Expansion microscopy

Microscopy & ex vivo · reads

Expansion microscopy is fluorescence imaging of fixed, post-mortem tissue that has been physically enlarged. Labels are linked to a swellable gel grown inside the specimen; after digestion or softening, the gel swells in water, about 4.5 fold in one round and about 16 to 20 fold with re-embedding. A confocal or light-sheet microscope images the expanded gel, reaching tens of nanometres. What it reads is the fluorescence of the anchored labels, an indirect structural proxy of the fixed tissue state; fixed tissue has no ongoing activity to record.

Spatial resolution

25.8 nm (point spread)

Temporal resolution

n/a

Field of view

400 mm³ (acquired volume)

Neurons covered

298 neurons (acquired volume)

Reads

fluorescence of gel-anchored labels (antibodies, fluorescent proteins) in expanded tissue; relative spatial distribution of labeled molecules; pre- and postsynaptic protein marker localization (e.g., Homer1/PSD95, Bassoon)

Writes

n/a

Destructive?

terminal

Prerequisites

anesthesia: Mouse protocols anesthetize animals before transcardial perfusion; the AAV-barcode protocol specifies terminal pentobarbital anesthesia.; surgery: After perfusion, mouse brains are harvested/dissected; fly brains are dissected before fixation.; other: Animal brains are fixed or post-fixed. ExRPath/15ExMPath instead use formalin-fixed post-mortem human brain tissue.; other: Mouse brains may be sectioned into 50 µm cryostat sections or 50–250 µm vibratome slices, depending on the protocol.; germline genetic engineering: Protocol-specific options include Thy1-YFP transgenic mice and split-GAL4 fly lines; these are not universal ExM requirements.; genetic engineering: The protein-barcode protocol specifically requires surgical AAV injection with analgesia at least three weeks before terminal perfusion; this is not universal.; other: Labels are applied by antibodies or gel-anchorable labels; labeling may occur before polymerization, before ExM, or after expansion depending on the protocol.; other: Specimens are embedded in swellable hydrogel. Human pathology protocols first use AcX to anchor proteins; the original method uses gel-anchorable labels.; other: Tissue is chemically softened after polymerization by proteolysis or by heat/detergent denaturation; archival human tissue uses high-temperature, pressurized treatment.; other: Water swelling expands the gel about 4–4.5×; ExRPath can re-embed and swell again to approximately 20×.

Theoretical spatial resolution

3 nm (point spread)

Theoretical temporal resolution

n/a

Description

Expansion microscopy physically magnifies fixed tissue so that conventional diffraction-limited fluorescence microscopes can resolve nanoscale structure. Labels such as fluorophore-conjugated antibodies or fluorescent proteins, or proteins treated with an anchoring reagent, are chemically linked to a swellable polyacrylamide/polyacrylate gel synthesized within the specimen. Proteolytic digestion, or heat and detergent softening, removes mechanical constraint, and dialysis in water swells the gel isotropically, about 4.5 fold in one round. Re-embedding in a second gel or other protocols reach about 16 to 20 fold. Unanchored lipids and other components are removed, leaving a gel nearly index matched to water. Effective resolution equals the microscope's resolution divided by the expansion factor, so spinning-disk confocal or lattice light-sheet imaging reaches tens of nanometres. In fixed mouse brain, images resolve dendritic spine morphology, myelination patterns and neurites segmented for neuron tracing. Paired presynaptic Bassoon and postsynaptic Homer1 or PSD95 puncta mark putative synapses that can be assigned to neurites, supporting dense connectomic reconstruction. In the whole Drosophila brain, imaging quantified bouton variation and synapse counts per region. In formalin-fixed post-mortem human brain, adapted anchoring and softening steps expanded tissue about 20 fold for immunostaining of nanostructure.

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