Light-sheet microscopy
Image cleared tissues or live embryos quickly with little photodamage.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
1Light only the plane you are looking at
In a light-sheet microscope the illumination and the detection come from different directions. A thin sheet of laser light enters the sample from the side, and a detection objective at right angles images that sheet onto a camera.
The sheet's trade-off
A focused Gaussian sheet is thinnest at its waist and spreads beyond it. Its usable length grows with the square of its thickness: about 50 µm for a 2 µm sheet, 200 µm for 4 µm, 800 µm for 8 µm. A thin sheet sections well but only across a narrow band.
Dual illumination
Light from two opposite sides lets each sheet cover only half the field, so a thinner sheet stays thin everywhere, and the far side is not dimmed by absorption along the way.
Shadows
Anything that absorbs or scatters casts a shadow along the sheet. Pivoting the sheet through a small angle during each exposure averages the shadows out.
2Live samples
Because the dose per volume is so low, light-sheet microscopy can follow whole embryos for hours or days: every cell of a zebrafish or fly embryo dividing and migrating. Samples are held in soft, low-concentration agarose, often inside fluorinated (FEP) tubes whose refractive index is close to water's, so the sheet and the image are not distorted.
3Cleared tissue
Tissue is opaque because lipids, proteins and water have different refractive indices, so light scatters at every interface. Clearing removes the lipids and soaks what remains in a medium of matching index.
Key terms
Practise these as flashcards →A thin plane of excitation light, made by a cylindrical lens or by scanning a focused beam, that lights only the focal plane of the detection objective.
The distance from the sheet's waist over which its thickness grows by √2; it sets the usable field for a given thickness.
Rocking the sheet through a small angle during each exposure so that shadows cast from different angles average out.
Removing lipids and matching the refractive index of what remains, so light passes through the tissue without scattering.
Imaging the cleared sample in a medium of the same index (RIMS ≈ 1.46, DBE ≈ 1.56), without which it turns turbid again.
Staining an intact organ with antibodies, which must diffuse in over days to weeks.
A fluorinated polymer tube with a refractive index close to water, used to hold live samples in the light path.
When to use it
- Long time-lapse imaging of whole living embryos, organoids or small organisms with minimal photodamage.
- Imaging entire cleared organs, such as a mouse brain, at cellular resolution in hours.
- Fast volumetric imaging where a point scanner would be too slow, such as whole-brain calcium imaging in larval zebrafish.
Compared with alternatives
Higher-NA optical sections of smaller samples; lights the whole depth for every slice, so slower and harsher for large or living volumes.
Simplest and fastest for thin samples, but with no sectioning at all.
The antibody labelling that whole-mount staining scales up to entire organs.
Light-sheet volumes run to terabytes; segmentation, registration and tracking are a project of their own.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Making whole fixed organs transparent for light-sheet imaging. CLARITY is aqueous (hydrogel, SDS, index ≈ 1.46) and keeps fluorescent proteins; iDISCO+ is solvent-based (methanol, DCM, DBE, index ≈ 1.56), faster and clearer but quenches them, so labels are antibodies.
- CLARITY: hydrogel embedding and SDS delipidation over weeks (days with electrophoresis); imaging in RIMS-type media.
- iDISCO+: methanol dehydration, whole-mount antibody staining over 1–2 weeks, DCM and DBE clearing in days.
- Solvents shrink tissue and dissolve many plastics and glues; aqueous methods tend to swell it.
- Both need the sample imaged in its own index-matched medium.