GloNeuro

Microscopy

Microscopy makes visible the cells of the brain: neurons, glial cells and the synapses that connect them. Much of what is known about the cellular organisation of the brain was first discovered with the microscope.

SubjectCells, synapses and molecules
ScaleFrom fractions of a millimetre down to a few nanometres
Applied toTissue sections, living animals, cultured cells and organoids

Methods by scale

Microscopy
about 1 nm to 1 cm
Neuroimaging
about 0.5 mm to the whole brain
Brain networks
single neurons to the whole brain
Electrophysiology
about 10 nm to the whole brain
1 nm10 nm100 nm1 µm10 µm100 µm1 mm1 cm10 cm1 m
  1. Ion channelabout 10 nm
  2. Synaptic vesicleabout 40 nm
  3. Neuron cell bodyabout 10–30 µm
  4. Thickness of cortexabout 2–4 mm
  5. Human brainabout 15 cm long
Spatial scale, logarithmic: each step along the axis is ten times larger than the last. Ranges and sizes are approximate.
The resolution limit of light microscopy. Light from a single point spreads into a blur about 200 nanometres wide. As two fluorescent points move closer, their blurs merge; below roughly 200–250 nm, for green light and a high-quality oil-immersion objective, they can no longer be told apart. Super-resolution methods and electron microscopy get round this limit.

Neurons are far too small to see with the naked eye, and brain tissue is almost transparent. Microscopes magnify tissue, and stains, dyes and fluorescent proteins make particular cells stand out against their neighbours.

Light microscopy shows whole cells and their branches across a small region of tissue. Electron microscopy reaches much finer detail, resolving individual synapses and the tiny vesicles that store neurotransmitters. Some modern methods go further still, imaging neurons while they are active inside a living brain.

By combining these approaches, researchers can describe what kinds of cells the brain contains, how they are shaped and arranged, and how they change during development, learning, ageing and disease.

The guided experiment Fluorescence microscopy of neurons: c-Fos immunofluorescence covers the staining of brain sections and the imaging of neuronal markers at different wavelengths.

Key techniques

Histological staining

Chemical stains make cells visible in thin slices of preserved brain tissue.

Tissue is fixed, cut into thin sections and treated with dyes. The Golgi method impregnates a small, random fraction of neurons with silver so that each appears complete, from its dendrites to its axon. Nissl stains bind RNA-rich material in cell bodies and reveal how neurons are arranged in layers and clusters.

Applications include mapping cortical layers and brain nuclei, examining tissue changes in neuropathology and counting and classifying cells.

Cajal’s drawings of Golgi-stained neurons, made more than a century ago, are still used in teaching today.

Fluorescence and confocal microscopy

Fluorescent labels make chosen cells or proteins glow, and confocal optics produce sharp optical slices.

Antibodies, dyes or genetically encoded proteins such as green fluorescent protein (GFP) label specific targets. Excited by light of one colour, they emit light of another. A confocal microscope scans a focused laser across the sample and uses a pinhole to block out-of-focus light, so thin optical sections can be stacked into a three-dimensional image.

Applications include identifying cell types by the proteins they express, visualising synapses, dendritic spines and glia and three-dimensional reconstruction of cells.

Several colours can be imaged at once, so different cell types can be compared in the same sample.

Two-photon microscopy

Imaging deep inside living brain tissue with infrared light.

Two photons of infrared light, arriving almost simultaneously, together excite a fluorescent molecule. This happens only at the focal point, so the image is naturally sectioned and scattered light causes little blur. Combined with calcium indicators, which brighten when neurons fire, the method records the activity of hundreds of neurons in a living animal.

Applications include recording the activity of neuronal populations during behaviour, following dendritic spines over days or weeks of learning and imaging blood flow and glial responses.

Infrared light penetrates tissue further and causes less damage than visible light, although imaging is still limited to roughly the outer millimetre of tissue.

Electron microscopy

Beams of electrons reveal structure at the scale of nanometres.

Electrons have a far shorter wavelength than visible light, so electron microscopes resolve much finer detail than any light microscope. Tissue is fixed, stained with heavy metals and cut into ultrathin sections. Synaptic vesicles, the synaptic cleft and the dense band of receptor proteins all become visible. Imaging thousands of consecutive sections allows every connection in a block of tissue to be traced.

Applications include identifying and characterising synapses, reconstructing complete wiring diagrams (connectomics) and studying myelin and subcellular damage in disease.

Volume electron microscopy made it possible to map the complete connectome of the adult fruit-fly brain.

Super-resolution microscopy

Overcoming the diffraction limit of light to map individual molecules.

Conventional light microscopes cannot separate objects closer than about 200 nanometres, because light spreads as it passes through the optics. STED microscopy shrinks the glowing spot with a second laser, while localisation methods such as PALM and STORM switch fluorescent molecules on a few at a time and pinpoint each one. Detail down to tens of nanometres becomes visible.

Applications include mapping the arrangement of proteins at synapses, revealing the periodic skeleton inside axons and following single molecules as they move.

These methods were recognised with the 2014 Nobel Prize in Chemistry.

Tissue clearing and expansion

Making whole brains transparent, or physically enlarging them.

Brain tissue scatters light because of its lipids and proteins. Clearing methods such as CLARITY remove the lipids while preserving structure, so an intact mouse brain can be imaged in three dimensions, often with a light-sheet microscope that illuminates one plane at a time. Expansion microscopy embeds tissue in a swellable gel and enlarges it several-fold, so ordinary microscopes can resolve finer detail.

Applications include mapping cells and their projections across a whole brain, examining human tissue samples in three dimensions and nanoscale imaging with standard microscopes.

Light-sheet microscopes image large cleared samples quickly while exposing them to relatively little light.

History of the field

  • Discovery
  • Technique
  • Application

  1. 1873Technique

    The “black reaction”

    Camillo Golgi’s silver stain fills a few neurons completely, revealing their full shape for the first time.

  2. 1888Discovery

    Neurons are individual cells

    Santiago Ramón y Cajal uses the Golgi stain to argue that the nervous system is built from separate cells, the neuron doctrine.

  3. 1950sDiscovery

    Synapses seen directly

    Electron microscopy reveals the synaptic cleft and the vesicles that store neurotransmitter.

  4. 1990Technique

    Two-photon microscopy

    Infrared excitation makes it possible to image deep into living tissue, later central to imaging activity in the living brain.

  5. 1994Technique

    Fluorescent proteins in neurons

    Green fluorescent protein is expressed in the neurons of a living worm, opening the way to genetically labelled cells.

  6. 2007Technique

    Brainbow

    Individual neurons are labelled in many distinct colours, so that intermingled cells can be traced.

  7. 2013Technique

    Improved calcium indicators and tissue clearing

    Improved genetically encoded calcium indicators make single action potentials visible, and CLARITY renders an intact brain transparent.

  8. 2014Technique

    Super-resolution recognised

    The Nobel Prize in Chemistry honours microscopy that breaks the diffraction limit, now used to map proteins inside synapses.

  9. 2015Technique

    Expansion microscopy

    Tissue is physically enlarged in a swellable gel so that ordinary microscopes resolve nanoscale detail.

  10. 2024Application

    A cubic millimetre of human cortex

    Electron microscopy reconstructs a fragment of human cerebral cortex containing tens of thousands of neurons and millions of synapses.

  11. 2024Application

    A whole adult fly brain

    The FlyWire consortium maps the complete wiring of the adult fruit-fly brain, roughly 140,000 neurons.

  12. 2025Application

    Structure meets function

    The MICrONS project links the recorded activity of mouse visual cortex neurons to their wiring, mapped in a cubic millimetre of tissue.

Strengths and limitations

StrengthsLimitations
The finest spatial detail of any method used in neuroscienceUsually examines small volumes of tissue
Identifies specific cell types, connections and moleculesMost methods need tissue samples or animal models
Can follow structural change over time in living animalsLight penetrates only a limited depth into living tissue

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