Microscopy Advances Accelerate Neuroscience Research

The intricacies of brain function are intimately tied to the complexity of brain structure, both of which we are only just beginning to understand. Microscopy, one of the most important tools for unraveling the interconnectedness of brain structures, is advancing at a quick pace. Advances such as super-resolution microscopy are enabling neuroscientists to approach brain imaging in ways that weren’t previously possible, visualizing the nervous system at subcellular and molecular levels, and raising hopes of future therapies for neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS). Here’s a look at recent neuroscience discoveries made possible by ongoing microscopy advances.

Imaging the brain without labels

Neuroscientists are using coherent Raman scattering (CRS) microscopy—which achieves subcellular spatial resolution without using imaging labels—to investigate mechanisms of neurodegenerative diseases, such as imaging the amyloid plaque pathology seen in Alzheimer’s disease,¹ the Lewy pathology (bodies and neurites) observed in Parkinson’s disease,² and the nerve degeneration that occurs during ALS.³ CRS microscopy “achieves chemically specific image contrasts by exciting the molecular vibrations of endogenous chemical compounds that are present in a sample,” says Volker Schweikhard, Application Manager in the Life Science Division at Leica Microsystems.

Leica’s STELLARIS 8 CRS microscope gives researchers the tools to achieve this in 2D cell culture, 3D organoid models, and 3D tissues. “Another emerging research field enabled by CRS is the optical imaging of metabolic activities in animals, including de novo lipid and protein synthesis in brain tissues,” says Schweikhard. “This approach looks particularly promising to study brain development and degeneration.”

Indeed, using CRS microscopy along with other imaging methods is already leading to new discoveries. For example, combining CRS with fluorescence microscopy uncovered a connection between pro-inflammatory states of microglia and aging-related brain dysfunctionalities.⁴ “And at the current rate of progress,” says Schweikhard, “the range of applications in neuroscience can be expected to grow even further.”

Imaging the brain with super resolution

Super-resolution microscopy, so named for its ability to resolve structures smaller than the traditional imaging barrier of the diffraction limit, has developed quickly in recent years. One of these methods, structured illumination microcopy (SIM), uses a spatially structured pattern of light to excite the sample, and a super-resolution image is computationally reconstructed from the resulting interference patterns. SIM platforms are quickly evolving to become more powerful and easier to use than their predecessors; Nikon’s N-SIM S Super Resolution Microscope is fast enough for super-resolution imaging of live cells.

A recent study led by Satoshi Gojo at the Kyoto Prefectural University of Medicine used Nikon’s N-SIM S system to localize exogenous mitochondrial DNA within endogenous mitochondria.⁵ Such a feat—finding specific DNA inside a subcellular organelle—was made feasible using super-resolution microscopy. “Mitochondrial dysfunction is at play in many disorders, including some forms of neurodegeneration, so I look forward to seeing where this discovery leads,” says Adam White, Product and Logistics Manager in Advanced Microscopy at Nikon Instruments.

Combining Nikon’s N-SIM with optogenetics allowed Kai Zhang’s group at the University of Illinois at Urbana-Champaign to explore finer details of the nerve growth factor (NGF) receptor signaling pathway.⁶ Normally, NGF activates tyrosine kinase A (TrkA) as well as another lower-affinity receptor. Zhang’s group used optogenetics—originally developed to modulate neuronal activity with light and genetic modifications—to zero in on TrkA activation alone, with the aim of precluding off-target effects that can occur when using NGF to study TrkA-mediated events. They used SIM to confirm dimerization of the optogenetic TrkA receptors in response to blue light stimulation, and suggest that their method be used to study the mechanisms of other receptor-mediated signaling pathways. “I find this an interesting application for optogenetics, as it demonstrates how these tools can augment more traditional methods for studying cell signaling,” says White.

Another type of super resolution, stochastic optical reconstruction microscopy (STORM), reconstructs a super-resolution image from stochastic emissions of individual fluorophores within a specimen. A group led by Erin Schuman at the Max Planck Institute for Brain Research recently used Nikon’s N-STORM system to study protein synthesis within dendrites during synaptic plasticity.⁷ “Local synthesis is required for synaptic plasticity, but little is known about the level and specific location of the necessary molecular tools,” says White. “This proves to be a perfect application for N-STORM.” Using metabolic labeling and DNA-PAINT (DNA point accumulation in nanoscale topology, a super-resolution imaging method using short DNA oligonucleotides labeled with fluorophores), Schuman’s group detected dendritic ribosomes and nascent proteins at single-molecule resolution, and found that local protein synthesis was correlated with the level of synaptic activity.

Single-molecule imaging within the brain

The brain’s unique spatial organization makes it a prime target for investigation by microscopy—especially as techniques mature in both resolution and molecular capabilities. Vizgen’s MERSCOPE platform uses MERFISH (multiplexed error-robust fluorescence in situ hybridization), a single-molecule imaging technology that can measure the copy number of up to 100s of 1000s of RNA molecules simultaneously, with subcellular resolution. “The MERSCOPE platform enables spatially profiling the expression of hundreds of genes across full tissue slices, revealing the exact three-dimensional coordinates of nearly all copies of the targeted transcript with better than 100 nm accuracy,” says George Emanuel, Scientific Cofounder, Director of Technology and Partnerships at Vizgen.

High-resolution spatial profiling of gene expression is a powerful mapping tool for any type of tissue. In the brain, researchers are dissecting complex interactions, such as the migration of microglial cells down blood vessels during development. “Only high-resolution transcriptome profiling is able to distinguish the microglia transcription from the transcription in the blood vessels,” says Emanuel. “This unveils a window into the composition of the brain that was not feasible with previous commercially available technologies and is particularly relevant in neuroscience research.”

Spatial profiling is turbo-charging cell atlas efforts in the brain by delivering positional information of individual cells within tissues (previous methods typically involved tissue dissociation followed by single-cell sequencing). Using MERFISH and a retrograde tracer to map neuronal connections from terminus to source, researchers can identify the subtypes of neurons involved. A group led by Xiaowei Zhuang at Harvard University recently used this method to generate a cell atlas of the mouse primary motor cortex, and investigated the projection patterns of intratelencephalic neurons.⁸ They also found that neurons from the primary motor cortex send and receive information to other areas in neuronal clusters.

Microscopy is advancing neuroscience still further to provide high-resolution images of live neurons and glial cells. A new study led by Robert Prevedel at the European Molecular Biology Laboratory uses several methods including adaptive optics 3-photon microscopy to obtain high-resolution images deep within live brain tissue.⁹ All of these results inspire future avenues of inquiry for neuroscientists armed with the latest microscopy techniques.

References

1. M. Ji, M. Arbel, L. Zhang, C. W. Freudiger, S. S. Hou, D. Lin, X. Yang, B. J. Bacskai, X. S. Xie, Label-free imaging of amyloid plaques in Alzheimer’s disease with stimulated Raman scattering microscopy. Sci. Adv. 4, eaat7715 (2018).

2. Shahmoradian, S.H., Lewis, A.J., Genoud, C. et al. Lewy pathology in Parkinson’s disease consists of crowded organelles and lipid membranes. Nat Neurosci 22, 1099–1109 (2019). 

3. Tian, F., Yang, W., Mordes, D. et al. Monitoring peripheral nerve degeneration in ALS by label-free stimulated Raman scattering imaging. Nat Commun 7, 13283 (2016).

4. Marschallinger, J., Iram, T., Zardeneta, M. et al. Lipid-droplet-accumulating microglia represent a dysfunctional and proinflammatory state in the aging brain. Nat Neurosci 23, 194–208 (2020).

5. Maeda, H., Kami, D., Maeda, R. et al. Generation of somatic mitochondrial DNA-replaced cells for mitochondrial dysfunction treatment. Sci Rep 11, 10897 (2021).

6. Khamo JS. et al. Optogenetic Delineation of Receptor Tyrosine Kinase Subcircuits in PC12 Cell Differentiation. Cell Chemical Biology, ISSN: 2451-9456, Vol: 26, Issue: 3, Page: 400-410.e3.

7. Sun, C. The prevalence and specificity of local protein synthesis during neuronal synaptic plasticity. Science Advances. 7 (38).

8. Zhang, M., Eichhorn, S.W., Zingg, B. et al. Spatially resolved cell atlas of the mouse primary motor cortex by MERFISH. Nature 598, 137–143 (2021).

9. Streich, L., Boffi, J.C., Wang, L. et al. High-resolution structural and functional deep brain imaging using adaptive optics three-photon microscopy. Nat Methods 18, 1253–1258 (2021).