Has Deep Brain In Vivo Imaging Come of Age?

Written by Adam Tozer

January 28, 2019

  • It is now possible to image sub-cellular structures like dendritic spines with in vivo imaging techniques
  • Microendoscopes can resolve dendritic spines in vivo but may require aspiration of tissue above your area of interest
  • Multimode fibers can resolve dendritic spines and cause less damage to brain tissue, but their field of view is dependent on the diameter of the fiber

WATCHING THE BRAIN IN ACTION WITH IN VIVO IMAGING TECHNIQUES

To understand the brain neuroscientists need to see it in action. They need the most detailed picture possible of how brain cells interact as the brain performs its daily functions. One way scientists can do this is by using in vivo imaging techniques.
Recently, several papers have described different approaches to in vivo imaging. Each has achieved success in resolving sub-cellular compartments such as dendrites and axons in different ways. Read on to understand the strengths and weaknesses of each approach.
Need some background? Read our fundamentals article on in vivo imaging

MULTIPHOTON IN VIVO IMAGING IS A LESS INVASIVE APPROACH, BUT DEPTH IS LIMITED

One way is to directly image the neurons as they behave. This can be done in the cortex by creating a cranial window and using a laser scanning multiphoton microscope to image the activity of underlying neurons expressing a reporter of neuronal activity like GCamp6, for example. The limitation here is that brain tissue is not transparent, so the depth at which a scientist can image is limited. Recently, Mriganka Sur’s group at MIT used a 3-Photon approach to image GCamp6 fluorescence in the visual cortex of awake mice in vivo.1 They were able to image all 6 layers of cortex and the sub-plate underneath.
Whilst the 3-Photon approach enables deeper visualization of brain tissue without having to aspirate and destroy tissue, the drawback is the mouse must remain head-fixed during the experiment.

MCI-Neuroscience’s Pryer endoscope facilitates deep brain multiphoton imaging, enabling scientists to resolve subcellular structures deep in the brain, such as in the hippocampus.

MULTIPHOTON MICROENDOSCOPY ENABLES DEEP BRAIN IMAGING IN FREELY MOVING ANIMALS

Microendoscopes are a technology that enables scientists to investigate cellular activity deep within the brain whilst the animal is free to move around. However, microendoscopes are challenging to implant and, if not implanted properly can prove difficult to infer information from, as movement artifacts can impact on imaging resolution. These drawbacks have meant that, until now, resolving structures smaller than the cell body has been difficult, if not impossible.
A recent paper, this time from Na Ji’s group at UC Berkeley, US, describes how the group incorporated a Bessel focus scanning module with a microendoscope to enable multiphoton imaging with sub-cellular resolution, in a freely moving animal.2
The Bessel focus scanning module increases the axial focal plane and improves the axial excitation of the microendoscope, which incorporates a gradient refractive index (GRIN) lens. This means the user can image a greater thickness of tissue per horizontal scan, enabling resolution of sub-cellular structures like dendrites and axons with the grin lens. This also means these structures can be imaged in 3D, in real time as the Bessel focus scanning module increases the volume imaging speed by 10 to 100-fold.

To image a volume requires multiple 2D scans of a Gaussian focus but a single 2D scan with a Bessel focus. Taken from figure 1, Meng et al. (2019), eLife, under CC BY 4.0
Using their volumetric approach, the scientists were able to image the population dynamics of groups of cells in real time, probing as deep in the brain as the lateral hypothalamus, some 5 mm down inside the brain tissue.
As with all microendoscopes, this requires the aspiration and removal of overlying tissue, a caveat of deep tissue in vivo imaging.

MULTIMODE FIBERS ENABLE DEEP BRAIN SUBCELLULAR RESOLUTION WHILST REDUCING INVASIVENESS

Scientists have been working to overcome the invasiveness of in vivo imaging, as the act of implanting the microendoscopes themselves could affect the physiology and behavior of the brain, and therefore the animal. Which would only serve to question the relevance of performing these experiments.
A recent paper from Nigel Emptage’s lab at the University of Oxford, UK, describes the use of multimode fibers (MMFs) to image sub-cellular structures.3 Using multimode fibers, the group were able to implant a fiber into the brain of a mouse and image dendritic spines. Their thin multimode fiber, only 50 µm in diameter enabled the scientists to image subcellular structures at depth, and they did this with minimal disruption of the tissue.

a Imaging was performed by lowering an MMF 1.8 mm into the brain of an anesthetized Thy1-GFP line M mouse to reach the dorsal striatum (top). Atlas depiction of the region of the striatum imaged in (c) adapted from the Allen Mouse Brain Atlas (bottom) with fiber placement in blue. b Fluorescence imaging was performed at multiple distances (depths, reference plane: 50 μm) from the distal facet of the fiber by calibrating the system to different focal planes. c Dendritic spines were clearly identifiable, and their three-dimensional structure became visible when varying the focal plane. Scale bar: 10 μm. d Post-mortem histological section of the mouse brain imaged in (c) showing the path of the fiber through the cortex. Scale bar: 200 μm. The inset shows that the structure of cortical neurons is preserved even around the margins of the fiber track. Credit: Emptage lab, Oxford University, Light: Science and Applications.

Taking their approach a step further, the team also imaged functional calcium responses in neurons in response to auditory stimulation, further supporting the utility of the fibers in in vivo imaging studies.
Whilst the fibers enable deep-tissue imaging with minimal tissue disruption, they do have some limitations. Namely, their field of view is proportional to the diameter of the fiber, making it difficult to image from multiple cells or structures simultaneously. Also, the group were unable to record from freely moving animals, as the movement would deform the fibers affecting light propagation and imaging.
Further Reading: Fundamentals of deep tissue in vivo imaging part 2

THE FUTURE FOR IN VIVO IMAGING LOOKS BRIGHT

With the trade-off of invasiveness versus depth of imaging, the perfect in vivo imaging solution might not yet exist. However, the technologies mentioned above show how scientists and engineers are working towards imaging solutions that will enable scientists to see the brain in action, at the highest resolution possible.
Even further reading: In vivo imaging in awake behaving animals, fundamentals part 3

References:

Yildrim et al. (2019) ‘Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy.’ Nature Communications
Meng et al. (2019) ‘High-Throughput Synapse-Resolving Two-Photon Fluorescence Microendoscopy for Deep-Brain Volumetric Imaging In Vivo
Vasquez-Lopez et al. (2018) ‘Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy.’ Light: Science and Applications