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 fluorescence microscopy

Types of Light-Sheet 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

The inverted light-sheet microscopes, InVi SPIM and TruLive3D Imager, feature the detection objective below the sample. Single view acquisition reduces image processing demands in the InVi SPIM, while dual sided-illumination and an extended sample chamber enable fast acquisition speed and multi-position imaging. This configuration is ideal for 2D and 3D cell culture applications as well as imaging small embryos at subcellular resolution.


Sample Holder

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

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.

Sample chamber InVi SPIM (Light-sheet Microscopy)
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TruLive3D Dishes

The NEW commercially available, ready-to-use TruLive3D Dishes have been designed to further facilitate sample mounting in the InVi SPIM and the TruLive3D Imager. Powered by ibidi, the dishes are easily exchangeable, sterile, customizable, disposable and biocompatible. Grow your samples on the foil or place them into the dish wells. For imaging, simply slide the TruLive3D Dish into the adapter attached to the microscope. The dishes seamlessly integrate into the large chamber of the TruLive3D Imager, which can fit up to three of them.

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 in Live-Cell Imaging

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.

Environmental and Temperature control for Live Imaging
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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
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.

  • 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
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.

Embryonic development in Drosophila
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.

Read article

Cleared-sample / Cleared-Tissue Imaging

Tissue clearing techniques have become a valuable tool for applications in 3D microstructure analysis of tissues (e.g. neuroscience, developmental biology, connectomics).

The different refractive indexes (RI) of the major components of biological tissue, i.e. water, lipids and proteins result in light scattering when light passes through the tissue. Tissue clearing modifies the optical properties of usually opaque samples to render them transparent while keeping their structure and fluorescent labels intact. After clearing, light can travel many millimeters through a specimen unrestricted from absorption and scattering, ideal for high-resolution microscopic imaging deep within the specimen.

Light-Sheet Microscopy leverages the optical advantages of cleared samples enabling fast, long-term, confocal-like optical sectioning and high-quality 3D imaging of cleared samples.

Sketch of a cleared mouse embryo

Methods for Optical Clearing

Tissue clearing methods homogenize the RI of a sample by removing, changing or replacing some components.

Clearing methods can be grouped into two categories:

  • Solvent-based clearing methods (e.g. uDISCO, 3DISCO, BABB)
  • Aqueous-based clearing methods
    • Simple immersion (e.g. SeeDB, FAST-Clear)
    • Hyperhydration (e.g. CUBIC, ScaleS)
    • Hydrogel embedding (e.g. CLARITY, PACT/PARS)

No single clearing method will work for all tissue types, tissue sizes and/or experiments.

Solvent-based clearing methods

Advantages Disadvantages
High quality clearing Toxic and/or corrosive
High-clearing speed Not suitable for lipid staining

Aqueous-based clearing methods

Advantages Disadvantages
Preservation of fluorescent protein emission Slow clearing
Preservation of lipids Not suitable for big samples
Preservation of tissue architecture

Challenges of Imaging Cleared Samples

  • Sample mounting
    • Mounting of large & mechanically delicate samples (e.g. mouse brain > 1cm3)
    • Reproducible positioning of soft samples on the microscope stage
  • Refractive Index
    • Mismatch between the RI of the sample and the RI of the immersion media of the objective lens
    • Low order aberrations cause significant image quality degradation
  • Working distance
    • Mismatch between RI of the cleared sample and the RI of the medium
    • Large samples require long working distances
    • Aberrations limit the long working distance of dry and water lenses
    • The working distance changes if the immersion media change

The MuVi SPIM CS and the LCS SPIM provide new solutions in sample mounting, sample size, and optics to enable best-in-class, high-quality 3D imaging of cleared samples.

These systems are optimized for 3D imaging of intricate tissue structures, relevant to study the brain or the central nervous tissue in neuroscience, to analyze organ development or to investigate tumor structure and genesis in oncology.

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If you have any question regarding your microscopes functionality or you want to schedule a maintenance visit, then get in contact with LUXENDO’s support team. We will ensure to get the right person involved to answer your request.

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