The field of neuroscience is constantly working to solve the mysteries of the brain. Investigations into how the brain works, how neurons are connected, what triggers certain areas, and how all of this relates to the onset of disease are integral to several initiatives and programs that aim to learn more about how the brain controls the way we are.
With so many different disciplines involved in brain research, from genetic analysis and cell biology to signaling chemistry and whole brain imaging, neuroscientists have the opportunity to work with an array of different techniques that allow for complex analyses from the level of a molecule to a whole organism.
Advances in neuroscience tools
Imaging technologies have become a primary staple in neuroscience labs. The ability to visualize a neuron firing or map a neural circuit can teach us a lot about brain structure and function. However, scientists struggle to create the right balance between generating images that are detailed enough without elongating imaging time. New technologies can create the right balance between resolution, speed, and sensitivity, without compromising one for the other.
New technologies can create the right balance between resolution, speed, and sensitivity, without compromising one for the other.
Improving new tools to enhance all three characteristics highlights dynamic elements of the brain as opposed to purely static imaging. “The ability to capture a dynamic event is critical,” explains Lynne Chang, Ph.D., strategic marketing manager at Nikon, discussing the trend toward imaging whole and live animals. “High-resolution imaging of single cells in the brain of a live animal while monitoring its behavior allows for the manipulation and study of neuron function and further optogenetics studies.”
A limiting factor to dynamics studies in light microscopy imaging is the light diffraction barrier, an innate optical property of a lens that defines the maximum resolution available from a light microscope. The development of super-resolution microscopy breaks this barrier, allowing the visualization of actual molecular interaction as opposed to general co-localization. By combining or coordinating this technology with other techniques such as live cell imaging or tissue clearing, neuroscientists can gain a more comprehensive understanding of the processes they are studying.
Color-coded maximum intensity projection of the central nervous system of an embryo, Drosophila melanogaster. The very compact and bright parts of the CNS as well the fine and less densely stained structures of the peripheral nervous system can be nicely imaged with low laser power. Image courtesy of Dr. Julia Sellin, AG Hoch, LIMES Institute, Bonn, Germany.
The recent 2017 Society for Neuroscience conference explored some of these new developments, covering what has been discovered along with what tools have been developed to aid in discovery. One such development, the Airyscan from Zeiss, can detect small structures, faint signals, and fast processes due to greatly improved resolution with optical sectioning. Airyscan breaks the diffraction barrier, allowing super resolution using pinhole plane imaging. By applying an array detector of 32 sensitive GaAsP detector elements where each one functions as a tiny pinhole, Airyscan can collect more light at higher resolution than single pinhole imaging. “This technology is enabling imaging of the same images previously scanned but with much more detail, seeing things we couldn’t before, and has applications in activating neurons, calcium tracking, and signaling studies,” explains Renee Dalrymple, product marketing manager at Zeiss.
Nikon’s N-STORM system, also featured at the meeting, provides a tenfold improvement in resolution compared to conventional light microscopes. “This technique enables researchers to understand macro-molecular structures and inter-molecular interactions at the single-molecule level,“ describes Chang.
Stitched image of a whole, cleared brain (iCUBIC clearing method) from a YFP-H transgenic mouse, captured with a Nikon CFI90 20XC Glyc objective lens. Image courtesy of Alan Watson, Ph.D. and Simon Watkins, Ph.D., Center for Biologic Imaging, University of Pittsburgh.
Another exciting new development showcased at Neuroscience 2017 is a new objective for biological microscopes, the CFI90 20XC Glyc, from Nikon designed for deep imaging of whole, cleared tissues. “Deep tissue imaging has always been challenging due to light scatter and non-uniform refractive index. Tissue clearing, which converts tissues, organs and even whole organisms into optically transparent samples, overcomes these challenges. The new CFI90 20XC Glyc combines an 8.2 mm working distance with 1.0 N.A. to enable ultra-deep imaging of cleared tissues with greater clarity,” explains Chang.
Combining tissue clearing methods such as CLARITY with Nikon’s new objective lens offers a greater level of sensitivity to support approaches such as neural network mapping. Being compatible with a wide range of immersion media and tissue clearing agents and accommodating a broad range of refractive indices allows labs to use one lens for multiple applications.
Logos Biosystems developed the X-CLARITY Tissue Clearing System to accelerate and standardize the tissue clearing process. Based on the original CLARITY method, the X-CLARITY consistently clears both thick and thin tissues while preserving the tissue ultrastructure. By providing a standard way to process whole tissues for volumetric imaging, the X-CLARITY enables innovative research into networks in a whole organism.
A comprehensive study into the brain
Combining several of these new technologies into one larger study, researchers can now create a comprehensive view of targeted brain areas or functions of interest. At Harvard University, neuroscientist Jeff Lichtman, a pioneer in connectomics, is part of the Machine Intelligence from Cortical Networks (MICrONS) initiative to chart the structure and function of every detail in a small piece of rodent cortex.
By creating in-depth diagrams of how each neuron is connected to other brain cells, the project will help reveal how the brain develops and functions as well as boost our understanding of diseases that have been linked to dysfunctional neural circuitry, such as autism. The overall goal of the project is to image, reconstruct, segment, and annotate 1 cubic mm volume of cortex, containing an estimated 100,000 neurons. Typically, SEM imaging alone would take decades to accomplish. However, achieving this part of the overall project within a five month window is made possible by new multi-beam imaging technology.
Through the Zeiss research partner program, the team collaborated to develop a unique scanning electron microscope, MultiSEM, to address higher throughput deep brain imaging. “This project could teach us about the natural development of neural circuitry, and provide a basis to understand what goes wrong in disease and dysfunction. Additionally, new algorithms for AI applications could be developed from the process,” explains Kyle Crosby, MultiSEM business development specialist at Zeiss.
The typical workflow involves light microscopy in live mice, followed by x-ray imaging and scanning electron microscopy of fixed and stained brains to first monitor and then visualize neuron activation and response based on an external stimulus. This data generates a circuit diagram based on functional information, and also creates a three dimensional topography of the entire brain by imaging the same structures at higher resolution.
In order to image so much in a reasonable amount of time, the MultiSEM scans ultrathin brain sections using 61 electron beams simultaneously, while automating workflows to run continuously. The sections are then reconstructed on a computer back to a 3D volume that can be further segmented virtually to analyze connected neurons. The entire project workflow solves a multiscale problem of how to image details in the brain using various techniques and tie together all the data sets using a single platform, making these large volumes easily approachable.
Integrative studies take the stage
Not only have new technologies enabled comprehensive studies of the brain using various techniques, but different technologies can be integrated into one approach to expand methods for new uses. Recent collaborative efforts between Nikon and the Allen Institute for Brain Science have resulted in the adaptable design of an open-architecture multiphoton system to broaden imaging applications to larger formats.
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A major limitation of a conventional multiphoton system configured on a microscope stand is the usable space between the lens and the platform. This limits the choice of specimens based on size alone. Researchers at the Allen Institute and Nikon partnered to develop a flexible multiphoton system to accommodate larger samples. The large-format Nikon A1R MP system does away with a standard confining frame instead opting for an open configuration to provide enough clearance for large specimens such as a moving mouse or primate brain. And with the use of the new high-definition 1K ultrafast resonant scanner that delivers high speed, resolution, and sensitivity, the multiphoton confocal microscope is well-suited for live, deep tissue and intravital imaging.
The Allen Institute uses this system to image the cortex of a mouse running on a track-style running wheel while receiving visual stimuli from a video monitor. Integrated imaging and behavioral data can be analyzed on a single platform providing two-photon time-lapse imaging combined with behavioral information such as running speed and eye tracking. With this approach, Allen Institute researchers aim to generate a physiological map of neural activity based on sensory representation, behavior, and cognition.
Generating multi-modal workflows, which combines several imaging methodologies, greatly improves our understanding of a biological process, whether it be by providing a larger contextual overview for a super-resolution scan of a small sample volume, or by providing behavioral information at the whole organism level for high-resolution experiments carried out at the single-cell level.
Image: Ultrathin mouse brain section.100 µm wide FOV, imaged in 1.3 seconds. Image courtesy of Jeff Lichtman at Harvard University.