Light-sheet microscopy
A fluorescence microscopy method that illuminates only the plane being imaged with a thin sheet of light sent in from the side, at 90° to the detection objective. Gaussian sheets are typically 2–6 µm thick over a 20–150 µm long field, so out-of-focus excitation and photobleaching are much lower than in widefield or confocal imaging.
Light-sheet microscopy (also called selective plane illumination microscopy, SPIM) is a form of fluorescence microscopy in which the excitation light enters the sample from the side as a thin sheet, and a separate detection objective, at right angles to the sheet, images the illuminated plane onto a camera. Only the plane in focus is excited, so each camera frame is an optical section without a pinhole, and the rest of the sample receives no light while that plane is recorded. Typical Gaussian sheets are 2–6 µm thick and stay thin over a field 20–150 µm long; scanning the sample (or the sheet and detection plane together) through the volume builds a three-dimensional image plane by plane.
Illumination geometry
The sheet is formed either statically, by a cylindrical lens that focuses an expanded laser beam in one axis only, or by scanning a thin round beam across the field with a galvanometer mirror during each camera exposure (the digitally scanned light sheet). In both cases the profile across the sheet is that of a focused Gaussian beam with waist radius , so the sheet thickness at the center is . The detection objective is usually a water-dipping lens of moderate to high numerical aperture.
Thickness and field of view
A Gaussian beam stays within of its waist over twice the Rayleigh range,
where is the vacuum wavelength and the medium index. Taking as the usable field length, the field grows as the square of the thickness. At 488 nm in water ( = 1.33):
| Sheet thickness | Field | |
|---|---|---|
| 2 µm | 8.6 µm | 17 µm |
| 4 µm | 34 µm | 68 µm |
| 6 µm | 77 µm | 154 µm |
Halving the thickness to improve sectioning shrinks the field fourfold. Whether the sheet sets the axial resolution depends on the detection optics: a water-immersion objective of NA 0.8 at 520 nm emission has an axial point spread function about 1.7 µm wide (full width at half maximum), so a 4 µm sheet improves contrast by rejecting background but leaves axial resolution to the objective, while with low-NA detection objectives the sheet thickness dominates.
Bessel and lattice sheets
Two families of beams relax the thickness-field trade-off. A Bessel beam, made with an axicon or an annular aperture in a pupil plane, has a narrow central core that does not spread over a long distance, but it carries a large fraction of its power in concentric side lobes; scanned to form a sheet, the lobes excite out-of-focus fluorescence unless removed by two-photon excitation, structured illumination or confocal slit detection on the camera. Lattice light sheets, introduced by Chen and colleagues in 2014, use a spatial light modulator and an annular mask to create a two-dimensional optical lattice whose interference suppresses the side lobes; dithered across the field, it gives a sheet of about 1 µm or less over tens of micrometers. Axially swept sheets, whose waist moves across the field in step with a rolling camera shutter, address the same trade-off.
Where it is used
Light-sheet microscopy is the standard method for long time-lapse imaging of living embryos and organoids, where phototoxicity limits confocal microscopy: a confocal scan exposes the whole depth for every plane recorded, while a light sheet exposes each plane only when it is imaged. It is also used for chemically cleared tissue, including whole mouse brains several millimeters across, where the speed of camera-based acquisition (an entire plane per exposure) matters as much as the light dose.
Pitfalls
- Stripes and shadows. Absorbing or scattering structures cast streaks along the illumination direction. Illuminating from two opposite sides, or pivoting the sheet by a few degrees during each exposure, reduces them.
- Sheet-focus mismatch. A sheet offset from the detection focal plane, or tilted relative to it, blurs one side of the image; the overlap drifts with temperature and with refractive index changes in the medium.
- Thickness varies across the field. The sheet is thicker at the field edges than at the waist for a Gaussian beam, so sectioning is not uniform.
Common questions
How does light-sheet microscopy differ from confocal microscopy?
Confocal microscopy illuminates through the same objective used for detection and rejects out-of-focus light with a pinhole, so the whole cone of light above and below the focus excites fluorophores. A light sheet confines excitation to the imaged plane from the start, and a camera records every pixel of that plane at once, which lowers the light dose and raises the speed.
What sets the axial resolution in light-sheet microscopy?
The thinner of two quantities: the sheet thickness and the axial extent of the detection objective's point spread function. With high-NA detection the objective usually dominates; with low-NA detection, used for large fields, the sheet thickness does.
References: J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt and E. H. K. Stelzer, "Optical sectioning deep inside live embryos by selective plane illumination microscopy," Science 305, 1007 (2004); B.-C. Chen et al., "Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution," Science 346, 1257998 (2014); B. E. A. Saleh and M. C. Teich, Fundamentals of Photonics, 3rd ed. (Wiley, 2019).