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Light sheet fluorescence microscopy


Light sheet fluorescence microscopy (LSFM) uses a thin sheet of light to excite only fluorophores within the focal volume. Light sheet microscopes (LSMs) have a true optical sectioning capability and, hence, provide axial resolution, restrict photobleaching and phototoxicity to a fraction of the sample and use cameras to record tens to thousands of images per second. LSMs are used for in-depth analyses of large, optically cleared samples and long-term three-dimensional (3D) observations of live biological specimens at high spatio-temporal resolution. The independently operated illumination and detection trains and the canonical implementations, selective/single plane illumination microscope (SPIM) and digital scanned laser microscope (DSLM), are the basis for many LSM designs. In this Primer, we discuss various applications of LSFM for imaging multicellular specimens, developing vertebrate and invertebrate embryos, brain and heart function, 3D cell culture models, single cells, tissue sections, plants, organismic interaction and entire cleared brains. Further, we describe the combination of LSFM with other imaging approaches to allow for super-resolution or increased penetration depth and the use of sophisticated spatio-temporal manipulations to allow for observations along multiple directions. Finally, we anticipate developments of the field in the near future.

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Fig. 1: Canonical implementations of light sheet fluorescence microscopy.
Fig. 2: Illumination and detection set-ups.
Fig. 3: Mounting examples for sample chamber-based light sheet microscopes.
Fig. 4: Exploring the three-dimensional space.
Fig. 5: Exemplary image processing flowchart.
Fig. 6: Stitching, fusion, deconvolution and rendering of light sheet fluorescence microscopy data.
Fig. 7: Light sheet fluorescence microscopy for mouse developmental biology.
Fig. 8: Light sheet fluorescence microscopy for evolutionary developmental biology.
Fig. 9: Light sheet fluorescence microscopy for biomedical imaging.


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Download references


The Ceratitis, Apis and Gryllus live imaging data were acquired by a cooperation of F.S. and E.H.K.S. with M. F. Schetelig (Justus-Liebig-Universität, Gießen, Germany), P. Siefert and B. Grünewald (Institut für Bienenkunde, Oberursel, Germany) and T. Mito (University of Tokushima, Japan), respectively. The human prostate biopsy images were kindly provided by A. K. Glaser and J. T. C. Liu (Department of Mechanical Engineering, University of Washington, USA) and N. P. Reder and L. D. True (Department of Pathology, University of Washington, USA). F.S. and E.H.K.S. thank S. Plath for his assistance in generating the computer-assisted design schemes.

Author information




Introduction (E.H.K.S.); Experimentation (E.H.K.S., F.S., B.-J.C., K.M. and R.F.); Results (F.S., F.P., S.P., K.M. and R.F.); Applications (E.H.K.S., F.S., B.-J.C., K.M. and R.F.); Reproducibility and data deposition (E.H.K.S., F.P., S.P. and K.M.); Limitations and optimizations (B.-J.C., S.P., K.M., S.P. and R.F.); Outlook (E.H.K.S., F.S., S.P., F.P., K.M. and R.F.); Overview of the Primer (E.H.K.S., F.S., B.-J.C., K.M., R.F., F.P. and S.P.).

Corresponding authors

Correspondence to Ernst H. K. Stelzer or Frederic Strobl.

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E.H.K.S. has shares in related patents. The other authors declare no competing interests.

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Nature Reviews Methods Primers thanks J. Liu, who co-reviewed with A. Glaser; P. Mondal; and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Focal volume

The planar volume in front of an objective, from which a sharp image can be obtained. It is proportional to the depth of field of the detection objective and the field of view.


Portmanteau term of ‘volumes’ and ‘elements’, referring to single points of a three-dimensional grid.

Diffraction limit

The maximum achievable optical/spatial resolution of an image recorded with an optical microscope, equivalent to about half the wavelength of the illumination light.

Illumination train

The optical path used for forming the light sheet and excitation of fluorophores.

Detection train

The optical path used for collection of the emission signal.

Coherent illumination

Illumination with light composed of wave sources of the same frequency, waveform and phase; scattered laser light retains the original phase.

Numerical aperture

(NA). A refraction index-based dimensionless number that states the maximum half-angle across which an optical element, such as an objective, emits and collects light. The NA influences both the lateral and the axial resolutions.

Gaussian beams

Pencil-shaped diffracting focused beams whose planar profiles perpendicular to the beam axis are described with two-dimensional Gaussian functions.

Bessel beams

Non-diffracting, self-reconstructing beams generated with an axicon or a spatial light modulator.

Airy beams

Non-diffracting, self-reconstructing beams generated with a spatial light modulator.

Free working distance

The distance between the front surface of an objective and the centre of the focal volume.

Volume of view

The volume covered by the field of view (along the x axis and y axis) and the free working distance of the detection objective (along the z axis).

Lateral resolution

Spatial resolution along the x axis and y axis.

Oversampling and undersampling

The collection of redundant and less than available spatial/temporal information, respectively.

Axial resolution

Spatial resolution along the z axis.

Point spread functions

The images of point sources, often used to describe the response or the resolution of an imaging system.

High-content imaging

Collection of a large amount of complementary information from the same specimen.

Air objectives

Objective lenses that operate in air or vacuum; these cannot achieve the high and very high numerical apertures of water or oil immersion objectives.

Water-dipping objectives

Objective lenses that operate in aqueous or organic media; these can have higher numerical apertures (NAs) than air objectives, but not the very high NAs of oil objectives.

Isotropic resolution

Identical resolution along the x axis, y axis and z axis.


Describes a metazoan species that lays eggs with no or partial embryonic development in the parent.

Fluorinated ethylene propylene

(FEP). A synthetic material with a refractive index close to that of water (1.33), available as threads, foils and tubes.


Describes a metazoan species in which the embryos develop within the body of the parent.


In developmental biology, the process of removing a developing embryo from the uterus of the parent for experimentation.


In developmental biology, the time period between fertilization of the oocyte and implantation of the blastocyst into the uterine wall.


Water-soluble anionic polysaccharide of bacterial origin with similar biophysical and optical properties to agarose.

Rolling shutter

The line-wise recording of scientific complementary metal–oxide–semiconductor sensors; in light sheet fluorescence microscopy it is synchronized with line-wise illumination to record anisotropic confocal images along the y axis.

Spectral unmixing

Data processing procedure in which the spectral signatures of voxels are divided into collections of constituent spectra.

Fourier transform

A mathematical operation that transforms a real intensity-based image into a spatial frequency-based array. These arrays are used to perform mathematical operations in spatial frequency space and inverse Fourier transforms are used to calculate real images.

Fluorescence correlation spectroscopy

A time correlation-based statistical imaging method for the quantification of fluorescence fluctuations that is used to measure diffusion coefficients or characterize reaction kinetics.

Fluorescence lifetime imaging

The imaging of the spatial distribution of the lifetimes of fluorophores in a specimen; fluorescence lifetimes are sensitive to their close environment.

Hot pixels

Dysfunctional pixels that appear white in every image independent of the actual signal.

Dead pixels

Dysfunctional pixels that appear black in every image independent of the actual signal.


In fluorescence microscopy image processing, the combination of multiple partially overlapping stacks recorded along the same direction to generate larger three-dimensional images.

Multi-view fusion

In fluorescence microscopy data processing, the calculation of a single three-dimensional image based on multiple image stacks recorded along multiple directions of an opaque specimen.


A mathematical approach to take advantage of a priori information regarding the properties of an optical system to emphasize certain spatial frequencies and, in consequence, improve the resolution of an image.


Describes parallelized image processing using blocks such as fractions of a line, area or volume to process data on multiple computation and graphic processing units.

Hierarchical data formats

File formats that utilize a file directory-like structure to organize the data within the file in a structured fashion.

Lossy image compression

A data size-reduction approach based on approximation and/or partial data discard; the original data cannot be completely reconstructed.


The rearrangement of bits to, for example, allow or simplify image processing.


Detection and highlighting of morphological features, for example cell membranes or nuclei, in a two-dimensional or three-dimensional image.


Multicellular, usually densely packed, three-dimensional tissue-like cell culture models.


A miniaturized and functionally simplified cell culture version of an organ.


Cells at the surface of plant roots that are responsible for the formation of root hairs.


Describes light that is able to induce photochemical reactions.

Beam divergence

In Gaussian beams, the increase in beam radius as a function of distance from the beam waist.

Point spread function engineering

The modification of the plane wave usually encountered in the entrance aperture of a lens to change the properties of the point spread functions. For example, Bessel beams are generated by point spread function engineering.

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Stelzer, E.H.K., Strobl, F., Chang, BJ. et al. Light sheet fluorescence microscopy. Nat Rev Methods Primers 1, 73 (2021).

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