Super-resolution
Stimulated emission depletion: a doughnut-shaped depletion beam shrinks the effective excitation spot of a scanning confocal. Resolution improves with depletion power, at the price of bleaching and smaller pixels.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
STED
Stimulated emission depletion: a doughnut-shaped depletion beam shrinks the effective excitation spot of a scanning confocal. Resolution improves with depletion power, at the price of bleaching and smaller pixels.
How it differs from the standard method
- Needs dyes that are depleted at the STED wavelength (e.g. far-red dyes with a 775 nm depletion laser).
- Pixel size must shrink with the spot (Nyquist ≈ resolution / 2.3).
- Index-matched mounting and doughnut alignment keep the centre dark.
1The limit, and four ways round it
A lens cannot focus light to a spot much smaller than half its wavelength: with the best oil objective, two points closer than about 200 nm laterally (and 500 nm axially) blur into one. Most of the cell's machinery is smaller than that.
STED
A scanning confocal with a second, doughnut-shaped beam that forces excited molecules back to the ground state by stimulated emission everywhere except the doughnut's centre. Resolution λ/(2NA√(1 + I/I_sat)) improves with depletion power.
SIM
The sample is lit with fine stripes at several angles and phases. Moiré fringes carry detail beyond the passband into it, and a reconstruction doubles the resolution. Fast and gentle, works with ordinary dyes, but prone to reconstruction artefacts.
STORM / PALM
Only a sparse few molecules are switched on in each camera frame; each is localised to a few nanometres from the centre of its blurred spot, and thousands of frames are added into an image of positions.
Expansion
Labels are anchored into a swellable gel, the tissue is digested, and the gel swells isotropically in water. An ordinary confocal then resolves what was 4× (or 10×) smaller.
2What each one costs
There is no free resolution. In STED, more depletion means a smaller spot but more light on every molecule, so bleaching rises steeply, and the pixels must shrink to sample the smaller spot, which adds dose again.
3The label sets a floor
A primary plus a secondary IgG holds the dye 10–20 nm from its target, and that displacement is part of every super-resolution image: a 25 nm microtubule looks 50–60 nm wide even with perfect optics. Smaller labels (nanobodies, directly labelled primaries, tags) matter as much as the microscope once resolution passes about 50 nm.
Key terms
Practise these as flashcards →The smallest resolvable distance for a lens, about λ/(2NA) laterally (~200 nm for visible light and a 1.4 NA objective).
Using a red-shifted beam to force excited molecules to emit at its wavelength, so only those at the doughnut's dark centre fluoresce normally.
How strongly the SIM stripe pattern is imprinted on the raw images; low modulation gives noisy, artefact-prone reconstructions.
The regularisation in SIM reconstruction: low values sharpen but amplify noise into artefacts; high values smooth.
How well a single molecule's position is known, roughly the PSF width divided by √(photons), worsened by background.
Oxygen-scavenging buffer with a thiol (e.g. MEA) that drives cyanine dyes into a reversible dark state so they blink.
Bright, stable beads used to measure and correct stage drift over long localisation acquisitions.
The ratio of gel size after and before expansion (~4× for proExM); resolution improves by the same factor.
The distance between the dye and its target set by the label (about 10–20 nm for primary plus secondary antibodies).
When to use it
- When the structure is smaller than ~200 nm: synaptic proteins, nuclear pores, cytoskeletal arrangements, organelle contacts.
- SIM for fast, multicolour, live-compatible imaging at about twice widefield resolution.
- STED for live or fixed imaging at 30–80 nm in a confocal workflow, with suitable dyes.
- STORM/PALM for the highest resolution and molecule counting in fixed samples; expansion when only a confocal is available or for thick tissue.
Compared with alternatives
Diffraction-limited but simpler, gentler and faster; choose it when 200 nm is enough.
Electron microscopy reaches nanometres with full ultrastructural context, but without specific multicolour labelling or live imaging.
The labelling step behind most super-resolution images; label size and density limit what any method can show.
Reports whether two proteins are within ~40 nm without resolving them optically.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Stimulated emission depletion: a doughnut-shaped depletion beam shrinks the effective excitation spot of a scanning confocal. Resolution improves with depletion power, at the price of bleaching and smaller pixels.
- Needs dyes that are depleted at the STED wavelength (e.g. far-red dyes with a 775 nm depletion laser).
- Pixel size must shrink with the spot (Nyquist ≈ resolution / 2.3).
- Index-matched mounting and doughnut alignment keep the centre dark.