In vivo label-free confocal imaging of adult mouse brain up to 1.3-mm depth with NIR-II illumination (original) (raw)
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In vivo label-free confocal imaging of the deep mouse brain with long-wavelength illumination
Biomedical Optics Express, 2018
Optical microscopy is a valuable tool for in vivo monitoring of biological structures and functions because of its non-invasiveness. However, imaging deep into biological tissues is challenging due to the scattering and absorption of light. Previous research has shown that 1300 nm and 1700 nm are the two best wavelength windows for deep brain imaging. Here, we combined long-wavelength illumination of ~1700 nm with reflectance confocal microscopy and achieved an imaging depth of ~1.3 mm with ~1micrometer spatial resolution in adult mouse brains, which is 3-4 times deeper than that of conventional confocal microscopy using visible wavelength. We showed that the method can be added to any laser-scanning microscopy with simple and low-cost sources and detectors, such as continuous-wave diode lasers and InGaAs photodiodes. The long-wavelength, reflectance confocal imaging we demonstrated is label-free, and requires low illumination power. Furthermore, the imaging system is simple and low-cost, potentially creating new opportunities for biomedical research and clinical applications.
Zebrafish, 2011
Magnetic resonance imaging (MRI) and computed tomography (CT) are noninvasive medical imaging techniques used for the detailed visualization of internal organs of the human body. Because CT uses X-rays for imaging, there is a risk of radiation exposure. In contrast, MRI uses radiowaves and magnetic fields for imaging; thus, there are no reported biological hazards. However, neither MRI nor CT is suitable as a noninvasive imaging tool applicable in small laboratory animals such as zebrafish embryos or larvae. The recently established micro-CT scanner is only suitable for scanning adult fish and a staining procedure is required for imaging. In addition, CT-based scanning is generally more suitable for skeletal imaging but not for visualization of soft tissues because of its lower contrast. In this study, we evaluated whether 633 nm HeNe laser-coupled confocal microscope allows simulating MRI/CT scan and imaging soft tissues such as brain and eye in zebrafish embryos/larvae. We show that the 633 nm HeNe laser can penetrate well into intact brain and eye of zebrafish. It represents a noninvasive imaging method with high resolution while not requiring contrast agents, enabling the detection of differential signals from normal and pathological organs such as brain and eye.
In Vivo Multiphoton Microscopy of Deep Brain Tissue
Journal of Neurophysiology, 2004
Although fluorescence microscopy has proven to be one of the most powerful tools in biology, its application to the intact animal has been limited to imaging several hundred micrometers below the surface. The rest of the animal has eluded investigation at the microscopic level without excising tissue or performing extensive surgery. However, the ability to image with subcellular resolution in the intact animal enables a contextual setting that may be critical for understanding proper function. Clinical applications such as disease diagnosis and optical biopsy may benefit from minimally invasive in vivo approaches. Gradient index (GRIN) lenses with needle-like dimensions can transfer high-quality images many centimeters from the object plane. Here, we show that multiphoton microscopy through GRIN lenses enables minimally invasive, subcellular resolution several millimeters in the anesthetized, intact animal, and we present in vivo images of cortical layer V and hippocampus in the ane...
Near-infrared laser scanning confocal microscopy and its application in bioimaging
Optical and Quantum Electronics
Near-infrared (NIR) fluorescence imaging is an important imaging technology in deep-tissue biomedical imaging and related researches, due to the low absorption and scattering of NIR excitation and/or emission in biological tissues. Laser scanning confocal microscopy (LSCM) plays a significant role in the family of fluorescence microscopy. Due to the introduction of pinhole, it can provide images with optical sectioning, high signal-tonoise ratio and better spatial resolution. In this study, in order to combine the advantages of these two techniques, we set up a fluorescence microscopic imaging system, which can be named as NIR-LSCM. The system was based on a commercially available confocal microscope, utilizing a NIR laser for excitation and a NIR sensitive detector for signal collection. In addition, NIR fluorescent nanoparticles (NPs) were prepared, and utilized for fluorescence imaging of the ear and brain of living mice based on the NIR-LSCM system. The structure of blood vessels at certain depth could be visualized clearly, because of the high-resolution and large-depth imaging capability of NIR-LSCM. Keywords Near-infrared (NIR) Á Laser scanning confocal microscopy (LSCM) Á NIR nanoparticles (NPs) Á In vivo Á Bioimaging & Jun Qian
A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices
Journal of Visualized Experiments, 2011
A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps 1-3 . With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks 4 . Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will 5-6 . Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates 7-8 , alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters 9 . Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Advances in light microscopy for neuroscience
Annual review of …, 2009
Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists.
Implantable Fluorescence Imager for Deep Neuronal Imaging
2021
Implantable Fluorescence Imager for Deep Neuronal Imaging Jaebin Choi This thesis describes the design, fabrication, and characterization of the Implantable Fluorescence Imager (IFI): a camera chip with a needle-like form factor designed for imaging neuronal activity in the deep brain. It is fabricated with a complementary metal oxide semiconductor (CMOS) process, allowing for hundreds or thousands of singlephoton-sensitive photodetectors to be densely packed onto a device width comparable to a single-channel fiber optic cannula (~100 µm). The IFI uses a combination of spectral and temporal filters as a fluorescence emission filter, and per-pixel Talbot gratings for 3D light-field imaging. The IFI has the potential to overcome the imaging depth limit of multi-photon microscopes imposed by the scattering and absorption of photons in brain tissue, and the resolution limit of noninvasive imaging techniques, such as functional magnetic resonance imaging and photoacoustic imaging. It competes with graded index lens-based miniaturized microscopes in imaging depth, but offers several comparative advantages. First, its cross sectional area is at least an order of magnitude smaller for an equal field of view. Second, the distribution of pixels along its entire length allows the study of multilayer or multi-region dynamics. Finally, the scalability advantage of silicon integrated circuit technology in system miniaturization and data bandwidth may allow thousands of such imaging shanks to be simultaneously deployed for large-scale volumetric recording. List of Figures 1.1 Brain Complexity, "Brain Fields," and Structural Length Scales Visa -Vis Cell-Body Location, Density, and Heterogeneity in the Rodent Brain. (A) Biophysical scales for electrical, neurochemical, and optical domain recordings and relative sizes of brain structures. (B) A~2 µm thick optical section of an adult rat brain slice, stained with a fluorescent nuclear stain, wet mounted, and imaged by large-scale serial two-photon microscopy. Beneath this image, we enumerate three "brain fields"-that is, domains of neural activity: the electrical, neurochemical, and mechanical. (C-E) Cellular nuclear density at multiple scales (C, 500 µm; D, 200 µm; E, 20 µm), from the macroscopic down to the level of individual cells.
Journal of Visualized Experiments, 2013
Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.