Imaging Of Mouse Brain Research Articles

Saturated two‐photon excitation fluorescence microscopy for the visualization of cerebral neural networks at millimeters deep depth

Optical imaging is a key modality for observing biological specimen with higher spatial resolution. However, scattering and absorption of light in tissues are inherent barriers in maximizing imaging depth in biological tissues. To achieve this goal, use of light at near-infrared spectrum can improve the present situation. Here, the capability of saturated two-photon saturated excitation (TP-SAX) fluorescence microscopy to image at depths of >2.0 mm, with submicron resolution in transparent mouse brain imaging, is demonstrated. At such depths with scattering-enlarged point spread function (PSF), we find that TP-SAX is capable to provide spatial resolution improvement compared to its corresponding TPFM, which is on the other hand already providing a much improved resolution compared with single-photon confocal fluorescence microscopy. With the capability to further improve spatial resolution at such deep depth with scattering-enlarged PSF, TP-SAX can be used for exquisite visualization of delicate cerebral neural structure in the scattering regime with a submicron spatial resolution inside intact mouse brain.

Enhancing axial resolution and background rejection in line-scanning temporal focusing microscopy by focal modulation.

Compared with two-photon point-scanning microscopy, line-scanning temporal focusing microscopy breaks the limitation on imaging rate and maintains the axial resolution, which makes it promising for various biomedical studies. However, for deep tissue imaging, it suffers from reduced axial resolution and increased background noise due to sample induced wavefront distortion. Here, we propose a spatio-spectral focal modulation technique to enhance axial resolution and background rejection by simply subtracting an aberrated image, which is induced by a spatial light modulator, from an unaberrated image. The proposed technique could improve the axial resolution by a factor of 1.3 in our implementation, verified by both simulations and experiments. Besides, we show that compared with spatial modulation alone, spatio-spectral modulation induces less peak intensity loss caused by image subtraction. We further demonstrate the performance of our technique on the enhanced axial resolution and background rejection by deep imaging of cleared mouse brains and in vivo imaging of living mouse brains.

High-throughput light sheet tomography platform for automated fast imaging of whole mouse brain.

Acquiring information of the neural structures in the whole-brain level is vital for systematically exploring mechanisms and principles of brain function and dysfunction. Most methods for whole brain imaging, while capable of capturing the complete morphology of neurons, usually involve complex sample preparation and several days of image acquisition. The whole process including optical clearing or resin embedding is time consuming for a quick survey of the distribution of specific neural circuits in the whole brain. Here, we develop a high-throughput light-sheet tomography platform (HLTP), which requires minimum sample preparation. This method does not require optical clearing for block face light sheet imaging. After fixation using paraformaldehyde, an aligned 3 dimensional image dataset of a whole mouse brain can be obtained within 5 hours at a voxel size of 1.30 × 1.30 × 0.92 μm. HLTP could be a very efficient tool for quick exploration and visualization of brain-wide distribution of specific neurons or neural circuits.

DeepNeuron: an open deep learning toolbox for neuron tracing

Reconstructing three-dimensional (3D) morphology of neurons is essential for understanding brain structures and functions. Over the past decades, a number of neuron tracing tools including manual, semiautomatic, and fully automatic approaches have been developed to extract and analyze 3D neuronal structures. Nevertheless, most of them were developed based on coding certain rules to extract and connect structural components of a neuron, showing limited performance on complicated neuron morphology. Recently, deep learning outperforms many other machine learning methods in a wide range of image analysis and computer vision tasks. Here we developed a new Open Source toolbox, DeepNeuron, which uses deep learning networks to learn features and rules from data and trace neuron morphology in light microscopy images. DeepNeuron provides a family of modules to solve basic yet challenging problems in neuron tracing. These problems include but not limited to: (1) detecting neuron signal under different image conditions, (2) connecting neuronal signals into tree(s), (3) pruning and refining tree morphology, (4) quantifying the quality of morphology, and (5) classifying dendrites and axons in real time. We have tested DeepNeuron using light microscopy images including bright-field and confocal images of human and mouse brain, on which DeepNeuron demonstrates robustness and accuracy in neuron tracing.

MRI to Assess Neurological Function.

This article describes a detailed set of protocols for mouse brain imaging using MRI. We focus primarily on measuring changes in neuroanatomy, and provide both instructions for mouse preparation and details on image acquisition, image processing, and statistics. Practical details as well as theoretical considerations are provided. © 2018 by John Wiley & Sons, Inc.

Enhanced Performance of a Molecular Photoacoustic Imaging Agent by Encapsulation in Mesoporous Silicon Nanoparticles.

Photoacoustic (PA) imaging allows visualization of the physiology and pathology of tissues with good spatial resolution and relatively deep tissue penetration. The method converts near-infrared (NIR) laser excitation into thermal expansion, generating pressure transients that are detected with an acoustic transducer. Here, we find that the response of the PA contrast agent indocyanine green (ICG) can be enhanced 17-fold when it is sealed within a rigid nanoparticle. ICG encapsulated in particles composed of porous silicon (pSiNP), porous silica, or calcium silicate all show greater PA contrast relative to equivalent quantities of free ICG, with the pSiNPs showing the strongest enhancement. A liposomal formulation of ICG performs similar to free ICG, suggesting that a rigid host nanostructure is necessary to enhance ICG performance. The improved response of the nanoparticle formulations is attributed to the low thermal conductivity of the porous inorganic hosts and their ability to protect the ICG payload from photolytic and/or thermal degradation. The translational potential of ICG-loaded pSiNPs as photoacoustic probes is demonstrated via imaging of a whole mouse brain.

Structural covariance of brain region volumes is associated with both structural connectivity and transcriptomic similarity

An organizational pattern seen in the brain, termed structural covariance, is the statistical association of pairs of brain regions in their anatomical properties. These associations, measured across a population as covariances or correlations usually in cortical thickness or volume, are thought to reflect genetic and environmental underpinnings.Here, we examine the biological basis of structural volume covariance in the mouse brain. We first examined large scale associations between brain region volumes using an atlas-based approach that parcellated the entire mouse brain into 318 regions over which correlations in volume were assessed, for volumes obtained from 153 mouse brain images via high-resolution MRI. We then used a seed-based approach and determined, for 108 different seed regions across the brain and using mouse gene expression and connectivity data from the Allen Institute for Brain Science, the variation in structural covariance data that could be explained by distance to seed, transcriptomic similarity to seed, and connectivity to seed.We found that overall, correlations in structure volumes hierarchically clustered into distinct anatomical systems, similar to findings from other studies and similar to other types of networks in the brain, including structural connectivity and transcriptomic similarity networks. Across seeds, this structural covariance was significantly explained by distance (17% of the variation, up to a maximum of 49% for structural covariance to the visceral area of the cortex), transcriptomic similarity (13% of the variation, up to maximum of 28% for structural covariance to the primary visual area) and connectivity (15% of the variation, up to a maximum of 36% for structural covariance to the intermediate reticular nucleus in the medulla) of covarying structures. Together, distance, connectivity, and transcriptomic similarity explained 37% of structural covariance, up to a maximum of 63% for structural covariance to the visceral area. Additionally, this pattern of explained variation differed spatially across the brain, with transcriptomic similarity playing a larger role in the cortex than subcortex, while connectivity explains structural covariance best in parts of the cortex, midbrain, and hindbrain. These results suggest that both gene expression and connectivity underlie structural volume covariance, albeit to different extents depending on brain region, and this relationship is modulated by distance.

Manganese-Doped Carbon Dots for Magnetic Resonance/Optical Dual-Modal Imaging of Tiny Brain Glioma.

Brain gliomas are life-threatening diseases with low survival rates. Early detection and accurate intraoperative location of brain gliomas is vital to improving the prognosis. Herein, we synthesized manganese (Mn)-doped carbon dots (CDs) as magnetic resonance (MR)/optical dual-modal imaging nanoprobes by a one-pot green microwave-assisted route. These ultra-small-sized Mn-doped CDs possess distinct excitation-dependent photoluminescent emissions, high r1 relaxivity, and low cytotoxicity. The in vivo MR imaging and ex vivo optical imaging of mouse brain with tiny glioma demonstrate that the Mn-doped CDs could lead to an enhanced MR T1 contrast effect in the tiny brain glioma region, disclosing the great promise of these Mn-doped CDs as MR/optical dual-modal imaging nanoprobes for detection and intraoperative location of tiny brain gliomas.

Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples.

We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.

Cerebral capillary flow imaging by wavelength-division-multiplexing swept-source optical Doppler tomography.

Swept-source-based optical coherence tomography (SS-OCT) has demonstrated the unique advantages for fast imaging rate and long imaging distance; however, limited axial resolution and complex phase noises restrict swept-source optical Doppler tomography (SS-ODT) for quantitative capillary blood flow imaging in the deep cortices. Here, the wavelength-dividing-multiplexing optical Doppler tomography (WDM-ODT) method that divides a single interferogram into multiple phase-correlated interferograms is proposed to effectively enhance the sensitivity for cerebral capillary flow imaging. Both flow phantom and in vivo mouse brain imaging studies show that WDM-ODT is able to significantly suppress background phase noise and image cerebral capillary flow down to the vessel size of 5.6 μm. Comparison between the wavelength-division-multiplexing SS-ODT and the spectral-domain ultrahigh-resolution ODT (uODT) reveals that SS-ODT outpaces uODT by extending the capillary flow imaging depth to 1.6 mm in mouse cortex. Thus, for the first time, quantitative capillary flow imaging is demonstrated using SS-ODT in the deep cortex.

A spherical aberration-free microscopy system for live brain imaging

The high-resolution in vivo imaging of mouse brain for quantitative analysis of fine structures, such as dendritic spines, requires objectives with high numerical apertures (NAs) and long working distances (WDs). However, this imaging approach is often hampered by spherical aberration (SA) that results from the mismatch of refractive indices in the optical path and becomes more severe with increasing depth of target from the brain surface. Whereas a revolving objective correction collar has been designed to compensate SA, its adjustment requires manual operation and is inevitably accompanied by considerable focal shift, making it difficult to acquire the best image of a given fluorescent object. To solve the problems, we have created an objective-attached device and formulated a fast iterative algorithm for the realization of an automatic SA compensation system. The device coordinates the collar rotation and the Z-position of an objective, enabling correction collar adjustment while stably focusing on a target. The algorithm provides the best adjustment on the basis of the calculated contrast of acquired images. Together, they enable the system to compensate SA at a given depth. As proof of concept, we applied the SA compensation system to in vivo two-photon imaging with a 25 × water-immersion objective (NA, 1.05; WD, 2 mm). It effectively reduced SA regardless of location, allowing quantitative and reproducible analysis of fine structures of YFP-labeled neurons in the mouse cerebral cortical layers. Interestingly, although the cortical structure was optically heterogeneous along the z-axis, the refractive index of each layer could be assessed on the basis of the compensation degree. It was also possible to make fully corrected three-dimensional reconstructions of YFP-labeled neurons in live brain samples. Our SA compensation system, called Deep-C, is expected to bring out the best in all correction-collar-equipped objectives for imaging deep regions of heterogeneous tissues.

Oscillating gradient diffusion kurtosis imaging of normal and injured mouse brains.

Recent advances in diffusion MRI employ multiple diffusion encoding schemes with varying diffusion direction, weighting, and diffusion time to investigate specific microstructural properties in biological tissues. In this study, we examined time-dependent diffusion kurtosis contrast in adult mouse brains and in neonatal mouse brains after hypoxic-ischemic (HI) injury. In vivo diffusion kurtosis maps were acquired with a short diffusion time using an oscillating gradient spin echo (OGSE) sequence at 100Hz and with a relatively long diffusion time (20ms) using a pulsed gradient spin echo (PGSE) sequence. In the adult mouse brain, we found that the cortex and hippocampus showed larger differences between OGSE kurtosis and PGSE kurtosis than major white matter tracts. In neonatal mouse brains with unilateral HI injury, the OGSE kurtosis map overall provided stronger edema contrast than the PGSE kurtosis map, and the differences between OGSE and PGSE kurtosis measurements in the edema region reflected heterogeneity of injury. This is the first in vivo study that has demonstrated multi-direction OGSE kurtosis contrasts in the mouse brain. Comparing PGSE and OGSE kurtosis measures may provide additional information on microstructural changes after ischemic stroke.

Rapid volumetric imaging with Bessel-Beam three-photon microscopy.

Owing to its tissue-penetration ability, multi-photon fluorescence microscopy allows for the high-resolution, non-invasive imaging of deep tissue in vivo; the recently developed three-photon microscopy (3PM) has extended the depth of high-resolution, non-invasive functional imaging of mouse brains to beyond 1.0 mm. However, the low repetition rate of femtosecond lasers that are normally used in 3PM limits the temporal resolution of point-scanning three-photon microscopy. To increase the volumetric imaging speed of 3PM, we propose a combination of an axially elongated needle-like Bessel-beam with three-photon excitation (3PE) to image biological samples with an extended depth of focus. We demonstrate the higher signal-to-background ratio (SBR) of the Bessel-beam 3PM compared to the two-photon version both theoretically and experimentally. Finally, we perform simultaneous calcium imaging of brain regions at different axial locations in live fruit flies and rapid volumetric imaging of neuronal structures in live mouse brains. These results highlight the unique advantage of conducting rapid volumetric imaging with a high SBR in the deep brain in vivo using scanning Bessel-3PM.

High-contrast differentiation resolution 3D imaging of rodent brain by X-ray computed microtomography

The biomedically focused brain research is largely performed on laboratory mice considering a high homology between the human and mouse genomes. A brain has an intricate and highly complex geometrical structure that is hard to display and analyse using only 2D methods. Applying some fast and efficient methods of brain visualization in 3D will be crucial for the neurobiology in the future. A post-mortem analysis of experimental animals' brains usually involves techniques such as magnetic resonance and computed tomography. These techniques are employed to visualize abnormalities in the brains' morphology or reparation processes. The X-ray computed microtomography (micro CT) plays an important role in the 3D imaging of internal structures of a large variety of soft and hard tissues. This non-destructive technique is applied in biological studies because the lab-based CT devices enable to obtain a several-micrometer resolution. However, this technique is always used along with some visualization methods, which are based on the tissue staining and thus differentiate soft tissues in biological samples. Here, a modified chemical contrasting protocol of tissues for a micro CT usage is introduced as the best tool for ex vivo 3D imaging of a post-mortem mouse brain. This way, the micro CT provides a high spatial resolution of the brain microscopic anatomy together with a high tissue differentiation contrast enabling to identify more anatomical details in the brain. As the micro CT allows a consequent reconstruction of the brain structures into a coherent 3D model, some small morphological changes can be given into context of their mutual spatial relationships.

Virtual craniotomy for high-resolution optoacoustic brain microscopy

Ultrasound-mediated transcranial images of the brain often suffer from acoustic distortions produced by the skull bone. In high-resolution optoacoustic microscopy, the skull-induced acoustic aberrations are known to impair image resolution and contrast, further skewing the location and intensity of the different absorbing structures. We present a virtual craniotomy deconvolution algorithm based on an ultrasound wave propagation model that corrects for the skull-induced distortions in optically-resolved optoacoustic transcranial microscopy data. The method takes advantage of the geometrical and spectral information of a pulse-echo ultrasound image of the skull simultaneously acquired by our multimodal imaging system. Transcranial mouse brain imaging experiments confirmed the ability to accurately account for the signal amplitude decay, temporal delay and pulse broadening introduced by the rodent’s skull. Our study is the first to demonstrate skull-corrected transcranial optoacoustic imaging in vivo.

Breaking the diffraction limit of light sheets allows fast isotropic imaging of large samples by ultramicroscopy

By breaking the diffraction limit of light sheets of low numerical aperture (NA) we were able to generate extremely long thin sheets of light with a thickness in the one micron range and a vastly increased Rayleigh range. We measured the thickness of the light sheets with different methods including standard point spread function measurement with beads. By using these light sheets in our ultramicroscope fast 3D imaging of whole mouse brains with objectives with a large field of view was possible. Due to the extremely low divergence of the light sheets mouse brains could be reconstructed from a single stack of optical sections with isotropic resolution. The light sheets used were essentially non-Gaussian generated by new optics we developed. Compared to a Gaussian light sheet of the same NA our new light sheet is much thinner. Thus the diffraction limit which holds also for low NA Gaussian light sheets was significantly surpassed. These optics will allow the application of ultramicroscopy to ever increasing samples beyond the whole mouse brain range.

In Vivo Superresolution Imaging of Neuronal Structure in the Mouse Brain.

this study proposes and evaluates a technique for invivo deep-tissue superresolution imaging in the light-scattering mouse brain at up to a 3.5Hz 2-D imaging rate with a 21×21μm2 field of view. we combine the deep-tissue penetration and high imaging speed of resonant laser scanning two-photon (2P) microscopy with the superresolution ability of patterned excitation microscopy. Using high-frequency intensity modulation of the scanned two-photon excitation beam, we generate patterned illumination at the imaging plane. Using the principles of structured illumination, the high-frequency components in the collected images are then used to reconstruct images with an approximate twofold increase in optical resolution. using our technique, resonant 2P superresolution patterned excitation reconstruction microscopy, we demonstrate our ability to investigate nanoscopic neuronal architecture in the cerebral cortex of the mouse brain at a depth of 120μm invivo and 210μm ex vivo with a resolution of 119nm. This technique optimizes the combination of speed and depth for improved invivo imaging in the rodent neocortex. this study demonstrates a potentially useful technique for superresolution invivo investigations in the rodent brain in deep tissue, creating a platform for investigating nanoscopic neuronal dynamics. this technique optimizes the combination of speed and depth for improved superresolution invivo imaging in the rodent neocortex.

Ultrastable and Biocompatible NIR‐II Quantum Dots for Functional Bioimaging

AbstractFluorescence bioimaging in the second near‐infrared spectral region (NIR‐II, 1000–1700 nm) can provide advantages of high spatial resolution and large penetration depth, due to low light scattering. However, NIR‐II fluorophores simultaneously possessing high brightness, good stability, and biocompatibility are very rare. Hydrophobic NIR‐II emissive PbS@CdS quantum dots (QDs) are surface‐functionalized, via a silica and amphiphilic polymer (Pluronic F‐127) dual‐layer coating method. The as‐synthesized PbS@CdS@SiO2@F‐127 nanoparticles (NPs) are aqueously dispersible and possess a quantum yield of ≈5.79%, which is much larger than those of most existing NIR‐II fluorophores. Thanks to the dual‐layer protection, PbS@CdS@SiO2@F‐127 NPs show excellent chemical stability in a wide range of pH values. The biocompatibility of PbS@CdS@SiO2@F‐127 NPs is studied, and the results show that the toxicity of the NPs in vivo could be minimal. PbS@CdS@SiO2@F‐127 NPs are then utilized for in vivo and real‐time NIR‐II fluorescence microscopic imaging of mouse brain. The architecture of blood vessels is visualized and the imaging depth reaches 950 µm. Furthermore, in vivo NIR‐II fluorescence imaging of gastrointestinal tract is achieved, by perfusing PbS@CdS@SiO2@F‐127 NPs into mice at a rather low dosage. This work illustrates the potential of ultrastable, biocompatible, and bright NIR‐II QDs in biomedical and clinical applications, which require deep tissue imaging.

Optical projection tomography for rapid whole mouse brain imaging.

In recent years, three-dimensional mesoscopic imaging has gained significant importance in life sciences for fundamental studies at the whole-organ level. In this manuscript, we present an optical projection tomography (OPT) method designed for imaging of the intact mouse brain. The system features an isotropic resolution of ~50 µm and an acquisition time of four to eight minutes, using a 3-day optimized clearing protocol. Imaging of the brain autofluorescence in 3D reveals details of the neuroanatomy, while the use of fluorescent labels displays the vascular network and amyloid deposition in 5xFAD mice, an important model of Alzheimer's disease (AD). Finally, the OPT images are compared with histological slices.

Perfluoroarene-Based Peptide Macrocycles to Enhance Penetration Across the Blood-Brain Barrier.

Here we describe the utility of peptide macrocyclization through perfluoroaryl-cysteine SNAr chemistry to improve the ability of peptides to cross the blood-brain barrier. Multiple macrocyclic analogues of the peptide transportan-10 were investigated that displayed increased uptake in two different cell lines and improved proteolytic stability. One of these analogues (M13) exhibited substantially increased delivery across a cellular spheroid model of the blood-brain barrier. Through ex vivo imaging of mouse brains, we demonstrated that this perfluoroarene-based macrocycle of TP10 exhibits increased penetration of the brain parenchyma following intravenous administration in mice. Finally, we evaluated macrocyclic analogues of the BH3 domain of the BIM protein to assess if our approach would be applicable to a peptide of therapeutic interest. We identified a BIM BH3 analogue that showed increased penetration of the brain tissue in mice.