The continuous imaging capability of the system was shown by imaging global CBF increases during CO2 inhalations and regional CBF decreases across two hemispheres during sequential unilateral and bilateral common carotid artery ligations. The longitudinal imaging capability was demonstrated by imaging CBF variations over a long recovery period of 14 days after an acute stroke. Compared to the 2D mapping method, the 3D imaging method reduces partial volume effects, but needs more computation time for image reconstruction. Results from this study generally agree with those reported in the literature using similar protocols to induce CBF changes in rats. The scDCT enables a relatively large penetration depth (up to ∼10 mm), which is sufficient for transcranial brain measurements in small animals and human neonates. Ultimately, we expect to provide a noninvasive noncontact cerebral imager for basic neuroscience research in small animal models and clinical applications in human neonates.

Repetitive transcranial magnetic stimulation protects mice against 6-OHDA-induced Parkinson's disease symptoms by regulating brain amyloid β1-42 level.

Repetitive transcranial magnetic stimulation (rTMS) is a technique protecting neurons against diverse neurodegenerative disorders by delivering magnetic stimuli into the brain through the intact scalp. In the current study, the protection effect of rTMS on Parkinson's disease (PD) and the associated mechanism driving the treatment were explored. The PD symptoms were induced using 6-OHDA in mice, and the effect of rTMS of two frequencies (1Hz and 10Hz) on the cognitive behaviors and neuron viability was detected. Afterwards, the level of Aβ1-42 and activity of MKK7-ERK-Fos-APP axis under the administration of rTMS were recorded as well. The intracranial injection of 6-OHDA impaired the cognitive behaviors of the mice in the test of Morris water maze as well as reducing the viability and number of neurons in PD mice. After the treatment of rTMS of both frequencies, the cognitive function of mice was improved and the neuron viability and number were restored in mice brain tissues. The administration of rTMS also increased the cerebrospinal fluid (CSF) level of Aβ1-42 in PD mice, which was accompanied by the suppressed levels of p-MKK7, p-ERK1/2, p-c-Fos, and APP. Moreover, the effect of rTMS on mice nerve system was all exerted in a frequency-dependent manner. In conclusion, the findings outlined in the current study affirmed the protection effect of rTMS against PD. The anti-PD function of rTMS was associated with the suppression of MKK7-ERK-Fos-APP axis, which subsequently resulted in the increased CSF Aβ1-42 level and decreased brain Aβ1-42 level.

Near infrared imaging of intrinsic signals in cortical spreading depression observed through the intact scalp in hairless mice

Brain cooling was inevitable in both thinned and intact skull windows in neuroimaging in vivo of mice. Thus we proposed the novel imaging method leaving intact scalp on the skull using the light at 670, 785, and 975 nm. In this study, we used hairless mice (Hos:HR-1) since the deterioration of image quality was resulted from the hair. Cortical spreading depression was induced by KCl application through small incision and burr hole on the frontal bone. Intrinsic optical signals through the intact scalp in the observation area were detected. Time course of the signal showed a triphasic feature which was consistent with the intrinsic optical signals through the intact skull. In three pairs of signal amplitudes at the different wavelengths, no significant differences were observed. Although the intact scalp weakened the amplitudes significantly, e.g., 4.0 from 6.9 at 975 nm, the signals during cortical spreading depression were sufficient to be detected.

Optimal focus evaluated using Monte Carlo simulation in non-invasive neuroimaging in the second near-infrared window

Adjusting the focal plane through the intact scalp of mice is crucial in novel angiography of cerebral vasculature using quantum dots emitting second near-infrared light at a wavelength of 1100 nm. Reagents were administered through the caudal vein. When we focused 0.4 mm below the scalp surface, based on the anatomical properties of mice reported previously, the intensity of clear fluorescence images observed transiently under a microscope became very weak within several seconds. The remaining time was extremely short to repeat adjustment of the focal plane. To investigate focus, photons exciting quantum dots at depths of 0.4, 0.8, 1.4, and 2.0 mm and emission photons were tracked in a four-layered Monte Carlo model including the scalp, skull, cerebrospinal fluid, and cortex. Based on the most near-ballistic photons emitted from quantum dots at 0.4 mm depth and specification of the microscope used, including numerical aperture and depth of field, the optimal focus plane was set.•Novel angiography for cerebrovascular structures was proposed using quantum dots with second near-infrared fluorescence.•Anatomical properties reported previously allowed focusing 0.4 mm below the surface of intact scalp before observation under fluorescence.•Clear images of cerebrovascular structures were attributed to many near-ballistic photons emitted from quantum dots at 0.4 mm depth.

Bright Aggregation-Induced-Emission Dots for Targeted Synergetic NIR-II Fluorescence and NIR-I Photoacoustic Imaging of Orthotopic Brain Tumors.

Precise diagnostics are of significant importance to the optimal treatment outcomes of patients bearing brain tumors. NIR-II fluorescence imaging holds great promise for brain-tumor diagnostics with deep penetration and high sensitivity. This requires the development of organic NIR-II fluorescent agents with high quantum yield (QY), which is difficult to achieve. Herein, the design and synthesis of a new NIR-II fluorescent molecule with aggregation-induced-emission (AIE) characteristics is reported for orthotopic brain-tumor imaging. Encapsulation of the molecule in a polymer matrix yields AIE dots showing a very high QY of 6.2% with a large absorptivity of 10.2 L g-1 cm-1 at 740 nm and an emission maximum near 1000 nm. Further decoration of the AIE dots with c-RGD yields targeted AIE dots, which afford specific and selective tumor uptake, with a high signal/background ratio of 4.4 and resolution up to 38 µm. The large NIR absorptivity of the AIE dots facilitates NIR-I photoacoustic imaging with intrinsically deeper penetration than NIR-II fluorescence imaging and, more importantly, precise tumor-depth detection through intact scalp and skull. This research demonstrates the promise of NIR-II AIE molecules and their dots in dual NIR-II fluorescence and NIR-I photoacoustic imaging for precise brain cancer diagnostics.