All about Light-Sheet Microscopy

Discover more details about Light-Sheet Microscopy and its advantages when it comes to fast 3D fluorescence imaging of biological samples.

Technology Details

What is “Light-Sheet Microscopy”?

Light-sheet microscopy is a fluorescence imaging technique, which utilizes a sheet of laser light to illuminate only a thin slice of the sample.

The basic technical principle is a wide-field fluorescence microscope, placed perpendicular to the light-sheet, that collects the fluorescence signal and images of the observed region by means of a full-frame camera. The orthogonal arrangement that decouples the illumination from the detection enables intrinsic 3D optical sectioning, as compared to other fluorescent imaging techniques like confocal and spinning disc microscopy. As a result, the method features drastically reduced overall acquisition duration, photobleaching effects and phototoxicity, as well as yields excellent signal-to-noise ratio and enables high temporal and 3D-spatial resolution.

Light-sheet fluorescence microscopy (LSFM) can be utilized to image a huge variety of fixed, live or cleared biological samples. Applications of light-sheet microscopy can range from imaging of subcellular structures and rapid inter- and intracellular processes to the acquisition of the long-term development of a model system, to the complete visualization of a macroscale cleared sample.

Confocal laser scanning vs. Light-Sheet

Types of Microscopes

Our microscopes can be subdivided into three classes based on the position and number of the illumination and detection objectives.

Horizontal Multiple-view Set-up

MuVi SPIM illumination and detection concept
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The multiview light-sheet microscope, MuVi SPIM, features the illumination and detection objectives along the horizontal plane of the microscope. The unique 4-axis concept enables two orthogonal views of the specimen without the need for rotation of the sample. Simultaneous acquisition from two detection sides enables unparalleled acquisition speed, correction of shadowing effects and high precision of data fusion.

Altogether, the horizontal multiple-view configuration is optimal for imaging large gel-embedded samples or cleared samples. It facilitates upright or inverted sample mounting and enables tile scan imaging.

Inverted Set-Up

InVi SPIM illumination and detection concept
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The inverted light-sheet microscope, InVi SPIM, features the illumination and detection objectives below the sample. This asymmetric configuration is ideal for imaging of 2D and 3D cell culture applications as well as small embryos at subcellular resolution. Single view acquisition reduces image processing demands.

Sample Holder

The V-shaped, 3 cm long sample holder is placed inside of the chamber. It is covered with FEP foil allowing separation of the sample from the chamber, enabling the use of small sample medium volume, without influencing imaging properties.

Sample chamber InVi SPIM
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Different strategies can be used to place your sample in the holder for imaging. 2D cells can be grown directly on the foil as in a “curved coverglass”. 3D spheroids or small embryos can be dropped into the trough and held by gravitation, allowing multiple samples to be arranged for multi-position imaging.

Up-Right Set-up

The upright light-sheet microscope, QuVi SPIM, features symmetric illumination and detection objectives on top of the sample for dual view acquisition. This configuration is suitable for samples mounted on slides or SBS plates and facilitates high content imaging of screening applications or widespread samples.

QuVi SPIM illumination and detection concept
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Environmental Control

The LUXENDO Light-Sheet microscopes provide precise and stable temperature and environmental control.

The systems (i.e. MuVi SPIM, InVi SPIM, and QuVi SPIM) can be equipped with environmental control. A Peltier based water cooling/heating system is available in the MuVi SPIM and the InVi SPIM (cooling is not yet possible in the QuVi SPIM). The immersion medium is kept at a homogeneous temperature, while the heated lid prevents condensation. Temperature can be adjusted between 20–37 °C for optimal incubations conditions.

In addition, the InVi SPIM and the QuVi SPIM also provide precise and stable environmental control (i.e. CO2, O2, N2, and humidity). Gas-concentration for the different components ranges between 0–15 % for CO2, 1–21 % for O2 and 20–99 % for H2O (humidity). The gas humidifier offers feedback control for precise regulation.

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Optical Sectioning

Optical sectioning refers to the generation of clear images of specific focal planes within a 3D structure. Good Z resolution enables the 3D reconstruction of a sample.

Fluorescence microscopy, e.g. confocal microscopy, spinning disk confocal and light-sheet microscopy, enable optical sectioning. Confocal Microscopy and Spinning Disk Microscopy image a specific focal plane by point scanning the sample and rejecting out of focus fluorescent signal with a pinhole(s). These techniques enable high-resolution image acquisition at the expense of photo-damaging effects and/or high time consumption.

Light-Sheet Microscopy offers intrinsic optical sectioning by the specific illumination of one particular focal plane. This is achieved by the orthogonal arrangement of the illumination and detection objective lenses as well as the projection of a thin light-sheet on the sample. Intrinsic optical sectioning significantly reduces photo-bleaching and phototoxic effects offers high acquisition speed and the possibility to perform long-term experiments.

Optical sectioning in light-sheet microscopy
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Why your next confocal should be a light-sheet microscope?

Light-Sheet Fluorescence Microscopy is the method of choice for long-term, high interval (minutes to days) live sample imaging.

Most of the models used in confocal microscopy are suitable for light-sheet microscopy. Due to its unique capabilities, additional challenging specimens are included in the spectrum of samples that can be acquired with a light sheet microscope.

Compared to confocal laser scanning and spinning disk confocal microscopy, light-sheet microscopy enables fast, high resolution, true volume, and in-depth imaging with the following major advantages:

Low Photobleaching

Photobleaching refers to the permanent loss of ability to fluoresce due to light-induced damage of the fluorophore molecules in a sample. Long-term exposure to light, especially in time-lapse studies, induces photobleaching, hindering the detection of the fluorescent molecules.

Photobleaching after several excitation and emission cycles
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A comparison of the photobleaching rates of light-sheet microscopy, spinning disk, and confocal microscopy reveals a reduction in photobleaching when working with light-sheet microscopy. The effect is already visible when imaging a single plane @ 100 fps, but the difference becomes particularly astonishing when comparing imaging of a stack of 40 µm (1 µm steps) @ 100 fps.

Comparison of photobleaching rates of light-sheet microscopy, spinning disk and confocal microscopy. Illumination intensity set to obtain 5 photons per fluorophore per frame.

  • Parameters
    • InVi SPIM: 62x/1.1NA, 2048 × 2048 pixel, pixel size 100 × 100 nm, light-sheet thickness 2 µm, illumination time per voxel 25 µs
    • Spinning disk confocal: 60x/1.2NA, 2048 × 2048 pixel, pixel size 100 × 100 nm, pinhole diameter 50 µm or 1.5 Airy units, pinhole distance 250 µm, illumination time per voxel 10 µs
    • Point confocal: CLSM with 10k resonant scanner; 60x/1.2NA, 200 × 200 pixel, pixel size 250 nm, pinhole diameter 50 µm or 1.5 Airy units, illumination time per voxel 0.5 µs
    • Fluorescence lifetime: 2.5 ns
    • 
Intersystem crossing rate: 2.5·106 s-1
    • Triplet lifetime: 5 µs
    • Bleach rate: 100 s-1 at 1 kW cm-2

    References

    Harms, G.S., et al. (2001). Autofluorescent proteins in single-molecule research: applications to live-cell imaging microscopy. Biophys. J. 80: 2396-2408.

    Im, K.B., et al. (2013). Diffusion and binding analysed with combined point FRAP and FCS. Cytometry A 89: 876-889.

Low Phototoxicity

Long-term imaging can have phototoxic effects on the sample, altering the normal behavior of the cells and the whole specimen.

Phototoxicity in cell culture over time
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Light-sheet microscopy stands out for its effective use of excited photons, which minimizes phototoxic effects. This contributes to prevent the generation of misleading and artificial results.

The study from Jemielita et al. (2013) brings out seemingly imperceptible phototoxic effects induced by long-term exposure to light. The comparison of light-sheet microscopy and spinning disk microscopy images revealed inappropriate bone development in zebrafish due to photo-damage in spinning disk microscopy.

Source article

Jemielita, M. et al. (2013) Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques. Biophotonics 6 (11-12): 920-8

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High Temporal and Spatial Resolution

Light-sheet microscopy enables high imaging speed and the possibility to capture a higher number of events. This is of particular relevance in fast occurring dynamic processes.

Drosophila embryonic development
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A study by Reichmann et al. (2018) carried out at EMBL serves as an example. It shows that during the first cell division in mouse embryos, the maternal and paternal chromosomes remain separated. Only light-sheet microscopy made these findings possible.

Source article

Reichmann, J. et al. (2018) Dual-spindle formation in zygotes keeps parental genomes apart in early mammalian embryos. Science, published online.

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